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A kinetic analysis of transcription initiation by the Bacillus subtilis sigma-43 RNA polymerase : the… Dobinson, Katherine Frances 1986

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A K I N E T I C A N A L Y S I S OF T R A N S C R I P T I O N I N I T I A T I O N BY THE B A C I L L U S S U B T I L I S S I G M A - 4 3 RNA P O L Y M E R A S E . THE E F F E C T OF THE DELTA S U B U N I T by KATHERINE B.Sc.  The  A THESIS  F R A N C E S DOBINSON  University  SUBMITTED  of  IN  British  PARTIAL  Columbia,  1980  FULFILLMENT  OF  THE R E Q U I R E M E N T S FOR THE DEGREE OF DOCTOR OF P H I L O S O P H Y in THE F A C U L T Y OF GRADUATE Department  We a c c e p t to  this the  of  Microbiology  thesis  required  THE U N I V E R S I T Y © Katherine  STUDIES  as  conforming  standard  OF B R I T I S H Dobinson,  COLUMBIA 1986  In  presenting  degree  at  this  the  thesis in  University of  partial  fulfilment  of  British Columbia, I agree  freely available for reference and study. I further copying of  this  department  or  publication of  thesis for by  his  or  her  that the  for  an advanced  Library shall make  it  It  is  granted  by the  understood  that  head of copying  my or  this thesis for financial gain shall not be allowed without my written  Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  representatives.  requirements  agree that permission for extensive  scholarly purposes may be  permission.  Date  the  t\W-ftk  ABSTRACT The  initiation  subtilis  sigma-M3  promoters have  and  of  RNA p o l y m e r a s e  the  effect  been i n v e s t i g a t e d  templates  for  4>29 A2 o r  G2 p r o m o t e r .  A2  promoter The  using  the  are  of  initiation with  polymerase initiated  alone  s h o w n by  than  A2.  the  two  promoters  the  of  delta  which  the  suggested  initiation  carried  the  form  assay  The the  of  the  to  that  were  the  while  formation required t h e RNA  on c o m p l e x  at  kinetic  affects  non-  be a s t r o n g e r  the  lowered  A2  was a l s o  the  promoter. of  investigated.  efficiency  complexes undergo  at  formation  parameters  the  promoter  single-round  but  the  was  formation  on t h e  G2 p r o m o t e r  formation  ii  which  The G2 p r o m o t e r  h a d no e f f e c t  delta  in  A2 p r o m o t e r  with  A2 p r o m o t e r  enzyme/promoter  promoter  examined  heparin-resistant,  subunit  the  were  assay  nucleotides,  on t h e  the  that  initiation  localization  G2 p r o m o t e r .  Delta  The  at  and  a single  was i n v e s t i g a t e d  complex  formation  which  was o b s e r v e d  delta  of  data  at  the  and e x t e n t effect  plasmids  transcription  to  complexes at  The  analysis.  complexes at  of  complex  kinetic  It  assay.  rate  vitro  run-off  transcription initiation  an iri  029  here.  was a b l e  of  phage  on  a competition  effect  Bacillus  subunit  RNA s y n t h e s i s  at  Bacillus  delta  initiating  complexes  also  The  the  the  two  The c l o n i n g  heparin.  presence of  at  by  the  events  heparin-resistant  the  by  reported  a single-round  inhibited  of  a n a l y s i s were  kinetics  multiple  of  transcription  the  with  transition(s) initiated, model  for  is  that  the  of  G2 p r o m o t e r s  not  efficiently  from  complex  but  delta  is  of  is  RNA s y n t h e s i s  delta  delta  delta,  complexes.  i i i  can at  were  proposed, from  essential  complexes  release  which  interpretations  release  associated with  and  initiation  other  effect  enzyme/promoter which  a complex  although the  postulated  to  for  in  thus  be  possible. which  it  A  is  the initiation.  interact the  can  weaker  slowing  with  both  Enzyme the  A2 p r o m o t e r the  formation  A2 do of  T A B L E OF CONTENTS Abstract Table  of  ii Contents  iv  List  of  Figures  vi  List  of  Tables  viii  List  of  Abbreviations  ix  Acknowledgements  x  1.  INTRODUCTION  II.  MATERIALS  1  AND METHODS  A.  P h a g e and  B.  Media  C.  Construction  D.  DNA-RNA H y b r i d i z a t i o n  E.  RNA P o l y m e r a s e  F.  Isolation  G.  Purification  H.  In  vitro  Plasmid  of  of  p328-5  1.  C l o n i n g and A2 P r o m o t e r  13 Analysis  14  Purification  15  Core/sigma of  the  18  Delta  Transcription  RESULTS  3.  12 13  III.  2.  DNA  Subunit  19  Assays  Localization  of  the  20  029 23  Purification Delta Factor  of  the  RNA P o l y m e r a s e  and 31  C h a r a c t e r i z a t i o n of the C o n d i t i o n s f o r the I n v i t r o S i n g l e Round T r a n s c r i p t i o n A s s a y  4.  C h a r a c t e r i z a t i o n of the E f f e c t of D e l t a G2 P r o m o t e r s  A.  Initiation  at  the  the I n i t i a t i o n R e a c t i o n o n I n i t i a t i o n a t t h e A2  A2 P r o m o t e r  i v  38 and and 54  B.  IV.  C h a r a c t e r i z a t i o n of a t t h e G2 P r o m o t e r  Methods  B.  C h a r a c t e r i z a t i o n of T r a n s c r i p t i o n Assay  D. E.  F. G.  Initiation  Reaction 89  DISCUSSION  A.  C.  the  for  Measuring I n i t i a t i o n the  In  Rates  96  vitro 98  C h a r a c t e r i s t i c s of H e p a r i n - r e s i s t a n t F o r m a t i o n a t t h e A2 P r o m o t e r  Complex  C h a r a c t e r i s t i c s of H e p a r i n - r e s i s t a n t F o r m a t i o n a t t h e G2 P r o m o t e r  Complex  100 105  The E f f e c t o f t h e D e l t a S u b u n i t on C o m p l e x F o r m a t i o n a t t h e A2 a n d G2 P r o m o t e r s  106  Model f o r Complex F o r m a t i o n G2 P r o m o t e r s  115  Comparison of the a n d G2 P r o m o t e r s  at  Sequences of  the  A2 a n d  t h e A2 119  REFERENCES  122  v  LIST T a b l e I.  OF TABLES  A s s a y f o r RNase and DNase a c t i v i t y delta preparation  i n the 41  Table I I .  Template c o m p e t i t i o n assay  Table I I I .  K i n e t i c parameters observed f o r d i f f e r e n t p r e p a r a t i o n s o f B a c i l l u s s u b t i l i s RNA polymerase . .88  vi  79  LIST Figure  1.  Restriction P328-5  OF F I G U R E S a n a l y s i s of  the  plasmid 26  Figure  2.  R e s t r i c t i o n map o f  Figure  3.  H y b r i d i z a t i o n of p328-5 t r a n s c r i p t s P 3 2 8 - 5 a n d $29 DNA r e s t r i c t i o n fragments  Figure Figure  Figure Figure Figure Figure  Figure  Figure  Figure  Figure Figure Figure  4. 5.  6. 7. 8. 9.  10.  11 .  12.  13. 14. 15.  plasmid  A s s a y f o r DNase a c t i v i t y polymerase preparation  in  p328-5.  27 to 30  t h e RNA 33  SDS-polyacrylamide gel electrophoresis o f p u r i f i e d B a c i l l u s s u b t i l i s RNA polymerase  37  SDS-polyacrylamide gel of p u r i f i e d d e l t a  40  Assay delta  for proteolytic preparation  electrophoresis activity  in  the 43  E f f e c t o f h e p a r i n c o n c e n t r a t i o n on t r a n s c r i p t i o n f r o m t h e A2 p r o m o t e r The e f f e c t o f enzyme c o n c e n t r a t i o n on t h e l e v e l o f t r a n s c r i p t i o n f r o m A2 p r o m o t e r E f f e c t of the i n i t i a t i o n time on t h e l e v e l o f t r a n s c r i p t i o n A2 p r o m o t e r  47 the 50  interval from the 53  The e f f e c t o f t h e e l o n g a t i o n t i m e i n t e r v a l on t h e l e v e l o f transcription f r o m t h e A2 p r o m o t e r  56  Nucleotide requirements for r e s i s t a n t complex formation promoter  59  E f f e c t of d e l t a t h e A2 p r o m o t e r Effect at the  heparina t t h e A2  on t r a n s c r i p t i o n  o f d e l t a on c o m p l e x A2 p r o m o t e r  from 65  formation  S e m i l o g a r i t h m i c p l o t of the data from A G - i n i t i a t i o n time courses i n F i g u r e 12a and b  vii  68  71  Figure  Figure  Figure  Figure  16.  17.  18.  19.  E f f e c t o f d e l t a o n t h e maximum l e v e l o f t r a n s c r i p t i o n f r o m t h e A2 p r o m o t e r a t d i f f e r e n t enzyme c o n c e n t r a t i o n s  73  E f f e c t o f d e l t a on t h e of i n i t i a t e d complexes  76  stability  Example of d a t a o b t a i n e d from i n i t i a t i o n time c o u r s e s f o r use tau p l o t a n a l y s i s  in  Tau p l o t f r o m a n a l y s i s o f c o m p l e x f o r m a t i o n a t t h e A2 p r o m o t e r  85 87  Figure  20.  Nucleotide requirements for stable c o m p l e x f o r m a t i o n a t t h e G2 p r o m o t e r . . . . 92  Figure  21.  E f f e c t o f d e l t a on i n i t i a t i o n t h e A2 a n d G2 p r o m o t e r s  from  Model for the i n i t i a t i o n at promoters  on  Figure  Figure  22.  23.  e f f e c t of d e l t a t h e A2 a n d G2  95  116  Promoter sequences of G2 p r o m o t e r s  viii  the  A2 a n d 120  LIST  OF A B B R E V I A T I O N S  b.p.  Base  pair  BSA  Bovine  serum  cpm  Counts  per  EDTA  Ethylenediaminetetra-acetic  kb  Kilobasepairs  kd  Kilodalton  PEG  Polyethylene  PMSF  Phenylmethylsulfonyl  rNTP  Ribonucleoside  triphosphate  SDS  Sodium d o d e c y l  sulphate  SSC  Standard  TCA  Trichloroacetic  albumin  minute acid  glycol  saline  fluoride  citrate acid  ix  ACKNOWLEDGMENTS  I  would  Spiegelman and  Harry and  for  guidance.  my o t h e r  two  Deneer,  good  for  always  for  your  of  to  his  thank  the  I  also  to  encouragement, in  the  lab,  George invaluable  and  in  advice  particular  the  Bacillus project,  V e r a Webb  you  for  your  discussions  would  onslaught there.  unfailing  thanks  on  love, like  to  C o l u m b i a Wesbrook  many h e l p f u l  also  like  of  baking  Finally,  a n NSERC p o s t g r a d u a t e  British  special  constant  cohorts  being  would  give  To my f r i e n d s  humour.  withstanding  I  like  support  to  to and  acknowledge scholarship Fellowship.  x  thank  in  times  James, of  my p a r e n t s ,  and  for  stress, thank  and  you  encouragement. the  financial  and U n i v e r s i t y  support of  INTRODUCTION  In is  Escherichia coli  mediated  by  responsible and  bacterial obtained coli  the  RNA.  Much o f  studies  RNA p o l y m e r a s e  a /3 a n d y 3 ' s u b u n i t kilodaltons, one  to  two  catalyzes  synthesis  Bautz  and  Sugiura Bautz,  which  essential promoter  shock  a sigma  that  steps  et  the  factor  The  transcription  binding  recognized  which and  cx s u b u n i t s in  cannot  accurately (Burgess  Burgess,  70,000  dalton  transcription  from  many E .  gene  product,  a number  by  al.  et  of  which  1984),  transcription  also  from enzyme.  process  involves  several  c a n be c o n t r o l l e d :  1  sigma  is coli  during  sigma-70  RNA s y n t h e s i s ,  1 969;  is  the  of  et  (1984)  proteins  a_l.,  and  size  polymerase  and  of  160,000  enzyme  RNA s y n t h e s i s  initiation  and  core  direct  not  composed  DNA t e m p l a t e s  G r o s s m a n e_t  T h e E..  core  Travers  with  (Neidhardt  to  coli.  11 k d  The but  been  the  inducing  response  E.  150,000  An a d d i t i o n a l  hptR  are  at  1969;  Recently,  that  1971).  on n a t u r a l a l . ,  has  43 k i l o d a l t o n  transcripts  specific  for  a l . ,  is  ribosomal  complex  approximately  associated  sites.  responsible  as  of  in  multi-subunit  1971).  is  for  demonstrated  heat  et  which  regarding  RNA s y n t h e s i s  two  transcription  messenger,  information  approximately  transcription  1969;  subunit  a  omega s u b u n i t s Berg  of  transcription  respectively),  1969;  al. ,  is (of  (Burgess,  initiate  of  the and  prokaryotes  RNA p o l y m e r a s e  transcription  RNA p o l y m e r a s e s from  other  a DNA-dependent  for  transfer  and  the functions  promoters  sequential promoter  elongation  and  termination.  In  prokaryotes  the  sequence of  in  the  initiation  of  transcription  in  the  regulation  of  gene  promoter  binding,  complexes been  and  studied The  the  current from  work  Walter  reaction (and  in  et  which  complex  of  and  for  .  the  of  formation  simple  the the  of  events  of  initiation have  therefore  initiation  involving:  1)  a  with  binding  site  the  to  the  binding  interacted  complex  from  has  s c h e m e , s u g g e s t e d by  specifically  the  DNA, 2)  a  localized  enzyme e f f e c t e d  RNA s y n t h e s i s  role  coli.  RNA p o l y m e r a s e  DNA a t  an i m p o r t a n t  transcription  a "closed"  involved  The m e c h a n i s m  these  E.  (1967 ),  whereby  the 3)  a_l  in  reaction  unwinding  in  relatively  reversibly)  melting  regulation  model  the  expression.  subsequent  intensively  evolved of  the  plays  events  form  complex  an  a  "open"  formed  in  step  2. Hinkle of  and C h a m b e r l i n  formation  a membrane a stable its  phage  filter  on t h e  binding  since  the the  rate  observed  that  the  would  expected  rate for  diffusion-controlled therefore  of  of the  of  polymerase in  which  membrane  reaction the  complex  interaction that  DNA t h a t  the  2  of  the  using  has  filters.  formed  first-order  They was  only  also  slower  depended upon components.  rate-limiting  by  Their  was d e p e n d e n t  formation which  kinetics  c a n be d e t e c t e d  followed  reaction  the  complexes  RNA p o l y m e r a s e .  a reaction  hypothesized  investigated  RNA p o l y m e r a s e  binding  concentration  be  assay  on n i t r o c e l l u l o s e  showed t h a t  kinetics,  T7 D N A / R N A  association with  retention  data  of  (1972b)  step  than  the It  was  i n RNA  synthesis  was  polymerase location  the  the  transcription to  associates  promoter  include with  and  and  c  complexes  existence (for  E-P  there of  polymerase/DNA  assay,  the  Walter  is  of  the  above)  al.  open  time  formation  (measured proceeded  addition  for  initiation  the  complexes et  no  a l . ,  1982;  evidence  that  of  did  initiation  of  RNA s y n t h e s i s  was  subsequent  to  the  of  which binding  a half-time  of  formed, not  rapidly  nucleoside from  and  stable  a filter  with once  of  fragment  in  These d a t a  3  support  Stefano  the  formation  to  1985).  the  1 minute.  promoter  (McClure,  complexes  was a p p r o x i m a t e l y  open  non-promoter  promoter  half-time  and  complex,  with  polymerase/laeUV5  the  enzyme  the  polymerase  However,  since  which  Hippel  present  minutes.  RNA u p o n  as  (1967).  and  von  0.2  synthesize  modified,  enzyme/DNA  on a r e s t r i c t i o n  promoter  the  0  evidence  1974;  the  that  et  to  DNA:  closed  closed  location  complexes  as d e s c r i b e d  approximately  at  in  ^ = - E - P  specific  by  for  the  the  observed  lacUV5  C  on  non-specific  Chamberlin,  promoter  (1979)  carried  E ^ = E - P  considerable  there  step  of  DNA, p r i o r  thus  the  interactions  DNA i m p e d e s Gralla  see  1985)  transient  is  the  was  sites  promoter-specific  reviews  McClure,  are  Q  on  release  The m o d e l  pathway  non-specific  hypothesized  While  sites  a preliminary  E-D^ r e p r e s e n t s  E-P  subsequent  site.  initiation  E + D ^ E - D ^ where  and  from n o n s p e c i f i c  of  follows,  binding  triphosphates  pre-formed suggested  rate-limited  at  promoter-specific  a  complexes that  the  step complexes.  Stefano formation  and G r a l l a  of  complexes with  The r e a c t i o n order rate  varied  formation  b y up  at  the  of  complex). formed  The r a t e  rate  complex.  orders  the  which  the  at  was c o n v e r t e d  to  a  it  functional  directly  refers  that  the  of  enzyme to  T7 A1 p r o m o t e r  the are  that  rate-limiting  Based  on t h i s  Stefano et  al.  formation  the  demonstrated  and s u b s e q u e n t  colleagues  1982 ) h a v e of  steps  (Kadesch  shown t h a t  open c o m p l e x e s at  to  and N a C l  changes i n  concentration, the  reaction  (i_e.  is  not  a DNA m e l t i n g  (1979,  postulated  addition  1980) the  occurs  of  the  phage  the  prior  to  reaction).  work  and M c C l u r e ( 1 9 8 0 ) ,  existence  4  to  template  suggesting  in  in  et  temperature  step  information,  and G r a l l a (1982)  a_l. ,  insensitive  25 a n d 3 7 ° C )  open complex  to  transcription.  promoters  formation  (between the  functional  independently  by C h a m b e r l i n and h i s  and  intermediate  quantitated.  Rosenberg et  association  here  were  be  M c C l u r e (1980)  1982 ;  a closed  c o m p l e x e s c a n be c o n s i d e r e d t o  complexes.  be s e p a r a t e l y  to  a  of  open  binding  the  ability  initiate  functional  the  which  complexes of  Operationally,  al. ,  (equivalent  functional  and  first  proceeded through  stability  which  promoters.  between  proposed, in  promoter  the  showed a  magnitude  the  The t e r m  Studies  promoters  depend upon  complexes to  could  of  intermediate at  lac  polymerase concentration  two  unstable  investigated  s e v e r a l mutant  different  complexes at  would  the  to  further  A m o d e l was t h e r e f o r e  kinetically  the  rates  d e p e n d e n c e on t h e  promoters.  and  (1980)  of Kadesch  a "pre-closed"  promoter  complex  complexes  (possibly  at non-promoter  which  recent  work  complex.  (1985)  a n d Roe e t a l . (1984)  an i n t e r m e d i a t e ,  high  the  strengths,  Based  on t h e a b o v e  following  model  E+P^F=^ E - P  The in  complexes which  promoter unwound. promoter  E-P  steps  initiate  the  and i t  It  at the  which can  i n the presence which  functional that  by a l t e r i n g  f r o m many  must  E . coli  holoenzyme  as s t r u c t u r e therefore determine  is  from  those  Hawley  i s not  of  lead  to the  complexes  is  the e f f i c i e n c y one, or  thus of  several  pathway.  that  1979;  with the  site  complex  i s conceivable  interactions initiated  complexes  E - P t h e DNA s t r a n d s o  is this  c a n be c h a n g e d  (as w e l l  promoter)  specifically  RNA s y n t h e s i s  t h e RNA p o l y m e r a s e  structure  formation:  The s e q u e n c e o f e v e n t s  Transcription only  complexes.  has proposed  (1985)  at the promoter  of p r o m o t e r - s p e c i f i c ,  i n the  and open  or  - . E - P J ^ F ^ ^ E - P Q  complex  are separated.  transcription  the presence  at low temperatures  i s associated  In t h e open  complex  suggested  McClure  v  to form a  E - P ' and E - P . a r e a l l c l o s e d  b u t t h e DNA h e l i x  ribonucleotides.  quite  data,  c  isomerizes  the closed  f o r complex  t h e enzyme  immediately  formation  between  ^ = ^ E - P '  C  only  polymerase  by Buc and M c C l u r e  has a l s o  observed  ionic  t o DNA/RNA  sites)  closed  of  Finally,  analogous  (/3/1 ' o ^ c r ) .  influence the rate  1983  5  requires Promoter  o f t h e DNA w h i c h  promoters  and M c C l u r e ,  promoters  for  surrounds  t h e enzyme/DNA  at which  transcription  ( s e e R o s e n b e r g and C o u r t , reviews).  E_. c o l i  promoters  approximately  35  transcription  start  sites  are  Court, alter  1979;  the  ejt  al. ,  at  two  This in  to  in  pair  vary  rates  could  transcription  of  to  in  -10  lac  the  for  considerably.  the  promoter  since  fragments therefore  the  of  DNA.  lead  to  the  enhance which the  1976). and et  1979).  lacUV5  and  by  1982) in  in  single have  the  differences  promoters  profound  is  in, v i v o  differ  Alterations  altered  (Silverstone  1980,  the  is  (Gilbert,  the  (Dickson  sequence  The d i f f e r e n c e s  all  of  at  which  to  to  closely  absence of  Gralla,  Gralla,  solely  and  which  s e q u e n c e TATGTT  and  these  example,  CAP p r o t e i n  promoters and  shown  RNA s y n t h e s i s ,  formation  (Stefano  the  (Rosenberg  been  lacUV5  Stefano  complex  centred  s e q u e n c e more  such that  direct  1976;  mutant  were  been  reaction in contained  promoter  changes  in  capacity.  Transcription in  the  absence of  structure  can  the  vivo  region  be a t t r i b u t e d  on i d e n t i c a l sequences  has  of  Mutations  promoter  The m u t a n t  able  changes  to  promoter  operon  -10  Gilbert,  shown  1980 ).  TATAAT c o n s e n s u s s e q u e n c e is  The r a t e s  base  the  the  other  respectively  in  homology  consensus sequences at  al. ,  transcribed  in  the  1970;  several  et  lac  1977).  promoter  vitro.  al.,  The  1975,  changed  TATAAT,  AMP c o m p l e x ,  sites  The  of  upstream  c o n s e n s u s sequence have  weakly  CAP/cyclic  regions  sequences such that  transcription. only  site.  Siebenlist  promoter  two  10 b a s e p a i r s  TTGACA a n d  resembles  is  and  have  common w i t h J!_.  in  Bacillus  coli,  but  subtilis  there  6  are  has  several  several  features  significant  differences core  as w e l l .  The B.. s u b t i l i s  assembly, s i m i l a r  a (1 a n d £>' s u b u n i t respectively), small  (11  Doi,  1982  differ  for  in  example,  two  with  the  kilodalton)  the  core  respects  (which  (an  inhibitor)  the  al. ,  E_. c o l i In  responsible  for  respectively these  analogous  determine enzyme, of  the  are  the  predominant  coli  a l . ,  in  determined  1977,  which  are  core  subunits. the  For  inhibitors  and  by  (see  subunits  rifampicin  the  beta  1969 ;  subunit  Iwakura  and b e t a ' s u b u n i t s  1978).  et  are  sensitivity,  The f u n c t i o n s  to  be d i f f e r e n t  has a number  of  sigma f a c t o r s  form  of  of  necessary for  for  the  the  from  of the  reviews).  In  has s e v e r a l r e g i o n s  7  (see  Losick  vegetative  of  considerably smaller  subunit  core  cells  associated with  designated sigma-55)  coli  that  transcription  sporulation  RNA p o l y m e r a s e i s  but E.  specificity  process of  1982  Sigma-43 i s  the  are  the  (formerly  with  coli  two  likely  which  sigma f a c t o r  homologous  the  enzyme t o  beta  of  subunits.  1981 ; D o i ,  kilodaltons.  size,  and  function  and s t r e p t o l y d i g i n  transcriptional  Pero,  subunits  uncertain  E.  consists  130 k d ,  elongation)  the  therefore  s e v e r a l of  factor  et  subtilis  and  E.  rifampicin  genes i n v o l v e d  sigma  are  of  the  the  B_. s u b t i l i s  E_. c o l i  Bacillus  of  alpha  that  ( R a b u s s a y and Z i l l i g ,  (Hailing  subunits  140 a n d  from  from  core,  omega p e p t i d e s  inhibits  enzyme  1973).  coli  are  Apart  streptolydigin  of  but  sensitivity  initiation  E.  43 k i l o d a l t o n  review).  other  the  (approximately  and 9 . 5  associated  to  RNA p o l y m e r a s e h a s a  only than  that  (Wong a n d D o i ,  a  43 the  70  are 1982;  kd  Gitt  et  a l . ,  1985).  transcription  from  sequence  as  has  observed  been  the  by  the  E_. c o l i  by  the  Bacillus  Moran  et  a l . ,  might  have  structure  43  is  to  mapping  also  transcription  Core/delta with  various  a l . ,  1978)  has  has  been  activity  upon  influence  the  to  colleagues  found  phage  SP01 m i d d l e  (Pero  et  a l . ,  transcription RNA p o l y m e r a s e  that  delta  The  very  or  in  required  DNA b y  (Spiegelman  et  8  data  has  peptide  1982). and  its  activity  role  in  templates  confer  vitro (Tjian  were  1978;  a l . ,  initiation  nevertheless P e r o and  does  her  transcription  host  been  extensively.  delta  of  protein  reported  phage-modified a l . ,  delta,  al.  sigma-  Spiegelman et  vitro the  subtilis  Doi,  low  1978;  P e r o e_t  and by  plasmid  protein  by  E_. c o l i  specificity.  Similar  SP82  enzyme  promoter  studied  does not  specific  genes  1975). from  have  and  in  been  and D o i ,  transcriptional  for  designated  (Wong  chromosomal  enzyme.  1980 ;  Bacillus  immunologically  therefore  that  efficiently  1 979,  Bacillus  counterpart  Williamson  suggesting  the  It  transcribed  very  described  the  sigma-43  shown  phage,  1977;  first  subunit,  both  no k n o w n  are  1982).  enzyme.  protein,  This  et_ a j . . ,  e_t a _ l . ,  that  directs  same c o n s e n s u s  which  requirements  E_. c o l i  from  the  (Moran  (Davison  associated with  analysis,  have  transcribed  suggesting  dalton  has  not  stringent the  that  some p r o m o t e r s  polymerase  more  RNA p o l y m e r a s e  promoters  are  be d i s t i n c t ,  Delta  et  that  RNA p o l y m e r a s e .  shown  in  E_. c o l i  enzyme  than  sigma-43  promoters  1982),  A 21,000 (1975),  The  for  and h o s t  Achberger  and  cell  Whiteley, seems  to  (1980)  1981).  overall  be d e p e n d e n t  observed  Bacillus delta.  The  that  the  chromosomal In  templates  the  of  very  was  delta,  et  core/sigma-43  from  essentially  however,  Dickel  sensitive  transcription  a n d 029  of  template.  activity  DNA was  contrast, 0e  upon  effect  to  the  al. on  inhibition  Bacillus  unaffected  by  by  phage  added  delta. Spiegelman study  the  et  effect  a_l.  of  delta  on  DNA.  They  complexes  with  SP82  of  less  DNA was  delta,  concentrations complex  of  by  presumably  at  studies  complex  have  employed  restriction with gel  the  $29  eluted  Achberger  has  polymerase promoter  requires  recognition  both and  that  in  from  at  enzyme/DNA DNA.  the  RNA  phage  complex  which  have  such  carry  (Dickel  inhibit  complexes, direct  which  DNA  complexes  and  analyzed  assays  to  promoters  that  et  reported  a_l. ,  phage  Bacillus  sigma  and  delta  transcription  for  are  1980 ) .  similar  findings  subtilis  RNA  SP82 DNA. the  by  have  formation  that  9  presence  polymerase  assay in  filters  of  to  More  sigma-43  to  all  the  binding  enzyme  the  appeared  the  assay  enzyme/DNA  form n o n - f i l t e r a b l e  (1981)  proposed  of  Delta  the  restricts  on  binding  filters,  on  results  sigma-43  formation been  by  fragments  and W h i t e l e y  complex It  the  The  delta  DNA r e s t r i c t i o n by  sites  filter  that  electrophoresis.  recognized  for  a modified  that  the  destabilizing  fragments  demonstrated  observed  to  formation  are  a filter  formation  tested.  non-specific  enzyme  the  bound  enzyme  formation  on  (1978 ) used  efficient  initiation  (Williamson  and D o i ,  and W h i t e l e y ,  1981);  solely  by  either  sigma-43  the  (1981)  required  for  the  the  coli  Whiteley  delta  1978;  or  sigma  functions  to  reduce  the  and D o i  delta-sigma  activity. the  are  interactions  and at  that  of  this  with and  sigma  is while  non-specific  sites  a model  will  on  complexes.  regulate  model  out  initiation,  proposed  which  carried  Achberger  such n o n - s p e c i f i c have  Achberger  in  regard  to  promoter  be c o n s i d e r e d  in  Discussion. The e f f e c t  interactions been  of  by  that  the  region  but  did  the  enzyme and p r o m o t e r ,  did  not  prior  DNA w h i c h  in to  previous  initiation. report  released was a l s o  core/sigma  by  from  et  aJL. , of  number  in  non-initiated alter  result  S p i e g e l m a n and  observed  that  non-initiated  11 o u t  of  complexes  10  that  between  of  it  (1979)  prior  to  present  Delta contacts  acted  supported  15 c o n t a c t s were  affect  RNA p o l y m e r a s e  pattern  Whiteley  enzyme/DNA complexes  not  contacts  suggesting  latter  I  complexes.  the  has  These  did  the  of  SP82  and DNase  1982).  by  the  This  phage  delta  in  complexes,  of  protection  presence  significantly  initiated  polymerase  promoter  was p r o t e c t e d  cause a decrease  however,  gene  (Achberger  the  formed  of  specific  methylation  analysis  revealed  upon  an e a r l y  investigated  studies  delta  with  footprinting  It  1980;  associated  suggested  binding  (1978)  The d e t a i l s  are  Bacillus.  recognition  DNA b y d e s t a b i l i z i n g  Williamson  was  in  al. ,  which  factor  further  promoter  et  functions  delta  have  Dickel  a that  delta  initiation. present in  in  formed only  contacts  in  initiated  Achberger of  the  with  et  al.  the  complexes. (1982)  RNA p o l y m e r a s e  initiation-specific complex  formation  subtilis  investigated.  m e c h a n i s m by w h i c h selectivity.  RNA p o l y m e r a s e  could  delta  The i n i t i a l  the  analogous  to  in  kinetics,  which  those  transcription  to  kinetics  used  would  The o t h e r  by  presented  development studies  goal  the  the  B.  of  manner  promoter-specific  11  promoters. by  of  the  previously effect  of  interactions to  E. for  analyze  in of  work  in  which  this an  the  delta  on  suggested the promoter  thesis  assay, transcription analysis  sigma-43  was t o delta  interactions.  been  and  coli  subtilis  this  of  efficient  transcription  be a p p r o p r i a t e  initiation  investigate of  the  be u s e d  work  less  initiation  has not  by  conformation  formation  in  weak  about  affects  f o c u s s e d upon  assay  or  RNA s y n t h e s i s  therefore  polymerase.  resulting  seen  proposed  the  premature  nonpromoters  approach  normally  altered  and p o l y m e r a s e / p r o m o t e r  a kinetic  are  was t h e r e f o r e  prevent  contacts,  at  that  delta  The i n f o r m a t i o n  transcription that  It  that  to  The m e c h a n i s m o f Bacillus  promoter  use  of RNA  the  affected  the  MATERIALS Phage  and  (both  P l a s m i d DNA  Bacteriophage  029  was g r o w n  the  the  hosts  Whiteley, as  phage  and  University  of  described previously  Purified heated then  phage were  at  65°C  treated  in  was d i s s o l v e d  sodium  in  Plasmid  procedure  with  of  strains  0.15  were  grown  bromide,  ethanol  water  O.lxSSC.  from  butanol  precipitated Small scale  transformants,  were  and  to  with  12  and  4°C  0.015 M  by  by  the  several  Spiegelman cleared The  lysate  by  isolated  remove  the in  ethidium distilled  preparations,  made a s d e s c r i b e d  (1981).  at  prepared  redissolved  plasmid  room  T h e DNA  as d e s c r i b e d  the  CsCl/ethidium bromide. with  at  chloroform.  were  (1972)  were  extraction  M NaCl,  over  Cleared lysates  treated  2 hours  dialyzed  as d e s c r i b e d by D o b i n s o n and  in  particles  phenol  water,  stored  C l e w e l l and H e l i n s k i  DNA w a s  or  by  and  3% s o d i u m a c e t a t e .  (SSC i s and  purified  (1977).  Proteinase K for  P l a s m i d DNA was p u r i f i e d  plasmid  p h a g e was Ito  L15  H.R.  1$ S D S , 20 mM EDTA  distilled  O.lxSSC  SR22 o r  from  The r u p t u r e d  presence of  ( 1 979 ) .  centrifugation  Falkow  50 u g / m l  pH 7 . 0 ) .  and  by Kawamura and  treated  bearing  modifications (1985).  obtained  Washington)  sterile  against  a_l.  were  10 m i n u t e s .  the  citrate,  Dunn e t  i n JB_. s u b t i l i s  The DNA was p u r i f i e d  precipitated  overnight  for  with  temperature.  screen  AND METHODS  by  Crosa  used and  to  Media  Bacillus  was g r o w n  Plasmid bearing  1967). in  subtilis  (Champe and  glucose  concentration  added  to  and F e C l  0.2%,  concentration  of  Benzer,  w a s 0 .2555, and C a C l  3  m e d i u m was s u p p l e m e n t e d  with  thymidine  and  LB were  (to  (Luria-Bertani)  prepared  Construction The phage kb  ug/ml)  10  p328-5  ligated  the  R.C.  Miller,  with  the  ligation  ampicillin  plates  with  (to  20  used  contains  calf  kb  in  follows:  the et  were  The  ug/ml), ug/ml).  cloning al.  procedure  (1982).  sequences from  of  resistant  E.  British and and  ampicillin.  were 20  by  picked  ug/ml to  Hindlll  pBR322,  coli  Columbia)  tested  but  Bacillus the  was  onto  LB  for  2.4  from  a  with and  (obtained  SF8  tetracycline.  13  was c u t  C o l o n i e s from  ampicillin  and  phosphatase,  spread  and  the  electroelution  intestinal  fragment.  mixture  ug/ml  containing were  thiamine  The v e c t o r ,  University  50  which  gel.  2.4  containing  plates  200  ( B a n d C) was i s o l a t e d  treated to  (to  grown  a  mM r e s p e c t i v e l y .  was c l e a v e d w i t h  029  ^% p o l y a c r y l a m i d e Hindlll,  as  Doi,  P328-5  of  Phage  fragment  were  added to  uridine  plates  and  casamino a c i d s  a s d e s c r i b e d by M a n i a t i s  plasmid  029.  strains  were  2  (Yehle  modified  1962)  mM a n d 0.1  0.01  M medium  Escherichia coli  M9 m e d i u m  the  in  from  transformed plates  the  growth  Several  sensitive  to  o n LB colonies  tetracycline which was  were  were  screened f o r the presence  larger  than  shown t o h a v e  analysis  the o r i g i n a l  the correct  a n d by RNA-DNA  of  vector.  plasmids  Plasmid  DNA s e q u e n c e b y  hybridization  p328-5  restriction  a n a l y s i s (see  Results). Restriction  e n z y m e s a n d T4 DNA l i g a s e  f r o m New E n g l a n d B i o l a b s , by  Boehringer/Mannheim  suppliers'  electrophoresis  out  according  to Maniatis  Hybridization major  in vitro  initiated products by  from  al.  analysis  except  (1982),  Plasmid separated  previously  carried  that  The [ e * - 3 P ] U T P reaction  were  separated  8% p o l y a c r y l a m i d e  and t h e  the g e l as described the e l u t i o n  the  labelled  2  bisacrylamide)  that  buffer  gel  run-off b y M a n i a t i s e_t  was a s d e s c r i b e d  (1977).  a n d 029 DNA r e s t r i c t i o n through  to a n i t r o c e l l u l o s e described  a l l  f r o m p328-5 was  a 7 M urea,  by e l e c t r o p h o r e s i s  transferred  were  was u s e d t o c o n f i r m  transcription  from  b y Maxam a n d G i l b e r t  o f p l a s m i d DNA,  procedures  synthesized  through  acrylamide:1.38$ eluted  to the  al.(1982).  t h e 029 p r o m o t e r .  of a standard  transcript  was s u p p l i e d  Analysis  transcript  electrophoresis  (40$  et  obtained  used a c c o r d i n g  Manipulation  and c l o n i n g  Hybridization  phosphatase  and a l l were  recommendations.  gel  DNA-RNA  alkaline  were  fragments  0.7$ a g a r o s e g e l s and  membrane  by Dunn a n d S a m b r o o k  14  were  filter (1980)  as except  that  the  neutralization  M NaCl.  at  onto  The n i t r o c e l l u l o s e  65°C  transferred BSA,  contained  T h e DNA w a s b a k e d  overnight. hours  buffer  in  to  6xSSC,  0.02?  hybridization  1 mM EDTA  and  0.5?  hybridization  The  membrane was w a s h e d a t  minutes,  then  detected  by  in  carried  2xSSC  filters  T h e m e m b r a n e was containing  for  6xSSC,  0.02?  RNA w a s a d d e d ,  for  18 t o  hours  in  2xSSC,  40 m i n u t e s . using  0.5?  at  65°C.  SDS f o r  Hybridization  3M H i  Lite  film  3  then  labelled 24  1.5  65°C,  The  65°C  autoradiography  at  pH 7 . 4 ,  membrane was p r e - s o a k e d  buffer  out  for  the  BSA.  SDS.  and  1 M Tris-HCl  40 was  (Kodak)  at  -70°C.  RNA P o l y m e r a s e  Purification  Purification Polymerase 168  (Iowa  et  al.  (1978)  sperm DNA. contained  by 2)  0.5  sedimenting  by:  1)  gradient.  Litman  portion  band were  of  PEG/dextran on B i o - G e l  the  pooled  4)  with  calf  gradient  The the  was  described  by  phase A  1.5m,  sedimentation procedure  thymus 15 t o  was  or  prepared salmon  30?  and  fractions  from the  fastest  glycerol  gradient  enzyme  and c h r o m a t o g r a p h e d  15  RNA  subtilis  The D N A - c e l l u l o s e was  (1968)  3)  Bacillus  as p r e v i o u s l y  The a b o v e  1)  The g l y c e r o l M NaCl.  from  DNA-cellulose,  s e v e r a l ways.  described  activity  over  a glycerol in  Corp.)  chromatography  chromatography  modified as  2)  (core/sigma/delta).  was p u r i f i e d  Grain Processing  partitioning,  through  Holoenzyme  (sigma-43)  Spiegelman  3)  of  over  heparin-  agarose  (Sigma Chemical C o . )  with  S e p h a r o s e 4B  al.,  1979)  gradient  to  (Pharmacia)  remove  fractions  conductivity  conductivity  to  diluted  that  meter).  of  containing  0.1  M NaCl  buffer  containing  0.1  M NaCl,  All well  as  the for  isolation pH 7 . 9 ,  with  buffers those  of  the  type  of  and  glycerol  then  m  unstable  and  used  lost  50  to  described  buffers.  the  purified  90$  of  the  its  containing  in  steps,  a  In  20 mM 2 -  be n o t e d to  particular, was u s e d  e n z y m e was in  glycerol  -12°C  in  here the when  during  very one  to  (obtained of  and  as  the  sensitive  purification at  of  10 mM T r i s - H C l  should  30$ g l y c e r o l  16  for  PMSF,  activity  grade  with  NaCl.  contained  It  the  10 v o l u m e s  below  50 u g / m l  T h e e n z y m e was s t o r e d above,  M  be p a r t i c u l a r l y  in  to  with  chromatography  described  analytical  BDH C h e m i c a l s ) w a s u s e d  CDM 2 f  8 to  (Sigma Chemical C o . )  procedure  Therefore,  and d e l t a .  0.6  above  the  M NaCl  T h e e n z y m e was e l u t e d  M EDTA,  in  0.1  c o l u m n was w a s h e d  with  10$ g l y c e r o l .  glycerol  isolation  weeks.  1  2 f  enzyme seemed to  Sigma grade the  the  procedures  10 mM M g C l  a  containing  the  containing  for  The  s a m p l e was a p p l i e d  c o r e / s i g m a and d e l t a ,  mercaptoethanol that  buffer  to  a Radiometer  and  M NaCl-containing buffer. step  buffer  buffer  et  activity.  had been e q u i l i b r a t e d  buffer  single  DNase  with  The d i l u t e d  column which  (prepared  a s d e s c r i b e d by D a v i s o n  was m e a s u r e d w i t h  heparin-agarose  0.35  heparin-sepharose  a contaminating  were  equal  (conductivity  or  both  two from  enzyme  buffer, 0.46  M NaCl.  Enzyme and  activity  Whiteley  was  (1974),  with  out  0.05  M NaCl,  with  0.8  mM G T P , ATP a n d  H]UTP u s i n g  029  DNA a s  mixtures ug/ml  yeast  Co.). text  were  precipitated  RNA ( T y p e  in  terms  of  Lowry  assay,  using  BSA ( F r a c t i o n The  estimated  on  the  preparation.  that  exponential The g e l s Blue  acetic and  of  stained  acid/25?  scanned  Instruments). to  sigma content  as d e s c r i b e d  for  48 h o u r s  the  sigma,  weight. estimated  The  The g e l s  areas  delta  and  sigma:alpha  molecular  by  were  in  with  alpha ratio  weight  of  17  the  was  peaks  1972)  was  _al.  al^. ,  10?  7.5?  (1982)  which  were  daltons  1974). Brilliant  acetic with  7-5?  acetic  acid  (Helena corresponded  determined  calculated  45,000  et  et  of  in  a  the  destained  subunits  by  standard.  Coomassie  briefly  the  14-20$  Scan d e n s i t o m e t e r under  in  on S D S -  a  (McGuire  then  the  Leduc  a solution  rinsed  a Quick  The  in  Chemical  a preparation  contained  acrylamide  ethanol,  with  in  as  electrophoresed  gels  Sigma  100  Strominger,  of  gels  of  (determined  and  0.25  reaction  concentrations  enzyme  separation  25? e t h a n o l .  enzyme  yeast,  Sandermann  was  2  presence  active  R (Sigma Chemical C o . )  acid/  the  of  gradient  were  TCA i n  concentration  the  M MgCl ,  The  Sigma C h e m i c a l C o . )  enzyme  slab  the  by  Assays  0.02  template.  V,  basis  The  polyacrylamide except  protein  Spiegelman  C T P , 8 uM UTP a n d  from Torula  stated  as m o d i f i e d  proportion  with  II-S  Unless otherwise are  M T r i s - H C l pH 7 . 9 ,  the  by  modifications.  carried  [  0.04  several  were  uCi  in  a s s a y e d as d e s c r i b e d  using for  the  by  an alpha  subunit  (Doi,  for  sigma subunit  the  alpha  1982)  subunit,  considered  to  and  which  a molecular  (Gitt is  et  a_l. ,  enzyme  preparation.  It  active  enzyme  preparations  this the  method  the  since  enzyme w h i c h  it  should  of  was n o t  of  1985 ).  a component  be r e p r e s e n t a t i v e  in  weight  of  The  the  the  43,000  be n o t e d  amount  core  amount that  daltons of  enzyme,  of  core  the  in  was a s s o c i a t e d w i t h  to  determine  s i g m a was  the  amount  may be o v e r e s t i m a t e d  possible  was  if  in  of by  all  of  fact  active.  Isolation was  depleted  of  of  C o r e / s i g m a . Holoenzyme  delta  by  D N A - c e l l u l o s e chromatography  glycerol  gradient  1981)  or  by D E A E - S e p h a d e x  1978)  of  heparin-agarose  purified  enzyme  (Achberger  chromatography purified  diluted column NaCl  glycerol  gradient  as d e s c r i b e d (2.5  (0.1  core/sigma  cm x  M to  8 cm).  0.6  activity  The e n z y m e f r a c t i o n apparatus column  prior  (described  Holoenzyme agarose  to  step  M)  and  Elution  eluting  with  carried  to  over  a  as and  gradient  from d e l t a ,  and  concentrated  chromatography  e_t a l . ,  a DNA-cellulose  a linear  0.35  out  combined  core/sigma  between  then  Whiteley,  (Spiegelman  were  applied  separated  was  was  fractions  above  and  of  enzyme.  The D N A - c e l l u l o s e p u r i f i c a t i o n follows:  (core/sigma/delta)  in  0.55 an  of the  M NaCl.  Amicon  heparin-agarose  above).  which  had  was d i l u t e d  been with  18  purified buffer  to  through  the  give  conductivity  a  heparin-  equivalent  to  applied  a DEAE-Sephadex  to  equilibrated was  then  that  with  of  buffer  washed w i t h was e l u t e d  containing  0.32  described enzyme.  prior The  above  delta  adding  of  the  had  been  0.1  containing  M NaCl. 0.1  step  and  The  M NaCl,  with  column  and  buffer  present  in  the  was d e t e r m i n e d  determing  the  the  core/sigma for  enzyme t o was n o t  experiments  by  the  the  with two  of  the  delta  by  minutes  at  transcription  37°C  reactions.  critical  however,  since  were  affected  if  not  and  method  sigma content  was r e c o n s t i t u t e d  with  core/sigma  the  that  step  omitted.  Purification Delta fractions  heated  of  at  is  Subunit  from  until  and  heat  the  be  The  further  The (M.  at  combined  low  Hilton,  al.,  by  the  fractions  speed to  were  remove  contained  personal  (30  to  the delta,  communication),  chromatography  1978).  19  4 of  became f l o c c u l e n t  supernatant  purified  (Spiegelman et  delta-containing  (from step  solution  centrifuged  stable  pooled  gradients  precipitate.  could  Sephadex  Delta  glycerol  65°C  resulting  the  purification).  60 m i n u t e s )  which  of  was p u r i f i e d  holoenzyme  that  column which  a single  delta  for  pre-incubation  results was  of  Core/sigma  to  M NaCl  containing  in  preparations  incubating  0.1  M NaCl.  amount  holoenzyme  containing  buffer  core/sigma  The  buffer  over  DEAE-  The amount by  running  amounts  blue, to  curve  preparations weight delta  with  (Doi,  volumes MgCl ,  and  the  of  the  the  the  of  weight  of  21,000  stained the  peaks  to  generate  present  in  the  using for  which  used  of  The m o l a r  daltons  with  described  comparison  curve.  known  as  were  delta  estimated  with  quantitated  BSA p e a k s  by  were  areas under  were  amount  standard  Transcription  the  delta  delta peak  concentration  an  a  of  estimated  the  delta  subunit  in  Assays  transcriptions  buffer  1 mM E D T A ,  2  concentration  was  were  containing 48.5  10 u M .  done  in  40 o r  40 mM T r i s - H C l  mM N a C l  GTP a n d CTP c o n c e n t r a t i o n s  and  were All  100  pH 7 . 9 ,  3% g l y c e r o l .  4 0 0 uM a n d  nucleotides  the were  ul 20 mM  The A T P , UTP obtained  from  Chemical Co. The s t a n d a r d initiated  solution  carried  (multiple  by t h e  containing  been prewarmed  were  The g e l s  BSA a n d d e l t a  was e s t i m a t e d  Run-off  were  was  1982).  vitro  Sigma  preparations  was s u b s e q u e n t l y c a l c u l a t e d  molecular  In  the  s c a n n e d and  The w e i g h t s  standard  in  BSA a s s t a n d a r d s .  corresponded above.  delta  s a m p l e s on S D S - p o l y a c r y l a m i d e g e l s ,  of  Coomassie  of  out  stopped  for by  for  addition  of  transcription RNA p o l y m e r a s e  reactions to  D N A , A T P , G T P , CTP a n d UTP w h i c h 2 minutes  10 t o the  round)  at  15 m i n u t e s  addition  of  20  37°C. at EDTA  Transcription  37°C  and  (final  the  a had was  reactions  concentration  25 mM) o r  electrophoresis  Tris-Borate, Unless  2 mM E D T A ) otherwise  transcription) solution minutes  at  37°C,  prewarmed) of  the  of  heparin  (1  to  enzyme,  on i c e  acrylamide, localized  had  or  5 ug/ml,  one  added  10 m i n u t e s ,  then  at  -20°C.  were  gels.  the  gel  2  to  addition  a  mixture [oc-3 p]UTP 2  allow  H e p a r i n was u s e d a t  the  reaction  were  sample b u f f e r Aliquots  from  a  multiple  reactions  The r u n - o f f  slices  to  CTP a n d  electrophoresed  cut  the  The e l o n g a t i o n  electrophoresis  frozen  for  been  after  prevent  promoter.  autoradiography, in  to  also  Ci/mmol)  transcripts.  reactions  radioactivity  (which  A m e r s h a m , 410  M  a  GTP w a s p r e w a r m e d  Sigma C h e m i c a l C o . ) ,  at  7M u r e a  by  as f o l l o w s :  and  EDTA o r  transcription  out  removed  of  0.1  (productive  s a m p l e s were  for  chilled  kinetic  carried  M urea,  ice.  times  events  with  on  various  concentration  stopped  (10  at  initiated  continued  the  RNA p o l y m e r a s e  5 uCi/reaction; of  chilled  D N A , ATP a n d  ( G r a d e I,  initiation was  were  was a d d e d and  elongation final  and  stated  assays  containing  sample b u f f e r  of  and  the  through  8%  transcripts  were  the  the  gel  quantitated  by  and  Cerenkov  counting. The  sequences of  and number  of  transcript  and  transcripts  the  U residues 22  in  the  synthesized  029 in  promoter-specific  them was known  G2 t r a n s c r i p t ) . from  the  21  promoters  (66  run-off in  the  The number could  RNAs  A2 of  therefore  be c a l c u l a t e d incorporated  from into  the the  amount run-off  22  of  radioactivity  transcripts.  which  was  RESULTS  C L O N I N G AND L O C A L I Z A T I O N OF THE 029 A2 PROMOTER The b a c t e r i o p h a g e which  i s able  and W h i t e l e y , most  to i n f e c t  review  several  stranded  Bacillus  G e i d u s c h e k and I t o ,  1975;  extensively  a double  029,  characterized  s e e G e i d u s c h e k and I t o ,  which  the l y t i c  temporally the  light  into strand  infection, strand  (Mosharaffa  In  et  vitro  polymerase  sigma-43  appear  1976).  divided  Loskutoff  from  t h e phage from et  maps o f $29  D a v i s o n e t a_l. ,  the heavy al.,1973;  promoters  a l , , 1 979 ; D a v i s o n ^ t sites  1980)  promoters a l ., have  been  and t h e  23  been  (Duffy  vitro  constructed 1976;  Kawamura and  approximate  been d e t e r m i n e d  1980 ; D i c k e l also  early  by t h e  and i n  et_ a _ l . ,  have  for  polymerase  in vivo  DNA h a v e  1 973 ; I n c i a r t e  of the e a r l y  promoter  from  t o be r e c o g n i z e d  Detailed  Ito,  Several  c a n be  acid  RNA i s t r a n s c r i b e d  associated Bacillus  et a l .,  et  of the  b y t h e B . s u b t i l i s RNA  be i n i t i a t e d  (Loskutoff  locations  1970;  transcription  Geiduschek,  1977;  cycle  (for  al.,1973).  These promoters  transcription  early  systems  Ribonucleic  RNA i s t r a n s c r i b e d  et a l .,  can only  predominant and  late  i s one o f t h e  1982),  o f t h e p h a g e DNA t h r o u g h o u t  whereas  Schachtele  RNA.  two c l a s s e s ;  (Hemphill  Transcription  1982).  in detail.  during  species  Bacillus genetic  029 g e n o m e h a s b e e n s t u d i e d i s synthesized  DNA p h a g e  et a l  tentatively  . ,  (Sogo  1980 ) . located  by  nucleotide al.,1981; One  o f t h e $29  early  t o t h e 029  fragment  into  transcription  studies  Plasmid from  contains  a single  fragment  (Ito,  that  the H i n d l l l - C  pBR322  and t o d e t e r m i n e Treating  enzyme H i n d l l l  (Figures  The s m a l l  029 H i n d l l l - C  fragment  fragment  plasmid.  When p 3 2 8 - 5 w a s t r e a t e d band  of approximately  comparison  with  $29  released.  Since  asymmetric  EcoRI s i t e  EcoRI  the o r i e n t a t i o n  shown i n F i g u r e  lane  the cloned  o u t on p 3 2 8 - 5  0.8  and t h e v e c t o r  the  cloned restriction two  into  EcoRI  to  into  the p o s i t i o n  intact,  with  fragment  that the  (Figure  kb ( e s t i m a t e d fragments)  of the  vector 1,  lane  by was  had a s i n g l e , (pBR322)  of the cloned  24  H i n d l l l and  indicating  restriction  2.  with  of the  at  $29  one end o f t h e  2) y i e l d e d  3),  had been c l o n e d ,  a small  of  had been c l o n e d  migrated  fragment  site  carried  (see Figure  Hindlll  near  p328-5 with  Hindlll-C  1)  fragment  site  the orientation  1 and 3,  Restriction  analysis  fragment  the plasmid  1980),  and used i n t h e  the H i n d l l l - C  was t h e r e f o r e  1)  endonuclease  below.  Restriction  verify  fragments.  characterized  described  1978).  had been  restriction  EcoRI r e s t r i c t i o n  (see Figure  which  The 2 . 4 kb H i n d l l l - C  029.  ( Y o s h i k a w a e_t  D a v i s o n e t a l . , 1979,  P328-5 C a r r i e s  Fragment  insert.  promoters,  Hindlll-C  pBR322,  genome  1982).  (Sogo e t a l . , 1979;  cloned  EcoRI  o f t h e $29  Y o s h i k a w a and I t o ,  localized  was  sequence a n a l y s i s  also  DNA m u s t  had one be a s  F i g u r e 1. R e s t r i c t i o n a n a l y s i s o f t h e p l a s m i d p 3 2 8 - 5 . The p l a s m i d w a s d i g e s t e d w i t h E c o R I ( l a n e 1 ) , Hindlll ( l a n e 2 ) o r E c o R I a n d H i n d l l l ( l a n e 3 ) , r u n on a 1 . 0 ? a g a r o s e g e l and s t a i n e d w i t h e t h i d i u m b r o m i d e . The s i z e s o f t h e c l o n e d DNA f r a g m e n t s a n d t h e o r i g i n o f e l e c t r o p h o r e s i s (0) are i n d i c a t e d .  25  1 2  3  kb  o  -2.4 "^1.6 -0.8  26  EH E 1 pBR322 0.8 kb  H B  1  *1  1.6 kb  p  Figure 2. Plasmid p328-5. A 2 . 4 kb f r a g m e n t f r o m 029 D N A , d e l i n e a t e d by H i n d l l l r e s t r i c t i o n s i t e s , was i s o l a t e d and c l o n e d i n t o t h e H i n d l l l s i t e o f p B R 3 2 2 . R e s t r i c t i o n s i t e s are d e s i g n a t e d as f o l l o w s : E - E c o R I , H - H i n d l l l , B-EamHI. The p o s i t i o n a n d o r i e n t a t i o n of the promoter (p) are i n d i c a t e d .  27  Location of  of  a^promoter  predicted al.  on t h e  from  (1979).  the  in  xylene  200  Materials  gel  to  and  to  C from  The  transcript  band  the  of  located  the  with  as  in  1.6  was c o n s i s t e n t  al.,  S c h a c t e l e et  RNA was  restriction  to  029 that on  RNA  fragment  the  the  1.6  kb  al. , was  previous  transcription  in  the  3). thus  The was  EcoRI  initiated  The  site  and  former  data  subsequently  p328-5  EcoRI-Hindlll  1 973 ; D a v i s o n e t  and  28  pBR322.  promoter  transcription  with  p328-5  towards  fragment.  from  DNA ( F i g u r e  sequences or either  through  hybridized  029  of  directed  kb  fragments  as d e s c r i b e d  EcoRI-treated  in  I'  to  vitro  (A2)  vitro  (for  been e l e c t r o p h o r e s e d  The p r o m o t e r in  was o b s e r v e d  comparison  1980). vivo  in  sigma-43  by  from  that  conclusion 1973 ;  the  subtilis  as e s t i m a t e d  digest  transcription  within  and  the  Hindlll-EcoRI  other  terminated  S o g o e_t  of  in  a Hindlll  2 or  and  as d e s c r i b e d  nitrocellulose,  EcoRI s i t e  Figure  been  The s i z e  had  to  kb  EcoRI  assay,  Bacillus  had  (1980)  with  location  dye.  The  1.6  with  the  fragment shown  bound  RNA s u g g e s t e d  near  the  al.  The  029  of  RNA t r a n s c r i p t  synthesized  had no h o m o l o g y  treated  was h y b r i d i z e d  and M e t h o d s .  exclusively  size  with  DNA w h i c h  and  et  a standard  tracking  ^29  fragment  12a and b ) .  transcript  P 3 2 8 - 5 and  was  (in  on P 3 2 8 - 5 .  Davison  bases long  cyanol  an a g a r o s e  of  a discrete  see F i g u r e s  The of  work  and M e t h o d s )  approximately the  Hindlll-C  vitro  RNA p o l y m e r a s e example  Promoter  When p 3 2 8 - 5  transcribed Materials  029  the  (Loskutoff a l . ,  1979 ,  s e q u e n c e d and  initiation  sites  e_t  the  F i g u r e 3 . H y b r i d i z a t i o n o f p328-5 t r a n s c r i p t s t o p328-5 a n d $29 DNA r e s t r i c t i o n f r a g m e n t s . ( L e f t ) Ethidium b r o m i d e s t a i n e d 0.7$ a g a r o s e g e l p r i o r t o b l o t t i n g . L a n e 1, 029 DNA d i g e s t e d w i t h H i n d l l l . L a n e 2 , p 3 2 8 - 5 d i g e s t e d w i t h H i n d l l l . L a n e 3> P 3 2 8 - 5 d i g e s t e d w i t h EcoRI and H i n d l l l . S i z e s o f t h e c l o n e d DNA f r a g m e n t s ( e s t i m a t e d b y c o m p a r i s o n w i t h t h e 029 H i n d l l l fragments) are i n d i c a t e d . (Right) Autoradiogram of the n i t r o c e l l u l o s e b l o t a f t e r h y b r i d i z a t i o n t o t h e p328-5 transcript. T h e l a n e s a r e t h e same a s i n ( L e f t ) . The o r i g i n o f e l e c t r o p h o r e s i s (0) a n d t h e p o s i t i o n o f t h e H i n d l l l - C band a r e i n d i c a t e d .  29  1  2 3 kb  30  123  determined  by  S1 n u c l e a s e m a p p i n g  (Dobinson  and S p i e g e l m a n ,  1985).  P U R I F I C A T I O N OF THE RNA P O L Y M E R A S E AND D E L T A Holoenzyme gradients used  for  (as  had been p u r i f i e d  described  qualitative  quantitative the  which  in  analysis  presence of  in  Materials  vitro  of  through  glycerol  and M e t h o d s )  transcriptions.  transcription  a contaminating  FACTOR  DNase  could  be  However,  was h a m p e r e d d u e activity  in  the  to  enzyme  preparations. The g l y c e r o l  gradient  activity  fractions,  was  agarose  as d e s c r i b e d  in  to  remove  the  in  Figure  4  glycerol  DNase shows  for  in  presence of  DNase  conditions Hindlll been  used  labelled  at  were  in  counting.  bands  Hindlll  After  the  gel  experiment  enzymes were  CTP u n d e r  from phage site  with 4,000  with  31  had  the  029  with  a 273  DNA, w h i c h  cpm D N A . prior  quantitated  were  same  2  glycerol  been  incubated  [oi? P]-dATP.  ethanol  were  which  chromatography  transcriptions,  the  attempt  in  one  a 5% p o l y a c r y l a m i d e ,  incubating  an  of  approximately  on  in  presented  The two  vitro  through  peak  heparin-  and M e t h o d s  heparin-agarose  fragment,  the  over  the  enzyme and enzyme w h i c h  precipitated  electrophoresis the  by  from  The a u t o r a d i o g r a m  A T P , GTP a n d  for  contained  reactions  and  results  activity.  restriction  reaction  gel  the  pooled  purified  Materials  activity.  purified  tested the  further  gradient-purified  subsequently  enzyme,  base had  Each  The to  nondenaturing by  gradient  Cerenkov enzyme  F i g u r e 4 . A s s a y f o r D N a s e a c t i v i t y i n t h e RNA polymerase p r e p a r a t i o n s . A 3 2 d_i b n j r e s t r i c t i o n f r a g m e n t o f 029 DNA w a s i n c u b a t e d w i t h g l y c e r o l g r a d i e n t p u r i f i e d enzyme ( l a n e 1 ) , o r H e p a r i n a g a r o s e p u r i f i e d enzyme ( l a n e 2 ) u n d e r c o n d i t i o n s t h a t were used f o r iri v i t r o t r a n s c r i p t i o n r e a c t i o n s . The r e a c t i o n m i x t u r e s were e l e c t r o p h o r e s e d t h r o u g h a n o n d e n a t u r i n g p o l y a c r y l a m i d e g e l and t h e r e m a i n i n g r a d i o a c t i v i t y i n t h e DNA b a n d s q u a n t i t a t e d a s d e s c r i b e d in the text. T h e l o w e r b a n d c o r r e s p o n d s t o t h e $29 r e s t r i c t i o n fragment i s o l a t e d p r i o r to e n d - l a b e l l i n g . p  32  e n  a  e  e (  2  1  1  33  with  the  on t h e from  DNA f o r  gel  the  was 2 6 4  gel  (lane  2).  prior  to  1). the  the  position  DNA t h e  amount  to  the  combined enzyme  with  the  course  entire  the  to  the  above  in  Materials  minutes.  Although  activity  correlate (see  well  Figure  band had more from could  the in  some o f  other  the  to  of  inhibitor  of  less  the  band.  fractions in  the  it  for  by  34  (described  out  for  10 t o  each  than  from  that  not  fraction edge  of  the  fractions  this  presence of that  was  did  leading  as  20  inhibitory  Although  fractions  was  resulting  gradient  was p o s s i b l e  latter  the  observation  the  that  been removed  protein,  the  clear.  gradient  assay  enzyme i n  from  for  initiation  had  glycerol  but  be a c c o u n t e d  the  when t h e  be some o t h e r  the  not  glycerol  Another  band  gel  was o b s e r v e d  was c a r r i e d  Some f r a c t i o n s  part  the  activity  amount  activity,  latter part  DNase  from  it  the  The r e a s o n  DNA i s  transcription  preparation.  with  5).  from  activity  appeared  profile  the  work  and M e t h o d s ) the  enzyme.  on t h e  was  cpm  as a s i n g l e  heparin-agarose,  productive  enzyme t h e r e the  over  lose  of  this  enzyme band  and p u r i f i e d  in  of  in  DNA b a n d s was 4 , 0 2 4  detected  banded  enzyme  radioactivity  bands were  of  the  of  purified  two  appeared  activity  have  DNA w o u l d  labelling,  reaction  the  the  DNA w a s i s o l a t e d  incubation  detectable recovered  the  to  longer  radioactivity  Although  During the  of  where  anomolous e l e c t r o p h o r e s i s  when  DNA was no  heparin-agarose  corresponding  subsequent  in  the  The amount  When t h e  with  slices  the  at  cpm.  incubated gel  (lane  10 m i n u t e s  discrepancy the  there  well.  DNase  was  some  In enzyme  an a t t e m p t from  subsequent the  the  total  leading  gradient  over  Methods.  solve  activity  of  that  2x10  material  described  Materials  from  the  an a c t i v i t y of  the  between  activity  after  ATP and  time  it  between  is  from  not  chromatography  responded  could  in  procedure  be u s e d  the  tau  clear  why  there in  a  TCA assay,  0.6  M NaCl  (75$)  was  enzyme the  as  was had  typical which  retained  presence  of  productive At  be a  different  from  holoenzyme  D N A - c e l l u l o s e or  not  in  the  present  difference  fractions  of  the  all  13 a n d the  in  with  delta  by  DEAE-sephadex and M e t h o d s .  different  since  a productive  delta  14).  35  by  (core/sigma/delta)  Materials  detectably  same m a n n e r  see F i g u r e s removed  had  enzyme t h a t  analysis.  should  the  as d e s c r i b e d  the  plot  column  and  peak.  when r e c o n s t i t u t e d  response  in  in  and  purification,  incubation  and  enzymes were  the  from  Materials  into  the  The r e s u l t i n g  assays  over  the  containing  of  for  10-13  transcription and  of  combined in  The r e c o v e r y  stage  a 10 m i n u t e  was r e m o v e d  purification  buffer  75$.  is  5 were  onto  vitro  taken  fractions  incorporated  and M e t h o d s )  this  fractions  as d e s c r i b e d  an i n  by  the  band were  Figure  (^H  10?cpm.  60 a n d  gradient  Delta  assay  x  enzyme w h i c h  glycerol  two  1.5  GTP a n d  transcription  in  the  was l o a d e d  cpm  7  column  recovery  ranged  DNA,  of  problem  example,  shown i n  precipitable  eluted  For  heparin-agarose,  The enzyme  in  the  edge o f  purification.  glycerol  purified  to  (for  enzymes  transcription  an e x a m p l e  Neither peptide  both  The  of  the  purification  from  holoenzyme.  In  F i g u r e 5 . (A) S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of p u r i f i e d B a c i l l u s s u b t i l i s RNA p o l y m e r a s e . Fractions 8 t o 17 f r o m a g l y c e r o l g r a d i e n t w e r e electrophoresed t h r o u g h a 14-20$ g r a d i e n t g e l , as d e s c r i b e d i n M a t e r i a l s and M e t h o d s . Enzyme p u r i f i e d f r o m the g l y c e r o l g r a d i e n t f r a c t i o n s 10 t o 1 3 , b y h e p a r i n a g a r o s e c h r o m a t o g r a p h y i s shown i n the l a n e d e s i g n a t e d std. T h e p o s i t i o n s o f t h e s u b u n i t s (/3 ,/3 «. ,v, £ ) a r e indicated. (B) A c t i v i t y p r o f i l e from the glycerol g r a d i e n t f r a c t i o n s shown i n ( A ) . The enzyme activity i n t h e g l y c e r o l g r a d i e n t f r a c t i o n s was a s s a y e d u s i n g [^HjUTP as d e s c r i b e d i n M a t e r i a l s and M e t h o d s .  36  std  37  comparison  to  core/sigma  preparations  (for  example  see Table  enzyme w h i c h designated  holoenzyme  amount  of  delta  was r e d u c e d  by  approximately  III).  For  has been d e p l e t e d  the  of  was p u r i f i e d  by D E A E - s e p h a d e x  by S D S - p o l y a c r y l a m i d e g e l  fractions  consistently to  contaminant day.  gels  migrate the  it  to  the  to  (see  to  be  the  50$  subsequently  delta  that  were  was f r e e  band  the  also  high of  in  and  visible  evident  6) of  This  on  in  the  and as shown i n RNase,  DNase  and  the  band d i d the  the  gels  not in  amount  varied  an  often  of  of  from  day  nondenaturing  molecular  delta.  with  was  electrophoresis  subunits  was n o t  Figure  a protein  same p o s i t i o n  the  gradient  66 k i l o d a l t o n s  delta.  an a g g r e g a t e  proteins  7 delta  55  contained  was c o n c l u d e d  was p r o b a b l y  preparations  of  RNA p o l y m e r a s e  relative  contaminating  Figure  which  The c o n t a m i n a n t  and  protein  weight  chromatography  detected  to  in  clarity,  will  from g l y c e r o l  molecular  relation  sake of  delta  apparent  column  present  core/sigma.  When d e l t a fractions  the  No  weight  other  delta Table  I  and  proteolytic  activity.  C H A R A C T E R I Z A T I O N OF THE C O N D I T I O N S FOR THE I N ROUND T R A N S C R I P T I O N  Studies in  general,  through  of  the  E.  coli  enzyme w h i c h  a "closed  "  state  ASSAY  RNA p o l y m e r a s e  associates with to  an  38  VITRO SINGLE  have  shown  a promoter  "open" complex  state  that,  proceeds which  F i g u r e 6. SDS-polyacrylamide gel electrophoresis of purified delta. The s a m p l e s w e r e electrophoresed t h r o u g h a 14-20$ g r a d i e n t g e l , as d e s c r i b e d i n M a t e r i a l s and M e t h o d s . Lane 1, H e p a r i n - a g a r o s e purified enzyme. Lane 2 , p u r i f i e d d e l t a p r e p a r a t i o n . The p o s i t i o n s of the s u b u n i t s are i n d i c a t e d .  39  1 2  40  TABLE Assay Delta  +  for  RNase  and DNase  Average  I  activity  cpm DNA  (xlO  - 5  )  in  the  delta  Average  preparation cpm RNA  (x10~  1.75  +  0.05  3.01  +  0.34  1.65  +  0.05  2.28  +  0.66  32  )  P - l a b e l l e d DNA o r i n v i t r o RNA s y n t h e s i z e d f r o m E c o R I t r e a t e d p 3 2 8 - 5 was i n c u b a t e d a t 3 7 ° C f o r 10 m i n u t e s w i t h d e l t a ( 1 3 - 1 5 pmol) o r enzyme s t o r a g e b u f f e r ( d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s ) u n d e r t h e same c o n d i t i o n s u s e d f o r in vitro transcriptions. T h e RNA o r DNA s a m p l e s w e r e t h e n e l e c t r o p h o r e s e d on p o l y a c r y l a m i d e g e l s , and t h e b a n d s i n t h e g e l w e r e e x c i s e d a n d q u a n t i t a t e d f o r r a d i o a c t i v i t y by Cerenkov c o u n t i n g . The v a l u e s a r e a v e r a g e s o f triplicate DNA a n d d u p l i c a t e RNA s a m p l e s + t h e s t a n d a r d d e v i a t i o n .  41  F i g u r e 7. Assay f o r p r o t e o l y t i c a c t i v i t y i n the d e l t a preparation. C o r e / < r was i n c u b a t e d a t 3 7 ^ C i n t h e p r e s e n c e ( • ) o r - a b s e n c e ( • ) o f d e l t a f o r 1 t o 40 m i n u t e s b e f o r e b e i n g added to a s t a n d a r d transcription reaction. The t r a n s c r i p t i o n s w e r e i n i t i a t e d by t h e a d d i t i o n o f 80 nM e n z y m e t o t h e t e m p l a t e DNA ( a t 1.7 nM) a n d r i b o n u c l e o t i d e s a n d c a r r i e d o u t f o r 10 m i n u t e s at 37°C. D e l t a was a d d e d t o c o r e / s i g m a a t a r a t i o o f 0.5 m o l . d e l t a : 1 m o l . enzyme.  42  8 O  6  oc Q.  10  20  30  TIME (min)  43  40  is  stable  heparin 1980;  and r e l a t i v e l y  (Walter  Stefano  solution, "closed"  et  and  bound  al . ,  at  the  productive  Stefano  and  Gralla  mutant  promoter-containing form  complexes for  triphosphates which will  is  in  terminates off  to  transcripts and  transcripts complexes kinetics studied  varying  end o f  are the  formed  t h e 029  to  of  the  of  the  the  time  of  quantitate the  using  run-off  transcripts  which  transcription  transcription the  rate  initiation by  the  round  number  into of  for  method  open The be  was m o d i f i e d  transcription  in at  RNA  the  assay  differ  from  in  three  ways.  First,  restriction  s y n t h e s i z e d from  44  the  assay.  assay of  run-  of  heparin.  B_. s u b t i l i s  promoter-containing were  The  such complexes can t h e r e f o r e  round  Gralla  of  Enzyme  addition  fragment.  the  addition  nucleoside  heparin  transcript  restriction  to  reaction.  radioactivity  The c o n d i t i o n s  of  of  before  of  G2 p r o m o t e r s  instead  the  by  and  allowed  incorporation  investigate  S t e f a n o and  developed  a single  to  heparin.  RNA p o l y m e r a s e  are  time  in  in  RNA p o l y m e r a s e  added to  at  free  by  of  a single  A2 a n d  polymerase.  are  is  assay  fragments  lengths  McClure,  products  formation  using  work  the  prior  The p r o d u c t i v e this  promoters,  c a n be u s e d t o  of  rate  by  DNA o r  inactivated  synthesize a run-off  the  on t h e  s t u d y JE. c o l i  restriction  and h e p a r i n  at  initiation  lac  an o p e n c o m p l e x  proceed  is  transcription to  1974;  Enzyme w h i c h  sites  promoters  ( 1980)  inactivation  Chamberlin,  non-specific  In  at  1967;  to  G r a l l a ,. 1 980 ) .  complexes at  interactions  resistant  plasmid  those  fragments, which  had  been  cleaved at  promoter in  of  interest.  my a s s a y s  Gralla the  Figures  12 a ,  polymerase complexes sequence  than  by  in  included  the  the  and  would  prevent  the  promoter the  complexes  at  were  of  suggested  (see  by  Stefano  the  addition  non-initiated  the  nucleotide the  A2  promoter  and S p i e g e l m a n , 1 9 8 5 ) , to  allow  promoter. work  thus  the  The  modified  initiated  non-  concentration  of  initiation  at  the  events level  of  transcription  A2  in  which promoter  transcription  concentrations. which  presence of  45  used  8).  heparin  the  heparin  100 u g / m l  of  in  the  complexes,  The c o n c e n t r a t i o n  an e x p e r i m e n t  ATP  enzyme  measured  as opposed to  heparin  (see  t h e RNA  on  different  of  binary  concentration  the  with  transcription  that  from  and  experiments  heparin-resistant,  Figure  heparin  formed  the  this  was 5 u g / m l ,  of  Since  reactions  Finally,  by m e a s u r i n g  results  of  heparin-resistant,  multiple  determined  the  used i n  A2 p r o m o t e r .  was  shows  of  level  (Dobinson  in  Gralla  of  out  simultaneous  initiated  formation  assays  The e f f e c t the  low  complexes at  assay  the  the  n o n - s p e c i f i c DNA  heparin-resistant,  pppAGA  complexes.  Stefano  from  a very  transcript  formation  more  from  promoter/polymerase  A2 p r o m o t e r .  rate  initiated  in  to  the  with  thus  carried  that  These data  initiated  of  used  showed  with the  downstream  Secondly, preliminary  form  of  composite  rather  assays  not  a n d GTP w e r e form  b).  was  did  begins  rate  the  1982).  resulted  site  There  and r i b o n u c l e o t i d e s  complexes  to  in  A2 p r o m o t e r  heparin  site  than  (1980,  029  a restriction  from  Figure  8  enzyme/promoter  ATP a n d  GTP f o r  ten  F i g u r e 8. E f f e c t o f h e p a r i n c o n c e n t r a t i o n on t r a n s c r i p t i o n f r o m t h e A2 p r o m o t e r . Holoenzyme ( c o r e / s i g m a / d e l t a ) was i n c u b a t e d w i t h t h e A2 p r o m o t e r t e m p l a t e , A T P a n d GTP f o r 10 m i n u t e s . Then h e p a r i n (at t h e i n d i c a t e d c o n c e n t r a t i o n s ) , CTP a n d UTP" w e r e a d d e d to the r e a c t i o n s to a l l o w s y n t h e s i s of r u n - o f f t r a n s c r i p t s from the complexes formed d u r i n g the i n i t i a t i o n i n t e r v a l . T r a n s c r i p t s w e r e s e p a r a t e d on d e n a t u r i n g g e l s and t h e r a d i o a c t i v i t y i n t h e A 2 s p e c i f i c RNA q u a n t i t a t e d a n d c o n v e r t e d t o t h e n u m b e r o f t r a n s c r i p t s s y n t h e s i z e d p e r A2 p r o m o t e r , a s d e s c r i b e d i n M a t e r i a l s and M e t h o d s . T h e e n z y m e c o n c e n t r a t i o n was 50 nM a n d t h e DNA c o n c e n t r a t i o n 1 . 7 n M .  46  HEPARIN CONCENTRATION  47  ug/ml  minutes.  Initiation  CTP a n d h e p a r i n figure)  to  the  (at  was t e r m i n a t e d  indicated  reaction  and e l o n g a t i o n  of  transcripts  s y n t h e s i z e d per  function  the  out  heparin  for  A2 p r o m o t e r  concentration  the  absence of  approximately  2.4  transcripts/promoter  transcription  concentration transcripts off.  In  occurred Based  at  experiment  above d a t a  experiments  concentrations promoters 1980;  shown i n  Roe e t  the  a l . ,  and  the  at  50 nM e n z y m e ,  used  Burgess,  in  as a  synthesized in  1 ug/ml  the  number  and t h e n  65%  8 the  plateau  per  promoter. chosen  10 t o  lower  studies  20 of  Stefano  of  leveled  concentration  1978;  a  heparin  transcripts  heparin  of  elongation  were  Figure  was 5 u g / m l ,  routinely  (Miller  0.8  The n u m b e r  in  approximately  UTP,  the  was d e t e r m i n e d  As t h e  was i n c r e a s e d f r o m 0 t o  approximately  on t h e  subsequent  reaction.  d e c r e a s e d by  the  heparin,  in  of  initiated  10 m i n u t e s .  In  standard  addition  concentrations  was c a r r i e d  reaction.  the  the  transcripts  of  by  fold E.  for than  coli  and  Gralla,  1985) . I  The e f f e c t from the of  the  assays Single  subsequently that  enzyme c o n c e n t r a t i o n  A2 p r o m o t e r .  kinetic 50 n M .  of  of  heparin  was d e t e r m i n e d  at  an enzyme  round  5 ug h e p a r i n / m l of  In  experiment  the  from 5 to  transcription  The amount  carried  rounds  on  out  transcription at  different  was s u f f i c i e n t  transcription  assays  to  be u s e d  concentration  were  enzyme i n p u t s  to  prevent  higher  concentrations  shown i n  Figure  9 the  reactions  T h e DNA w a s i n c u b a t e d 48  to  show  multiple  at  80 nM e n z y m e .  in  of  enzyme.  contained  with  the  F i g u r e 9. The e f f e c t o f enzyme c o n c e n t r a t i o n on t h e l e v e l o f t r a n s c r i p t i o n f r o m t h e A2 p r o m o t e r . C o r e / s i g m a / d e l t a ( a t t h e i n d i c a t e d c o n c e n t r a t i o n s ) was i n c u b a t e d w i t h E c o R I - t r e a t e d p 3 2 8 - 5 DNA ( 1 . 7 n M ) , ATP a n d GTP f o r 10 m i n u t e s t o a l l o w t h e f o r m a t i o n o f i n i t i a t i o n complexes. H e p a r i n , (5 u g / m l ) , CTP a n d UTP were t h e n added to a l l o w e l o n g a t i o n from initiated complexes. The l e v e l o f c o m p l e x f o r m a t i o n (activity) i s e x p r e s s e d as the i n c o r p o r a t i o n o f [ P ] U T P i n t o A2 transcripts .  49  50  enzyme,  A T P a n d GTP f o r  heparin  (5  ug/ml)  were  enzyme  concentration  amount  of  approached  of  Initiation Promoter.  In  which  it  would  initiated  of  than  this  transcription,  and E l o n g a t i o n to  carry  was n e c e s s a r y sufficient  to  c o m p l e x e s and 2)  5 to  level  As  the  30 nM  the  At  enzyme  of  transcription  particular obtained  experiment  at  an  enzyme  transcripts/promoter  of  out  to  C T P , UTP a n d  reactions.  30 nM t h e In  the  increased.  80 n M , w a s 0 . 7  order  be  the  steadily  a maximum v a l u e .  concentration  work  to  before  was i n c r e a s e d f r o m  greater  maximum l e v e l  this  added  transcription  concentrations  the  10 m i n u t e s  Transcription the  kinetic  determine allow  1)  completion  the  the  analyses time  maximal of  from  A2  in  intervals  formation  of  run-off  transcripts. The e n d p o i n t round  of  transcription  A2 p r o m o t e r  template  for  lengths  varying  heparin, which  formed  elongation  not  concentration  rapid  (Figure chosen  with of  during  reaction  but  10).  of  time  investigated.  the  time  allow the  course  of  to  initiation for  Initiation  for  the  complexes  10 m i n u t e s .  At  reaction  after  the  five  reaction formed  an was  minutes  time  initiation  c o m p l e x e s were  the  adding  The  initiation  elongation  51  then  reaction.  complete  endpoint  single  ATP a n d GTP  from  initiation  a 10 m i n u t e  the  incubating  20 m i n u t e s ) ,  elongation  10 nM t h e  the  by  for  RNA p o l y m e r a s e ,  (1  was e s s e n t i a l l y Therefore  reaction  was d e t e r m i n e d  was c o n t i n u e d  as a c o n v e n i e n t  The  initiation  assay  CTP a n d UTP t o  had  enzyme  the  was reaction.  was  also  for  10  Figure 10. E f f e c t o f t h e i n i t i a t i o n t i m e i n t e r v a l on t h e l e v e l o f t r a n s c r i p t i o n f r o m t h e A2 p r o m o t e r . C o r e / s i g m a / d e l t a ( 1 0 nM) was i n c u b a t e d w i t h E c o R I t r e a t e d p 3 2 8 - 5 DNA ( 1 . 7 n M ) , ATP a n d GTP f o r v a r i o u s l e n g t h s o f t i m e b e f o r e h e p a r i n , CTP a n d UTP w e r e a d d e d to the r e a c t i o n . T h e e l o n g a t i o n r e a c t i o n was c a r r i e d o u t f o r 10 m i n u t e s . - T h e o r d i n a t e r e p r e s e n t s t h e i n c o r p o r a t i o n of [ P ] U T P i n t o A2 t r a n s c r i p t s (activity) . 3  52  53  minutes time  prior  allowed  varied  to  the  addition  for  the  subsequent  elongation  20 m i n u t e s .  Figure  between  elongation complete  reaction  within  synthesized increased minutes  1 and  in  1 minute  did  the  was v e r y  not  of  heparin,  rapid.  and  since  change as the  elongation  all  of  the  CTP a n d U T P . reaction  11 s h o w s  number  incubation  reaction  of  C H A R A C T E R I Z A T I O N OF THE I N I T I A T I O N  transcripts was  out  for  subsequently  the  was  time  was c a r r i e d  experiments  was  that  Transcription the  The  10  described.  R E A C T I O N AT THE A2  AND G2 PROMOTERS A.  Initiation  at  Nucleotide at  A2.  were  the  A2  requirements  The o b s e r v a t i o n  not  formed  A2 p r o m o t e r  Promoter  that  that  the  complexes at  initiating  ribonucleotide(s).  might  be s l o w  inactivate formation compared 2)  in to  to  enzyme.  the  formation assay  to  the  require  The d a t a  3)  in  rate  formation  complexes  A T P , GTP a n d with  and  the  a productive  Materials  54  the also  the  (heparin-  presence  of  suggested  extent were  of  can complex  1) A T P , of  complex  transcription in  that  therefore  The r a t e  and M e t h o d s )  the  complex  heparin  presence of  CTP.  with  stable  which  nucleotides in  of  initiation  at  The k i n e t i c s  formation  was f o l l o w e d  (described  A2 m i g h t  absence of  complex  A T P a n d GTP o r  formation  a heparin-resistant  relative  the  complex  w h e n RNA p o l y m e r a s e was i n c u b a t e d  suggested  transition  stable  heparin-resistant  resistant)  the  for  which  the  rate  F i g u r e 1 1 . The e f f e c t o f t h e e l o n g a t i o n t i m e interval on t h e l e v e l o f t r a n s c r i p t i o n f r o m t h e A2 p r o m o t e r . RNA p o l y m e r a s e a n d t h e A 2 p r o m o t e r t e m p l a t e w e r e i n c u b a t e d f o r 10 m i n u t e s w i t h A T P a n d GTP t o f o r m i n i t i a t i o n complexes, prior to the addition of heparin, CTP a n d U T P . T h e e l o n g a t i o n r e a c t i o n was t h e n c a r r i e d out f o r v a r i o u s l e n g t h s o f t i m e ( i n d i c a t e d on t h e figure). The i n c o r p o r a t i o n o f [o*32 -| p t r a n s c r i p t s was p l o t t e d a s a f u n c t i o n o f t h e l e n g t h o f time allowed f o r e l o n g a t i o n . The p r o m o t e r c o n c e n t r a t i o n w a s 1 . 7 nM a n d t h e e n z y m e c o n c e n t r a t i o n 50 n M . p  55  U T  i  n  t  o  A  2  level of  of  complex  [ot32p-]  function to  the  n T  of  p  i  the  products  combinations  the  or  of  reactions  Figure  in  the  A2 t r a n s c r i p t low  RNA p o l y m e r a s e inactivation  Incubating (Figures  transcripts together. composed  than This  of  the  ATP t h e  pathway  to  implying  Complexes will  be  the  in  be  the and  the  presence The  the  in  the  is  of  indicated carried  presence of  was  those  which  was  gels  complex  times  formation  under  of  in  above.  a  remaining  by  the  as  polymerase  formed  for  or  a state  when  the  result enzyme, It  the  B5)  ATP  synthesized  conditions sensitive  that  and  proceed  at  the to  are  that  A2  ATP a n d  A-initiated  AG-initiated  57  A T P , were  the  formed  were  in  for  any  10  more  incubated  complexes,  through  complexes at presence of  yield  template  assumed here to  template  not  ternary  A2 p r o m o t e r is  the  did  enzyme and  designated  called  ATP a n d  lane  showed  able  ternary in  b,  complexes which  GTP w i l l  of  an a r r o w )  enzyme w i t h  12a and  subsequently  Similarly, and  formed  that  quantitated  heparin.  enzyme i s form  out  nucleotides by  incorporation  autoradiograms  listed  carried  the  heparin  complexes  (indicated  heparin-resistant. of  from  of  When c o m p l e x  remains  by  addition  the  nucleotides  absence of  levels,  minutes  shows  were  by was  core/sigma+delta  legend.  out  very  the  12  (b)  the  addition  synthesized  the  in  the  measured  transcripts)  between  Figure  core/sigma  (as  run-off  0  and  (a)  formation  t  time  reaction  nucleotides. of  n  formation  the  in  the  the  not presence initiation  promoter. the  promoter  complexes. presence  complexes.  of  ATP  Figure 12. Nucleotide requirements for heparinr e s i s t a n t c o m p l e x f o r m a t i o n a t t h e A2 p r o m o t e r , (a) A u t o r a d i o g r a m of a p o l y a c r y l a m i d e g e l of the t r a n s c r i p t i o n p r o d u c t s from the assay f o r complex formation. C o r e / s i g m a ( 9 n M ) was i n c u b a t e d w i t h DNA ( 1 . 7 nM) a n d t h e f o l l o w i n g ribonucleoside t r i p h o s p h a t e s : A ) no n u c l e o t i d e s , B ) A T P , C ) ATP a n d G T P , D) A T P , CTP a n d G T P , a n d i n i t i a t i o n a l l o w e d f o r 15 s ( l a n e 1 ) , 30 s ( l a n e 2 ) , 45 s ( l a n e 3 ) , 60 s ( l a n e 4 ) o r 10 m i n ( l a n e 5 ) . H e p a r i n and the ribonucleotides which were not p r e s e n t d u r i n g complex f o r m a t i o n were added to a l l o w s y n t h e s i s of t r a n s c r i p t s . Transcripts w e r e s e p a r a t e d on d e n a t u r i n g g e l s a c c o r d i n g t o M a t e r i a l s and M e t h o d s . (b) Complex f o r m a t i o n with c o r e / s i g m a + d e l t a ( 9 n M ) a n d 1 . 7 nM p 3 2 8 - 5 . Lanes are t h e same a s i n ( a ) . (c) P l o t of data from initiation t i m e c o u r s e ( 1 5 t o 60 s e c o n d t i m e p o i n t s ) s h o w n i n (a). (•) complex f o r m a t i o n i n the absence of n u c l e o t i d e s or p r e s e n c e o f A T P . ( A ) c o m p l e x f o r m a t i o n w i t h ATP a n d GTP. (•) c o m p l e x f o r m a t i o n w i t h A T P , GTP a n d C T P . The o r d i n a t e r e p r e s e n t s the amount o f [O<3 P]TJTP i n c o r p o r a t e d i n t o t h e A2 t r a n s c r i p t s . 2  58  a  B 1  2  3  4  5  3  4  5  1  2  3  4  1 2  3  4  The gel  and  transcripts quantitated  The a m o u n t  of  shown i n  as d e s c r i b e d  radioactivity  transcripts  was p l o t t e d  for  complex  formation  the  number  of  A-initiated  (Figure  (equivalent  this  experiment). The a d d i t i o n  a 10-fold  resistant  against  complexes formed  low  to  of  complexes  in  c o m p l e x e s were  inactivation  than  nucleotides these  or  results  A-initiated  is  the that  the  the  complexes a f t e r  for  the  of  or  A-initiated  The a d d i t i o n  of  CTP t o  the  presence of  consistently extent  a n d 20%.  of It  ATP a n d  is  clear  per  promoter  reaction of  in  ATP.  and  to  was  in  resulted  the  that to  the  heparin  absence  The i m p l i c a t i o n  which  non-initiated  of of or  heparin-resistant,  is  added  it  over  is  to  the  probably observed  from  the  reaction  resulted  activity  observed  The i n c r e a s e  different  was  experiments,  between  although  approximately  complexes formed  61  that  heparin-  resistant  initiation  GTP.  if  showed  complexes.  increase varied  not  allowed  complexes  transcription  activity  observed with the  the  time  suggesting  although  level  in  of  a 60 s e c o n d r e a c t i o n  12c),  to  the  the  These data  heparin  non-initiated  in  the  low  increase  amount  maximum y i e l d  converted  responsible  into  initiation  extent  significant,  a further  Methods.  non-initiated  presence of  not  in  and  much m o r e  is  the  Materials  complexes formed  complexes are  AG-initiated reaction  in  from  12c).  Figure  AG-initiated  were  cut  transcripts  the  (see  the  during  0.05  GTP t o  increase  in  12a were  incorporated  heparin-resistant  very  in  Figure  in  the  10  presence  of  than  AG-initiated  are  complexes In is  are  the  able  to  promoter McClure sigma  A T P , GTP a n d  synthesize  It  al.  (1982)  the  have  time  shown, using  into  introducing the  at  The E f f e c t The p r o d u c t i v e  to  heparin  of  the  the  the  of  effect any  could  be a t t r i b u t e d  rate  delta  to  of  sephadex  chromatography,  is  into  Hippel  which to  essentially possible  in  to  by  from  avoid be  involved  an e l o n g a t i o n  the  A2  a s s a y was u s e d  the  that  therefore  might  at  et  through  reactions  to  formation  between  state.  Promoter.  transcription  at  A2.  kinetics  different  enzyme w h i c h  by D N A - c e l l u l o s e o r  as d e s c r i b e d i n  62  that  after  von  C T P was  variability  (core/sigma)  It  on c o m p l e x  transcription  depleted  by  h a v e moved  on I n i t i a t i o n  of  template,  has been s y n t h e s i z e d  steps  differences  preparations, delta  state.  an i n i t i a t i o n  transcription  A2  Hansen and  enzyme m o v e s ,  initiation  Delta  the  been i n c o r p o r a t e d  phase.  rate-limiting from  from  hypothesized  an e l o n g a t i o n  RNA p o l y m e r a s e  RNA p o l y m e r a s e  have  enzyme would  that  enzyme  coli  transcript  To e n s u r e not  E.  point  subsequent any  GTP t h e  a polyd(A-T)  an e l o n g a t i o n  the  transition  investigate  the  this  a 14 b a s e  from  more  Spiegelman, 1985).  9 nucleotides  into  A2 p r o m o t e r  omitted  whether  A T P , CTP and  has been f u r t h e r  that  initiation  in  and  8 or  irreversibly, the  resistant  a 14 b a s e t r a n s c r i p t  d i s s o c i a t e s from  approximately  by  complexes or  presence of  (1980)  RNA.  more  formed.  (Dobinson  the  CTP a r e  Materials  had  been  DEAEand  Methods, which  was c o m p a r e d w i t h  had b e e n r e c o n s t i t u t e d Standard,  out,  multiple  as d e s c r i b e d  in  determine  in  the  the  if  the  absence  delta  made was a l s o this  transcription Although it  were  formation  of et  the  at  A2 was  promoter  at  carried  with  core/sigma  time  to  transcription  A2 t r a n s c r i p t  which  Figure  delta  possible  stable a l . ,  13),  lower  indicating  inhibited  produced  than  transcripts  in  the  were that  some s t e p  to  determine  Delta  in  the  at  in  Achberger  the  delta  investigated  complexes.  function  of  the  time  addition  of  heparin  by  to  determine the  the  reaction.  In  Figure  14)  approximately  if  it  delta  was  of  was m e a s u r e d a s a  this 0.8  prior  to  the  particular transcripts  core/sigma following  63  and  formation  formation  initiation,  to  s y n t h e s i z e d by  the  inhibited  on c o m p l e x  Complex f o r m a t i o n for  pathway  1981)  A2 w a s b e i n g  inhibiting  allowed  of  experiment  inhibit  and W h i t e l e y ,  The e f f e c t  of  presence  enzyme/DNA complexes  from  initiation  the  has been shown t o  non-specific  transcription  from  the  transcription  transcription  therefore  (see  was d e p r e s s e d i n  step(s)  1980;  that  were  rate  were  of  considerably  transcription  inhibiting  promoter  delta.  affected of  core/sigma  pathway.  same w a y .  experiment  lengths  delta  was  (see  inhibited.  was p o s s i b l e in  the  above which  being  (Dickel,  and  was n o t  described  delta  purified  and M e t h o d s ,  varying of  from  transcriptions  The amount  slower  particular  delta  for  A2 p r o m o t e r .  of  round  addition  presence of  with  Materials  and c o r e / s i g m a + d e l t a  from  transcription  a 60  per  Figure 13. E f f e c t o f d e l t a on t r a n s c r i p t i o n f r o m t h e A2 p r o m o t e r . S t a n d a r d , m u l t i p l e round transcription r e a c t i o n s were c a r r i e d o u t w i t h c o r e / s i g m a (•) and core/sigma+delta (•) as d e s c r i b e d i n M a t e r i a l s and Methods, except t h a t the times a l l o w e d f o r the r e a c t i o n s w a s v a r i e d b e t w e e n 1 a n d 20 m i n u t e s . Delta was a d d e d t o c o r e / s i g m a a t a r a t i o o f 0 . 4 m o l d e l t a : 1 mol enzyme. The i n c o r p o r a t i o n o f [ c < 3 2 p ] n x p i n t o the A2 t r a n s c r i p t s i s e x p r e s s e d a s a c t i v i t y .  64  second delta  initiation. the  level  initiation  When c o r e / s i g m a was r e c o n s t i t u t e d  of  complexes formed  dropped  by  50$  to  during  a 1  approximately  0.4  with  minute  transcripts/promoter. The i n h i b i t o r y respect  to  the  time  transcription. (with  the  level  of  delta  must  the  of  coli  formation proceeds  of  pseudo-first excess  of  the  of  unoccupied  have  formation  h a d no e f f e c t  at  some p o i n t  led  precise  could  not  be  interactions  by  to  the  proposal  open  that  and  such promoter  that sites  exhibits  1980;  H a w l e y and M c C l u r e ,  of  is  allowing  (as  promoter  have  open complex  c a n be m e a s u r e d b y which  (1980)  into  formation  enzyme  the  for  sites  remaining  66  t  E  amount  varying  The f r a c t i o n  E  at  run-off  described above). (CC _c ]/C )  1980;  further  formation  quantitating  incorporated complex  of  the  and G r a l l a ,  rate  the  complexes  conditions  initial  step  above.  intermediate  and G r a l l a  A2  the  under  Stefano  the  from  during  the  was a f f e c t e d  on  kinetics  after time  complex  promoter-specific  open c o m p l e x e s at order  to  with  the  transcription  described  an u n s t a b l e  radioactivity  lengths  of  specific  was a d d e d t o  some p o l y m e r a s e / p r o m o t e r  promoters  transcripts  which  assay  studies  1980).  shown t h a t these  the  (Stefano  McClure,  was v e r y  complexes, although  pathway  through  formation  be p r e s e n t  RNA p o l y m e r a s e  of  delta  To i n h i b i t  initiation  from  Kinetic  delta  CTP a n d U T P ) d e l t a  therefore  initiation  E.  which  transcription.  determined  the  at  of  When a d d e d s u b s e q u e n t  heparin,  formation in  effect  of  after  a  Figure 14. E f f e c t o f d e l t a o n c o m p l e x f o r m a t i o n a t A2 . RNA p o l y m e r a s e was i n c u b a t e d w i t h t h e A2 p r o m o t e r t e m p l a t e , ATP a n d GTP f o r t h e i n d i c a t e d t i m e intervals. A l i q u o t s of the r e a c t i o n were then added to h e p a r i n , CTP a n d UTP t o a l l o w e l o n g a t i o n o f t r a n s c r i p t s from complexes formed d u r i n g the i n i t i a t i o n reaction. (•) c o r e / s i g m a ; (•) core/sigma+delta; (A) core/sigma present d u r i n g f o r m a t i o n of i n i t i a t i o n complexes, delta a d d e d t o t h e r e a c t i o n w i t h t h e h e p a r i n , CTP a n d U T P . T h e t e m p l a t e DNA was 1 . 7 nM a n d t h e e n z y m e c o n c e n t r a t i o n 30 n M . D e l t a was a d d e d t o c o r e / s i g m a a t a r a t i o of 0.8 mol d e l t a : 1 mol c o r e / s i g m a .  67  A  TIME (sec)  68  binding of  time  of  t  radioactivity  synthesized reaction  incorporated  follows  into  the  Since  first  reaction  is  (Stefano  and  show  the  a n c  loss  linear  Gralla,  during of  available the  that  formation  of  AG-initiated  reactions (Stefano  could and  0.006  s~  The  1982)  K 0  b  final rate  effect  of  synthesized  in  measured  various  at  formed  k o  b  in  experiment  the  )  of  complex  delta  for  binding plot  Figure  complexes at  A2.  the  the  also  The  s l o p e s of o  of  f  o  b  s  the  show  the  rate  pseudo-  the  was a d d e d  indicating  lines  reaction.  core/sigma  r  complexes  A2  is,  K  delta  s~^  12,  initiation  15.  pmole  was 0 . 0 1 9  S  from  The d a t a  velocity  3.6  that  formed,  In  per and  as w e l l  as  delta  formation.  on t h e  a productive  are  of  taken  at  Figure  the  number  of  s  from  core/sigma+delta,  the the  (  experiment  core/sigma.  E  sites  core/sigma inhibited  be c a l c u l a t e d  pmole  for  to  constants  15,  kinetics.  complexes  a measure of  particular  depressed  delta  Gralla,  intuitively,  depressing  order  of  initiation rate  (C )  binding  on a s e m i l o g a r i t h m i c  Figure  order  binding  course  in  which  transcripts  promoter  time  amount  radioactivity  presented  the  the  the  1982).  addition  this  run-off  The d a t a the  from  an e x t e n d e d  when g r a p h e d  that  this  of  kinetics  pseudo-first  first  RNA a f t e r  the  order  the  maximum a m o u n t  followed  at  into  complexes formed j  f c  reaction.  incorporated  from  (C )  s e c o n d s c a n be c a l c u l a t e d  level  of  A2  transcription  transcript  assay  was  also  enzyme c o n c e n t r a t i o n s .  The r e s u l t s  of  shown  the  of  in  Figure  69  16.  In  absence  Figure 15. S e m i l o g a r i t h m i c p l o t of the d a t a from the A G - i n i t i a t i o n t i m e c o u r s e s shown i n f i g u r e s 12a and b. The o r d i n a t e r e p r e s e n t s t h e f r a c t i o n o f u n o c c u p i e d promoter s i t e s r e m a i n i n g a f t e r the p e r i o d of initiation i n d i c a t e d , c a l c u l a t e d from the l e v e l of transcription (Cj.) o b t a i n e d a f t e r i n i t i a t i o n t i m e = t , and t h e maximum l e v e l o f t r a n s c r i p t i o n ( C _ ) o b t a i n e d a f t e r 10 minutes of i n i t i a t i o n . T h e p r o m o t e r c o n c e n t r a t i o n was 1 . 7 nM a n d t h e RNA p o l y m e r a s e w a s 9 n M . (•) c o r e / s i g m a , ( • ) c o r e / s i g m a + d e l t a (3.6 mole d e l t a / m o l e enzyme).  70  71  F i g u r e 16. E f f e c t o f d e l t a o n t h e maximum l e v e l o f t r a n s c r i p t i o n f r o m t h e A2 p r o m o t e r a t v a r i o u s e n z y m e concentrations. C o r e / s i g m a (at the indicated c o n c e n t r a t i o n s ) was i n c u b a t e d i n t h e p r e s e n c e ( • ) or a b s e n c e of d e l t a (•) u n d e r the c o n d i t i o n s d e s c r i b e d p r e v i o u s l y for f i g u r e 9. D e l t a was a d d e d t o c o r e / s i g m a at a r a t i o of 0.5 m o l . d e l t a : 1 m o l . enzyme. The l e v e l of complex f o r m a t i o n ( a c t i v i t y ) i s e x p r e s s e d as the i n c o r p o r a t i o n o f [ < * 3 P ] U T P i n t o A2 t r a n s c r i p t s . 2  72  ENZYME CONCENTRATION (nM)  73  added d e l t a enzyme  the  amount  concentration  approximately  0.9  concentration  of  level  of  obtained  (at  transcripts/promoter nM.  36  tested  the  at  of  formation  Delta  on t h e  would  tested of  allowing  the  by  for  elongation from  the  of  the  enzyme  of  transcription  nM)  36  Stability  the  was  would  functional  initiation  delta  of  could the  initiated  AG-initiated  complexes i n  lengths to  of  time  occur.  by  Enzyme  heparin  complexes would number  and  the  before  complexes during  be i n a c t i v a t e d  the  inhibit  c o m p l e x e s was  reaction  decrease in  Initiated  initiated  varying  initiated  incubation  corresponding  of  incubating  heparin  dissociated  the  be b y d e s t a b i l i z i n g  The s t a b i l i t y  presence  of  enzyme added d e l t a  maximum l e v e l  One m e c h a n i s m b y w h i c h  complexes.  all  of  an  presence of  was l o w e r and the  at  the  transcripts/promoter.  0.6  The E f f e c t  therefore  In  i n c r e a s e d as  a maximum  an enzyme c o n c e n t r a t i o n  approximately  Complexes.  transcription  i n c r e a s e d , to  transcription  concentrations  complex  of  which  the the  loss  of  be o b s e r v e d a s a of  transcripts  per  promoter. An e x a m p l e complexes  in  the  When p r e - f o r m e d presence complexes  of  of  a time  presence of initiation  heparin  the  decreased with  delta  reduced  delta  to  the  course  the  level  enzyme d i d  of  loss  heparin  is  of  shown i n  c o m p l e x e s were  fraction  of  initiated  incubated  core/cr  not  complexes formed, appear 74  to  alter  in  17. the  and c o r e / c r +  increased incubation of  Figure  time. the  the  While  addition  of  stability  of  F i g u r e 17. E f f e c t o f d e l t a on t h e s t a b i l i t y of i n i t i a t e d complexes. C o r e / o - ( • ) o r c o r e / c r + <J ( • ) was i n c u b a t e d w i t h E c o R I - t r e a t e d p 3 2 8 - 5 DNA ( 1 . 7 n M ) , ATP a n d GTP f o r 10 m i n u t e s t o a l l o w t h e f o r m a t i o n o f i n i t i a t e d complexes. H e p a r i n was t h e n a d d e d and a t t h e t i m e s i n d i c a t e d s a m p l e s were removed to a l l o w e l o n g a t i o n f r o m i n i t i a t e d c o m p l e x e s w h i c h had not d i s s o c i a t e d d u r i n g the h e p a r i n t r e a t m e n t . Promoter c o n c e n t r a t i o n w a s 1 . 7 nM a n d t h e e n z y m e c o n c e n t r a t i o n was 20 n M . D e l t a was a d d e d t o c o r e / s i g m a a t a r a t i o o f 0.5 m o l . : 1 mol. enzyme. C o m p l e x f o r m a t i o n was q u a n t i t a t e d a s t h e n u m b e r o f A2 t r a n s c r i p t s s y n t h e s i z e d per promoter. The s o l i d l i n e r e p r e s e n t s t h e l o s s o f a c t i v i t y d e t e c t e d i n t h e p r e s e n c e o f h e p a r i n and t h e dashed l i n e r e p r e s e n t s the l o s s of a c t i v i t y d e t e c t e d i n the absence of h e p a r i n .  75  TIME  (min)  the  complexes or  heparin. in  the  17 t h e  stable  result and  Therefore  initiation  Figure less  their  delta  must  reaction.  to  direct  affect  In  the  inactivation  some o t h e r  experiment  c o r e / s i g m a c o m p l e x e s seemed to  than  was n o t  the  sensitivity  core/sigma+delta  consistently  difference  was  observed  therefore  in  not  parameter  presented  be  complexes.  by  slightly  However,  repeat  in  this  experiments  considered  to  be  significant.  The  Stability  of  Non-initiated  Promoter.  Competition  to  the  measure  stability  A2 p r o m o t e r ,  with  determine  delta  if  increasing  the  that  available  at  8.5  of  nM a n d  competitor the  were  with  the  DNA e x c e s s , an a c t i v e  to  bind  enzyme w h i c h  of  five  carried  after  the  five  d i s s o c i a t e d from competitor  also  At  15 s e c o n d i n t e r v a l s  s a m p l e s were  bind out  removed  77  by  complexes.  To all  ensure the  concentration 5 nM.  The  recognized A2  reaction,  A2 p r o m o t e r . in  after  and added t o  the the  by  template  binding  the  template  to  under  of  the  minute  the  was a d d e d  to  to  the  c o r e / s i g m a was  a promoter  was a d d e d  attempt  formation  A2 p r o m o t e r  of  nM.  assay  enzyme c o n c e n t r a t i o n  also  A2  an  minutes.  sites  concentration 8.5  the  was c a r r i e d  the  in  non-initiated  promoter  with  the  complexes at  complex  DNA f o r  RNA p o l y m e r a s e )  a n d RNA p o l y m e r a s e ,  using  out  as f o l l o w s :  experiment  DNA ( w h i c h  sigma-43  of  stable  out  p328-5  sufficient  enzyme,  conditions  intention  carried  carried  non-initiated  inhibited  was  incubated there  the  of  were  dissociation rate  The e x p e r i m e n t first  assays  Complexes at  The  reaction  was  competitor  ATP a n d  GTP t o  allow form  enzyme w h i c h initiation  promoters. minutes  elongation  as  to  either  complexes, thus  The i n i t i a t i o n  before  complexes  was b o u n d  heparin,  from  the  trapping  reaction  initiated  competition  a decrease in  the  number  The from  competitor  the  al.,  1979,  This  plasmid  using  the  run-off  029  1980;  c a n be  plasmid  RNA o f  distinguished  number  from  II.  initiation synthesized  be n o t e d  0.2  enzyme five 0.08  A2.  together from  for  A2 d r o p p e d  although  was v e r y  were the  low  to  the  (obtained  1979;  Davison  into  of  the  G2  promoter  c a n be  experiment was to  minutes 0.02  are  of  the  the  10  gels.  shown  in  with  minute  were  a n d A2 the  a  easily  incubated  templates  level  of  transcripts/enzyme from  G2.  transcription  o b s e r v e d d e c r e a s e was  78  et  pEMBL.  on p o l y a c r y l a m i d e  prior  of  a  Columbia)  which  synthesized  level  and  RNA p o l y m e r a s e y i e l d s  competitor  five  be  A2  transcripts.  transcripts/enzyme  When t h e  allow  Transcription  (core/sigma)  minutes  ten  detected  cloned  120 b a s e s ,  competition  for  would  G2  downstream  the  of  a l . ,  1985)  sigma-43  to  to  The l o s s  British  A2 t r a n s c r i p t  transcripts  that  A2 p r o m o t e r  the  one  for  from  transcription whereas  the  of  of  out  construct  enzyme H i n d l l l .  with  reaction,  incubated  a l . ,  linearized  When t h e  A2 t e m p l a t e  of  (Sogo et  approximately  The r e s u l t s Table  G2 p r o m o t e r Garvey et  added  reaction  DNA w a s a p l a s m i d  restriction  linearized  were  the  enzyme a t  A2 t r a n s c r i p t s  G.B. Spiegelman, University  carrying  the  in  of  promoters  was c a r r i e d  complexes.  the  increase  the the  CTP a n d UTP w e r e  during  corresponding  of  It from  should the  TABLE Template Assay Conditions 1  2  3  Template  II  competition Pre-binding  assay Transcripts/enzyme A2 G2  A2  -  0.21  A2/G2  -  0.05  +  0.08  A2/G2  +  0.02  0.29  A2/G2  +  0.02  0.21  A2  0.38  The e f f e c t o f a c o m p e t i t o r (G2) t e m p l a t e on t r a n s c r i p t i o n f r o m t h e A2 t e m p l a t e i n a s i n g l e r o u n d t r a n s c r i p t i o n a s s a y , was m e a s u r e d u n d e r t h e f o l l o w i n g c o n d i t i o n s : 1) S t a n d a r d p r o d u c t i v e t r a n s c r i p t i o n c o n d i t i o n s ( 1 0 m i n u t e AG i n i t i a t i o n f o l l o w e d b y a 10 m i n u t e e l o n g a t i o n r e a c t i o n ) w i t h A2 a l o n e o r A2 a n d G 2 . 2 ) The i n i t i a t i o n r e a c t i o n was p r e c e d e d by a 5 ' 1 5 " b i n d i n g r e a c t i o n d u r i n g w h i c h t i m e A2 o r A2 a n d G2 w e r e i n c u b a t e d w i t h t h e RNA p o l y m e r a s e . 3 ) A2 was i n c u b a t e d w i t h t h e RNA p o l y m e r a s e f o r 5 m i n u t e s , t h e n G2 w a s a d d e d a n d t h e b i n d i n g r e a c t i o n c o n t i n u e d f o r a n a d d i t i o n a l 15 s e c o n d s b e f o r e a l l o w i n g i n i t i a t i o n to o c c u r .  79  reproducible. effectively when  the  T h e G2 t e m p l a t e  with  two  the  A2 p r o m o t e r  templates  were  reaction.  T h e G2 p r o m o t e r  competitor  when i t  complexes.  Within  added the  to  two  were  the  very  was t h e  While  that  it  is  In  possible  limitations time  in  of  intervals  dissociation particular delta  of  of  demonstrate  the  less  at  the  A2  competition about  promoters.  When t h e  A2 t e m p l a t e  transcripts  were  the  with  at  the  the  rate  (it  not  delta,  was n o t  of  complexes  two  be  This  from  A2  complexes would due  to  enough  to  to  sample  the  if  the  complexes i t of  at  this  determine  increasing  state  be  the  Although  equilibrium  so  ascertained.  be d e t e c t a b l e .  by  result  dissociated  increased  sensitive  at  templates  enzyme.  not  was  an  formation  did  non-initiated  promoter. assay revealed  complex  prior  polymerase  G2 t e m p l a t e  possible  15 s e c o n d s )  the  efficient  A2 p r o m o t e r s  could  very  enzyme  to  A2/RNA  distribution  A2-polymerase binary  rapid  the  a s an  non-initiated  than  would  complex  available  preformed  presence of  assay  information  the  that  a s s a y was n o t  instability  The  the  rate  affected  complexes  the  compete  core/sigma dissociated  a dissociation  more u n s t a b l e  acted  to  incubated  fact,  the  same a s w h e n t h e  enzyme bound  rapidly.  rapidly  also  A2 c o m p l e x e s t h e  that  for  15 s e c o n d s a f t e r  simultaneously  indicated  seemed t o  added s i m u l t a n e o u s l y  was a d d e d  promoters  thus  formation  two at  additional the  RNA p o l y m e r a s e was n o t to  initiation,  s y n t h e s i z e d per  80  A2 a n d G2 incubated  approximately  enzyme  (in  pieces  the  with  0.2  absence  A2 of  of  the  G2 c o m p e t i t o r ) .  60$  (to  0.08  enzyme  were  suggesting under  transcripts/enzyme) pre-incubated  that  the  conditions  complexes  could  transcripts  from  the  enzyme  plot  kinetic  of  order  proposed  of  from  by  only  the  five  II).  the  the  rapidly  observed  that  G2 p r o m o t e r  This  than  observation  al.  G2 p r o m o t e r from  and  minutes,  very  was a l s o from  by  initiation  Davison et  RNA s y n t h e s i s  (1980)  was much  A2 p r o m o t e r  lacUV5 and  that  the  that  in  more at  low  _  p c  is  the  above,  Figure  complexes at  analogous  Gralla  (1980)  formation  (such  to  a  the  A2  A2 f o l l o w e d  open  promoter  preliminary  15 s h o w e d t h a t  complex  +  of  and M c C l u r e  the  pseudoformation  P ^ = ^ E - P  an u n s t a b l e  ^ = f r  c  (closed) complex  open complex  is  pathway.  81  (1980)  open complexes at  as l a c U V 5 ) p r o c e e d s as  open p o l y m e r a s e / p r o m o t e r to  in  at  at  promoter.  E E  initiation  As d e s c r i b e d  presented  kinetics,  promoters  closed  Table  report  initiation  Stefano  where  It  synthesized  transcription  analysis  E_. c o l i  coli  for  template  heparin-resistant  (see  analysis).  formation first  the  decreased  concentrations.  Kinetics  the  were  transcription than  together  be f o r m e d .  a previous  efficient  (Tau  where  A2 p r o m o t e r  supports  when  thus  enzyme was i n a c t i v a t e d  not  more  vitro  The enzyme a c t i v i t y  the  E  _ p  the  some E..  follows:  o  intermediate,  and  have  E-P  transition  rate-limiting  step  is  the  from  the  Q  in  the  Based  on t h e above model S t e f a n o and G r a l l a  a composite rate assay to i n v e s t i g a t e at mutant l a c p r o m o t e r s . transcription  and  used  open c o m p l e x f o r m a t i o n  T h i s assay combines  the productive  r a t e a s s a y , i n which t h e r a t e o f complex  f o r m a t i o n i s measured a t d i f f e r e n t tau p l o t  (1982)  a n a l y s i s of McClure  equilibrium  enzyme i n p u t s , w i t h t h e  (1980)  to calculate  c o n s t a n t s which c h a r a c t e r i z e  of polymerase/promoter  open  the rate  the formation  complexes.  The S t e f a n o and G r a l l a m o d e l now a p p e a r s  t o be  oversimplified  w i t h r e s p e c t t o t h e c u r r e n t model ( M c C l u r e ,  1985).  data support the hypothesis that the  Recent  f o r m a t i o n o f open c o m p l e x e s three k i n e t i c a l l y  p r o c e e d s t h r o u g h a minimum o f  significant  and M c C l u r e , 1 9 8 5 ; S p a s s k y however t h a t t h e a d d i t i o n a l  s t e p s (Roe e t a l . ,  et a l , 1985).  and  temperature.  carried is  model  t h e S t e f a n o and G r a l l a m o d e l f o r open  c a n be w r i t t e n  *E-P  fi  where E-P^ i s t h e A G - i n i t i a t i o n m  a y n o t be c o m p a r a b l e a t the lacUV5  a s s a y , a t t h e A2  as f o l l o w s :  E+p^=^E-P  formed  analysis  f o r m a t i o n a t mutant l a c p r o m o t e r s , t h e i n i t i a t i o n  promoter  #  strength  (1982) t h e t w o - s t e p  p a t h w a y , a s m e a s u r e d by t h e A G - i n i t i a t i o n  (E-P )  by t h o s e  sufficient.  By a n a l o g y w i t h complex  of i o n i c  For the purposes of the k i n e t i c  o u t by S t e f a n o and G r a l l a  therefore  I t s h o u l d be n o t e d  intermediate reported  a u t h o r s was o n l y d e t e c t a b l e a t e x t r e m e s  1984; Buc  ±  complex.  The i n t e r m e d i a t e  to the closed  promoter  82  ( i t could  complex  that i s  f o r e x a m p l e be an  open the  complex  i n which  promoter).  whose from  E-P  conversion  single  step  binding  ±  of  and G r a l l a ,  is  that  although  is  an i n i t i a t e d adding  introduce  reveals work  kinetics kinetic plot  bond 1980 ,  complex  It  of Stefano at  which  therefore,  open  rate-  the A G - i n i t i a t i o n assay  The  and t h e e f f e c t  therefore  reaction  should not  mechanism as the r a t e  were  the  of the  a non-initiated  (1982).  as a  (McClure, 1980;  are potentially  assumed t h a t  t h e A2 p r o m o t e r  parameters  and s y n t h e s i s  the r e a c t i o n  and G r a l l a  that  the A G - i n i t i a t i o n  than  is  possible  The a s s u m p t i o n  of  rather  steps  t h e same b a s i c  and i s w r i t t e n  is rate-limiting 1982).  transition complex  is unlikely  nucleotide  A T P a n d GTP t o  i s also  The  of other It  from  intermediate  the i n i t i a t e d  irreversible  the endpoint  additional  limiting.  to  was n o t m e a s u r e d .  phosphodiester  complex,  as the u n s t a b l e  because the formation  the i n i t i a t i n g  dissociates  rate-limiting.  s  intermediate  Stefano  the  defined  s  t o be e s s e n t i a l l y  intermediates  first  a i  to E - P i  the unstable  assumed  t h e enzyme r e a d i l y  assay  used  in  initiation  of d e l t a  investigated  on t h e  using  the tau  analysis. An e x a m p l e  initial  rate  of  of  the data  initiation  polymerase  inputs,  unoccupied  promoter  logarithmic complex  scale  formation.  calculating  is  for  the measurement  complex  formation,  shown i n F i g u r e  sites  18.  ({C _c }/c ) E  as a f u n c t i o n The t a u p l o t  the p s e u d o - f i r s t  t  E  of  at  different  The f r a c t i o n  was p l o t t e d  the time  rate  allowed  constants  of  on a for  a n a l y s i s was c a r r i e d  order 83  of the  o u t by  (k fo ) 0  S  F i g u r e 18. (a) A u t o r a d i o g r a m of a p o l y a c r y l a m i d e g e l of the p r o d u c t s from the assay f o r i n i t i a t i o n complex f o r m a t i o n , a t t h r e e RNA p o l y m e r a s e c o n c e n t r a t i o n s . E n z y m e was a d d e d t o D N A , ATP a n d GTP a n d initiation a l l o w e d f o r 15 s ( l a n e 1 ) , 30 s ( l a n e 2 ) , 45 s ( l a n e 3 ) , 60 s ( l a n e 4 ) o r 10 m i n ( l a n e 5 ) . H e p a r i n , CTP a n d UTP w e r e a d d e d t o a l l o w s y n t h e s i s o f r u n - o f f t r a n s c r i p t s f r o m t h e c o m p l e x e s f o r m e d a t t h e A2 promoter d u r i n g the i n i t i a t i o n i n t e r v a l . Transcripts w e r e s e p a r a t e d on g e l s as d e s c r i b e d i n M a t e r i a l s and Methods. (b) S e m i l o g a r i t h m i c p l o t o f d a t a f r o m an i n i t i a t i o n time c o u r s e . The o r d i n a t e r e p r e s e n t s t h e f r a c t i o n of a v a i l a b l e promoter s i t e s (tC -C ]/C ) r e m a i n i n g a f t e r the p e r i o d of i n i t i a t i o n , c a l c u l a t e d as in f i g u r e 15. T h e p r o m o t e r c o n c e n t r a t i o n was 1 . 7 n M . RNA p o l y m e r a s e ( a c t i v e e n z y m e ) c o n c e n t r a t i o n s w e r e : 2 2 . 9 nM ( • ) , 1 9 . 4 nM ( A ) , 1 5 . 8 nM ( A ) , 1 0 . 6 nM (•), 9 . 7 nM ( O ) . E  84  t  E  9.7nM  10.6nM  12.3nM  1 2 3 4 5 1 2 3 4 5 1 2 3 4 5  I5  30 45 Time (sec.) 85  60  from  the  s l o p e s of  concentration,  the  lines  as d e s c r i b e d  obtained above.  at  e a c h enzyme  Tau v a l u e s  (tau=1/k  N obs  were  plotted  plots  s u c h as  constants (1982).  the  were  1/enzyme  one  is  derived  - 1 / K ^  concentration  shown i n  on t h e  where  f  Figure  as d e f i n e d  The y i n t e r c e p t  intercept for  against  k  by  tau is  2  the  rate  Stefano  and  Gralla  plot  the  of  the  intermediate  (E-P  #  E-Pj_), K «  is  the  equilibrium  for  the  intermediate  .1982)  and  reaction  the  overall  E + P  forward  *-E-P  Tau p l o t s  were  ±  i  s  Table  K  each enzyme.  Q n  values  various active  for  preparations enzyme i n  between  the  2  for  respect  enzymes, as d e s c r i b e d  examination stained  delta of  with  approximately The d a t a preparations  the  content  and  complex  constant  Gralla,  (K  calculated  of  of  estimate to  the  was a l s o  ) for  in #4  k  the  enzyme K «  2 >  the  have  determined  Materials  the  fraction  and  of  comparison therefore  sigma content  was n o t  and  a  sigma i n  facilitate  showed t h a t  same a m o u n t  presented (with  x  for  of  each  four  Methods.  determined,  e n z y m e on a n S D S - p o l y a c r y l a m i d e g e l  coomassie blue the  the  constant  stable  different  values  to  the  the  Stefano  five  The  The d e l t a  Although  the  constant  the  to  enzyme. five  rate  dissociation  The c o n t e n t  was u s e d  content  to  and  2  A  each p r e p a r a t i o n  with  1/k  k /K «.  I I I shows  preparations.  been n o r m a l i z e d  rate  generated  preparations.  1980 ;  is  forward  conversion  (McClure,  generate  19 a n d  the  A  to  the  in  of  Table  exception  8(6  delta  the as  enzyme  #3),  had  #5.  I I I show t h a t of  had  i  all  similar  K^^  of  -40  -20  0  20  40  60  1 / [ENZYME] (>iM- ) 1  Figure 19. Tau p l o t a n a l y s i s of i n i t i a t i o n complex f o r m a t i o n a t t h e A2 p r o m o t e r f o r o n e o f t h e e n z y m e p r e p a r a t i o n s (#1) s h o w n i n T a b l e I I I . Each p o i n t r e p r e s e n t s a tau v a l u e , c a l c u l a t e d from the initial r a t e o f c o m p l e x f o r m a t i o n f o r t h e c o r r e s p o n d i n g enzyme c o n c e n t r a t i o n , a s d e t e r m i n e d by t h e r u n - o f f transcription assay. The l i n e was c a l c u l a t e d b y l i n e a r regression.  87  TABLE Kinetic  III  parameters observed for d i f f e r e n t preparations B a c i l l u s s u b t i l i s RNA p o l y m e r a s e  Sigma Content {%)  Delta Content (%)  k-2„ (s" )  (nM)  K . on (x10 M" s"  1  88  66  0.049  47  1.0  2  48  57  0.083  21  4.0  3  48  60  0.035  6  5.8  4«  66  ND  0.238  55  4.3  5»  73  31  0.104  62  1.7  Enzyme  1  K .A„ .*  of  6  1  1  A t a u p l o t a n a l y s i s ( d e s c r i b e d i n t e x t ) was c a r r i e d o u t f o r f i v e d i f f e r e n t enzyme p r e p a r a t i o n s . The k i n e t i c p a r a m a t e r s 2 > A* > on c a l c u l a t e d from the t a u p l o t s as described in text. P r e p a r a t i o n s i n d i c a t e d by t h e a s t e r i s k s were d e p l e t e d o f d e l t a as d e s c r i b e d i n M a t e r i a l s and Methods. T h e d e l t a c o n t e n t o f #4 was n o t d e t e r m i n e d .  k  K  a  n  d  K  w  e  r  e  88  values k 2  whereas  values  are  that  the  (1982)  the  from  range for  seem t o  of  slow  K  content  is  Table  suggest  that  content  of  to  Q n  as  the  B.  Characterization  the  r  0  delta  x  s" •  10  lac  steps.  M" s" . 1  All  1  of  by  Stefano  not  rate  constant  k  of  any  delta  did  between  k  presented 2  and  suggesting  The r e l a t i o n s h i p The d a t a  values  introduced  The c o n t e n t  clear.  The  1  these  promoters,  a s s a y have  A  the  5.8  calculated  K ».  less  of  .238  mutant  kinetic  affect  to  1 to  values  several  and d e l t a III  from  .035  modifications  significantly not  ranged  Q n  ranged  within  Gralla  K  might  2  in  increase  decreases. the  Initiation  R e a c t i o n at  the  G2  Promoter The  G2 p r o m o t e r  has not  characterized  as  transcription  system,  of  the  in  specific that  vitro  the  A2 p r o m o t e r nor  transcription  the  restriction  decided G2  to  which  is  the  out  the  located  in  specific  120  130  vitro initiation  The s i z e bases)  initiated  site  Garvey et  carry  in  extensively  been mapped.  was b e i n g  site  communication;  as  (approximately  G2 p r o m o t e r Hindlll  has  transcript  transcript  been  did  accurately basepairs  (G.B. Spiegelman,  al,  1985).  an a n a l y s i s  of  It  was  complex  of  site the  G2-  suggest from  the  upstream  of  personal  therefore formation  at  the  promoter.  Nucleotide  requirements  the  G2 p r o m o t e r .  Complex  the  same a s s a y u s e d  to  for  stable  formation  define  89  the  at  complex  formation  G2 was a n a l y z e d  requirements  for  at  with  stable  complex the in  formation  absence of the  al.,  at  ATP a n d  A2 p r o m o t e r .  nucleotides  presence of  1980;  the  1)  Complex f o r m a t i o n  was c o m p a r e d  GTP, the  initiating  G.B. Spiegelman, personal  GTP.  The l e v e l  of  complex  was m e a s u r e d a s a f u n c t i o n  the  additions  of  RNA p o l y m e r a s e  nucleotides  to  the  the  to  rate  the of  complexes ATP a n d Figure  complex  GTP w e r e 20. at  Delta  suggested  formation  investigated  that  which  to  promoter  the  the  pppGAAG  directly  ATP a n d  of  is  able  to  compare  form  the  step,  included from  in  at  complex  the  in  G2 w a s  assay,  with the  The (j>29 02  formation  The e f f e c t  G2 b e g i n s  of  formation those  the  complex  at  delta  above, at  from  initiation with  at  therefore  described  communication)  a ternary  90  as shown  G2 P r o m o t e r .  complex  data  GTP o r  initiation.  formation of  of  heparin-resistant  the  rate  the  template  number  complexes at  (G.B. Spiegelman, personal  polymerase  between  and  G2  require  at  investigate  RNA s y n t h e s i z e d  time  same a s w h e n  different.  composite  GTP w e r e  of  kinetics  were  the  binding  complex  the  2)  (transcripts the  final  the  does not  initiation  had been used t o  order  Since  of  with  the  on I n i t i a t i o n  G2 a n d A2 p r o m o t e r s  on t h e  the  formation  G2 a p p a r e n t l y  of  and  in  the  of  or  et_  A T P , C T P , GTP a n d U T P )  essentially  characterization  promoter  In  formation  A2,  ( Davison  and h e p a r i n  with  heparin,  included  Unlike  The E f f e c t initial  of  o b s e r v e d were  complexes  the  addition  formation  reaction.  When h o l o e n z y m e w a s i n c u b a t e d (prior  rNTP  formation  promoter)  remaining  complex  communication)  per  the  to  in  A2.  the step.  sequence t h e RNA  the  G2  A2  Figure 20. N u c l e o t i d e requirements f o r s t a b l e complex f o r m a t i o n a t t h e G2 p r o m o t e r . T h e RNA p o l y m e r a s e ( c o r e / s i g m a / d e l t a ) was i n c u b a t e d w i t h H i n d l l l - t r e a t e d G2 p r o m o t e r t e m p l a t e ( 1 . 7 nM) i n t h e a b s e n c e o f nucleotides (•), i n t h e p r e s e n c e o f GTP ( A ) , o r w i t h ATP a n d GTP ( • ) , f o r the times i n d i c a t e d . H e p a r i n and the r e m a i n i n g n u c l e o t i d e s were then added to a l l o w the s y n t h e s i s o f t r a n s c r i p t s f r o m c o m p l e x e s w h i c h had formed d u r i n g the complex f o r m a t i o n r e a c t i o n . Complex f o r m a t i o n i s e x p r e s s e d as the number of t r a n s c r i p t s s y n t h e s i z e d p e r G2 p r o m o t e r . The e n z y m e c o n c e n t r a t i o n was 11 n M .  91  15  30 45 TIME (sec)  92  60  promoter, the  presence of The  in  similar  Figure  time  allowed  of  delta  with  per for  the  promoter  Delta  promoter,  whereas the  A2 p r o m o t e r  complex  formed  on c o m p l e x f o r m a t i o n the  effect  A2 p r o m o t e r .  at  of  at  A2 ,  delta  G2 i s  in  prior  had no e f f e c t final  The number  9 3  the  on c o m p l e x  level  was i n h i b i t e d  to  of  of of  addition  of  formation  at  complex  formation  by a p p r o x i m a t e l y  shown  was m e a s u r e d  was m e a s u r e d a s a f u n c t i o n  initiation,  heparin.  the  ternary  As a c o n t r o l  simultaneously transcripts  the  ATP a n d G T P .  effect 21.  to  50$.  the  the at  G2  Figure 21. E f f e c t o f d e l t a o n i n i t i a t i o n f r o m t h e 029 A2 a n d G2 p r o m o t e r s . C o r e / c r was i n c u b a t e d w i t h ( o p e n symbols) or w i t h o u t ( c l o s e d symbols) d e l t a (at a r a t i o of 0.9 m o l . d e l t a : 1 m o l . enzyme) i n the p r e s e n c e of A T P , GTP a n d t h e A2 o r G2 p r o m o t e r f o r 15 t o 60 s e c o n d s p r i o r to e l o n g a t i o n . Formation of A G - i n i t i a t e d c o m p l e x e s i s e x p r e s s e d as t h e number o f transcripts s y n t h e s i z e d p e r A2 o r G2 p r o m o t e r . ( A ) G2 p r o m o t e r , ( • ) A2 p r o m o t e r . P r o m o t e r c o n c e n t r a t i o n s were 1.7 n M . E n z y m e c o n c e n t r a t i o n was 49 n M .  94  95  DISCUSSION  Methods nature  of  for  the  measuring  RNA s y n t h e t i c  straightforward example,  in  polymerase necessary complex  purpose  analysis  order forms  to  have  Three  are  the  to  pathway  of  the  rates.  precludes  the  complexes  at  methods filter  binding  have  process.  can  For  which it  RNA  is  quantitate  of  been  assay  by  promoters  independently  which  multi-step  a  mechanism  an a s s a y s y s t e m w h i c h reaction  The  transcription  investigate  specific  formation  reaction.  initiation  the  elongation  devised  (Hinkle  the  for  and  this  Chamberlin,  v  1972a), 1976: rate  the  abortive  McClure et assay The  not  that  therefore  to  or  the  ternary  method  formation  has at  Chamberlin,  1972a,b)  (for  see D o i ,  review Work  E.  coli  by  and  filters.  the  E. and  coli at  the  McClure,  transcription  b a s e d on  the  RNA p o l y m e r a s e / D N A whereas  The  complexes of  of at  DNA  binary  for  (Hinkle  phage  can  This  analysis  T7 p r o m o t e r s  some J3. s u b t i l i s  (non-  promoters  elongation.  extensively  phage  complexes  uncomplexed  formation  absence  been used  and  productive  is  (initiated) in  (Johnston  1980).  filters  of and  promoters  1982). his  colleagues  M c C l u r e e_t a l . ,  promoters  the  Gralla,  stable  M c C l u r e and  1976 ;  assay  technique  highly  be m e a s u r e d  particular  McClure,  and  on membrane  bind  initiated)  complex  1978)  filter-binding  retained  does  al.,  (Stefano  observation are  initiation  1978 ) h a s  RNA p o l y m e r a s e 96  (Johnson  will  shown  and  that  at  reiteratively  some  synthesize  and  initiation enzyme.  release d i -  products),  Abortive  without  initiation  rifampicin  or  absence of  elongation  of  initiated  transcripts  The  and  abortive  used  to  are  rate-limiting  the  steps  1980)  fragments  (Cech et  While  the  measurement quantitate  of  products  and  two  in  the  to  identify  a l . ,  1980).  complex  formation,  complexes which is  RNA p o l y m e r a s e  conditions,  be a b l e  productive  to  the  would,  complex  Results), only  under  assay  formation specific  run-off  quantitated.  This  assay  was t h e r e f o r e which  97  do a l l o w  the  pathway DNA  the  necessarily transcripts ,  initiation  assay  appropriate transcripts.  however,  does described  promoter/polymerase  yield  method  study  b e c a u s e , as  which  and u s e f u l  to  initiation  full-length  rate  and  initiation  complete  the  interactions  simple  1981).  quantitated  do n o t  abortive  synthesize  transcription  functional  they  can produce  assumed w i t h  1978;  which  above  allow  four  promoter-containing  outlined  of  abortive  Reznikoff,  at  the  would  Cech,  c a n be  rate  of  presence  which  transcription  methods  that  (see  the  although  Munson and  steady-state  it  above  in  ( M c C l u r e and  1980;  although  measure  favoured  (abortive  release  phenomenon has been e x p l o i t e d  (McClure,  The  is  transcripts,  transcription  This  the  concomitant  ribonucleotides  present  Gralla,  measure  occurs.  tri-nucleotides  c a n be s y n t h e s i z e d e v e n when a l l  ribonucleotides Carpousis  or  transcripts  could  are  considered  be a d a p t e d  to  for  be the  a  study  of  functional  complex  formation  at  Bacillus  promoters.  Characterization T h e 029  conditions. pBR322  directs  EcoRI-treated  the  in  to  bases  promoter is  at  the  initiated  subtilis  general assay  the  and  standard  to  from  and  capable  of  the  promoter  conditions  for  the  productive  the  effect  to  study  was d e t e c t e d heparin  of  increasing  A1 t o ATP a n d  inhibited  The  of  Figure  8  to  (0.8  at  0.05  as  GTP a n d  at  low  then  of  Stefano  the  Since  delta  A2  1.  the  specific  to  define  transcription  the  shown  transcription  was u s e d  preformed  the  rate  peptide  on  13. s u b t i l i s  little  as 5 u g / m l  added  transcription  (see  in  the  concentrations  (1980, up  to  in  12a the  U T P , CTP a n d  transcription  promoter)  and G r a l l a  98  to  Figure  formed  concentration  observed per  RNA  transcripts/promoter),even  heparin,  a heparin  transcripts  heparin  vivo  When c o m p l e x e s w e r e  transcription  o b s e r v e d by  maintained  A5).  from  initiating  complexes very  concentrations  maximally level  was a d d e d  concentrations  been  interactions.  (approximately  lanes  presence  of  polymerase/promoter  promoter  RNA h a s  Spiegelman, 1985).  is  from  conditions  be i n i t i a t e d in  into  transcript,  This  which  assay  was c l o n e d  a discrete  A2,  polymerase/A2  that  length  same s i t e  When h e p a r i n  in  which  from  and  and b,  transcription  and M e t h o d s .  RNA p o l y m e r a s e  specific  at  vitro  of  under  (Dobinson  transcription  in  synthesis  Materials in  the  A2 p r o m o t e r  p328-5,  described be 2 3 7  of  of  was  1 ug/ml.  experiment was s i m i l a r 1982) 20  shown to  and was  ug/ml.  Because  very  little  transcription  complexes  formed  that  RNA p o l y m e r a s e  free  initiated  state  inactivation  in  at  by  curve  complexes  very  suggested  stable  by  heparin.  was 20 u g / m l  and  A2 i s  Although  the  highest  is  as  insensitive  complexes  at  many E.  coli  100 u g / m l  heparin  (Stefano  assays  concentration heparin at  is  sufficient  carried  initiation out  at  concentration initiation, Figures It  to  enzyme c o n c e n t r a t i o n s The  was  time  interval  used  in  course  most  19,  is  This  multiple  80 nM ( s e e described  important  c h o s e n as  even at  low  of  experiments. by  dependent for  the  was s u f f i c i e n t l y  formation,  to  the  tau  on t h e  to  to  is  attack  as are  c a n be 19 8 0 ,  for in  which  the  enzyme  concentration initiation Figure in  9).  Figure  10 was  the that  lowest  of  analysis  enzyme  of  events  10 n M , t h e  plot  ensure  resistant  subsequent  The r a t e  for  open  1982 ).  the  enzyme c o n c e n t r a t i o n s ,  99  heparin  AG-initiated  in  concentration.  some e x p e r i m e n t s  endpoint  long  the  concentration  which  an enzyme c o n c e n t r a t i o n  therefore  reaction  time  up  to  in  heparin  ug/ml)  8).  prevent  as d e m o n s t r a t e d  18 a n d  to  f r o m an e x p e r i m e n t  w a s 50 nM ( F i g u r e  a non-  AG-initiated  and G r a l l a , (5  in  insensitive  that  promoters,  concentration  was d e t e r m i n e d  the  appeared  RNA p o l y m e r a s e w h i c h  possible  A2 a r e  region  heparin  it  at  The h e p a r i n  that  was  it  sensitive  that  therefore  complexes  to  very  The p l a t e a u  also  such complexes at  both  from  nucleotides  and enzyme w h i c h  A2 w e r e  in  tested  absence of  heparin.  concentration are  the  was o b s e r v e d  that  the  initiation complex had gone  to  completion.  The r e a c t i o n  was e s s e n t i a l l y  minutes,  as shown i n  interval  was c o n s i d e r e d  to  time  the  The  investigated  course for  concentration the  Figure  of  the  10, be  used i n  the  therefore  a suitable  elongation  following  complete  minute  endpoint.  reaction  reason.  assay  a 10  by 5  was  also  T h e UTP  was o n l y  10 uM a n d  elongation  reaction  does not  require  concentrations  (Bremer,  1967)  has been shown t h a t  concentrations  the  particular slowing 11,  sites  the  RNA p o l y m e r a s e  on  the  overall  elongation  of  it  tends  DNA t e m p l a t e  elongation  initiated  high  although  to  reaction.  was v e r y  used i n  kinetic  period  therefore  sufficient  to  allow  of  heparin-resistant  all  initiated  low  at  et  transcripts the  at  al.. ,  As s e e n i n  elongation  1974), Figure  rapid. assays  The was  transcripts  to  completed.  Characteristics at  pause  (Gilbert  10 m i n u t e  be  nucleotide  the  even  A2 p r o m o t e r .  in  the  absence of  efficient.  In  transcripts  per  standard heparin rather  Transcription  the  experiment  promoter  reaction  (an  was o m i t t e d surprising  since  seemed t o  that  either  the  that  a large  fraction  rate  be r a p i d  of  not  of  of  Figure  (Figure  This  promoter, be  100  in  very  8 only  2.4  a 10  result  minute when  was  initiated 11).  It  would  complexes i s  enzyme d o e s n o t that  formation  1 base/second)  from  initiated  The o b s e r v a t i o n  A2  seem t o  reaction.  elongation  formation  the  synthesized during  average the  did  shown i n  were  from  transcripts  transcripts.  heparin,  from  complex  the  synthesize presence  appear  slow  or  complete of  heparin  0.8  suggest  that  completed, due  to  transcripts/promoter most  implying  a slow  Stefano from  rate  with  observed complexes enzyme  limiting  in  open  and 4)  addition 1976;  of  3)  in  1)  complexes  stable;  to  lacUV5  must  the  They  inactivates (Sippel  and  proposed that  initiate  slow  also  pre-formed  which  transcription the  proceeded  therefore  could  promoter  one m i n u t e .  RNA s y n t h e s i s  binding  the  et_al., are  to  be  the  rate-  the  transcription. observed for  formation  the  recognition at  of the  from  DNA h e l i x to  ribonucleotides  the  was  transcription  reaction  rifampicin,  of  transition  which  coli  RNA f r o m  transition  which  von H i p p e l  transcription  of  the  initiation  promoter.  involves:  complex  of  process leading  complex,  unwound  very  rate  RNA p o l y m e r a s e , 2 )  closed  by  be d u e  the  The b a s i c  E.  approximately  complexes which  could  the  the  reaction  slow  of  were  observed that  initiated  the  A2 p r o m o t e r at  rate  S t e f a n o and G r a l l a  the  synthesis  (1979)  synthesis  has not  By a n a l o g y ,  complexes  slow  transcripts  transcription  of  1968).  of  the  s y n t h e s i z e d does  initiation.  round  the  step  formation  of  was i n h i b i t e d  that  Hartmann,  that  a  that  initiated  complexes at  a single  slowly,  the  and G r a l l a  pre-formed  during  of  were  initiation the  at  review  1982, McClure,  formed  r e l e a s e of  at  the  site  in  promoter state,  by a  to  an  site  is  upon  see C h a m b e r l i n , 1985)  many E__. c o l i  The  open  promoters  RNA p o l y m e r a s e f r o m  101  site  c l o s e d complex  an i n i t i a t i o n (for  promoter  promoter  the  o f RNA  these  are  complexes  is  slow,  resistant  to  attack  ribonucleotides properties  are  and by  not  that  the  promoter.  2.5  initial from led  binary  to  ATP ( D o b i n s o n  enzyme w i t h therefore ternary  the  complex  12 c o m p l e x e s  the in  ATP a n d  formed  at  with  initiated  complexes.  ATP w e r e  In  the  to  synthesize  resistant  the  presence  1985)  the  complexes  to  1985).  were  the  RNA by  as  1978).  little  The  transcription therefore  for  heparin-  formed  to  A2  promoter the  should  and  the  the  enzyme in  as were  RNA p o l y m e r a s e  (Dobinson  Figure  Figure  enzyme  heparin  conditions  (see  the  As shown  when  GTP t h e  these  ATP  unwound  promoter.  pppAGA  for  heparin-resistant  A2 p r o m o t e r  102  tRNA  Incubating  and  form a  DNA i s  trimer  form  complexes  nucleotide  ATP a n d  and u n d e r  subtilis  have  A2.  as s e n s i t i v e  of  not  requirements  template  the  does  Pero,  these  (1976 )  a tyrosine  binary  the at  enzyme  al..  inhibited  Spiegelman,  which  incubated  Spiegelman,  into  initiating  associated with  able  heparin  A2 p r o m o t e r  enable  and  of  coli  c a n be d e s t r o y e d  (Talkington that  and  at  quite  However,  E_.  et  RNA p o l y m e r a s e  formation  preferred  all  is  absence  1978).  of  complexes  complexes  the  some B a c i l l u s  an i n v e s t i g a t i o n complex  in  Kupper  (j)29 A2 p r o m o t e r / p o l y m e r a s e  The  is  heparin  the  Burgess,  complexes  observation  resistant  is  even  complexes.  Furthermore,  ug/ml  in  characteristic  polymerase/promoter as  and  12. c o l i  heparin-resistant  enzyme  heparin  (Miller  polymerase/promoter reported  the  was non-  is  and  heparin12).  Thus  it  would  appear  complexes  at  that  linkage  phosphodiester  unusual  but  resistant  it  is  of  complexes  the  at  There  (V. are  observation not  the  enzyme does  not  would  complexes  which  protected  from  complexes  is  is  or  initiate  be  heparin-  requires  the  recently  heparin-resistant promoter  requires  for  the  complexes  at  The  possibility  first  that  at are  the  simply  the  the  A2  promoter  also  that The  sensitive  sensitivity  a matter  is  promoter.  of  of  to the  whether  of  the  binary  complexes  1)  the  enzyme  forms  dissociate attack to  or  2)  direct  so  shown  coli  e_t al.  the  enzyme  attack  by  to  open  enzyme i s in  not  the  heparin  and  in  the  presence  of  (1977),  for  example,  have  RNA p o l y m e r a s e  103  that  the  transcription  Pfeffer  E.  of  been  explanations  not  if:  ribonucleotides. the  may  of  open.  occur  heparin  first  promoter  also  complexes  sensitivity  readily  the  communication).  however,  susceptible  rapidly  of  of  synthesis  also  form open complexes  closed  also  tRNA  has  rrnB  heparin.  complexes is  It  possible  the  formation  ( 1 976)  personal  suggest,  could  that  a_l.  A-initiated  The u n u s u a l  cannot  et  formation  require  tyrosine  subtilis  to  the  the  non-initiated  that  complex  the  heparin-resistant  characteristic  formation  Webb,  resistant  non-initiated  heparin  the  three  are  the  This  unique;  at  B.  that  observation  least  a dinucleotide. that  heparin  not  of  and m i g h t  by K u p p e r  demonstrated  nucleotides  at  bonds.  complexes  characterized synthesis  formation  A2 r e q u i r e s  phosphodiester two  the  in  open  complexes  at  the  phage  T7 A1 p r o m o t e r  heparin.  The  low  initiated  complexes  accounted  for  if  level at  the  of  which  the  was  complexes enzyme  consistently  nucleotides  or  in  comparison with Figure was  12a and  losing  formation  been  to  1972b,  loses  to  of  protect  can form  the  stable  polymerase  lacUV5  observation incubated  that  with  hypothesis not  the  that  enzyme,  complexes promoter  Bacillus  form  stable  complexes,  promoter  unless  both  formation  is  in  reaction.  104,  the  in (see  the  enzyme  complex  the  presence  the  formed  only  RNA  probably of  if  DNA h a s the  DNA, s u c h as  by  the  (Stefano, enzyme  (Hinkle  E_. c o l i  assays,  although  the  of  been r e p o r t e d  The  GTP a r e  which  time  that  that  thus  of  in  lower  vitro  as d e f i n e d  ATP a n d  number  formation  subtilis  be  rapidly  absence  ATP was  enzyme.  A2 t e m p l a t e  the  the  1981)  t h e _B_. s u b t i l i s  the  in  in  A-  nucleotides  reaction  complexes with  open  the  previously  during  by  transcription.  suggested  Stefano,  the  the  or  therefore  c o m p l e x e s was  incubation  has  heparin-resistant at  result  activity  denaturation  shown  enzyme  It  of  attack  binary  would  that  complex  during  reaction.  and C h a m b e r l i n ,  due  presence  direct  from  initiate  incubated  This  activity  polymerase  to  a 1 minute b).  open  a 10 m i n u t e  were  the  in  observed  after  template  to  was a d d e d w i t h  polymerase  detected  and  A2 p r o m o t e r  enzyme  when h e p a r i n  It  susceptible transcription  the  inactivated allow  is  E_. c o l i  1981).  lost  the  The  activity  consistent  with  included  in  when  with  RNA p o l y m e r a s e above,  RNA  the the  the  does 029  A2  complex  The  stability  of  the  non-initiated  investigated  with  a competition  was  that  the  observed  promoter could  be d e t e r m i n e d .  hypothesis promoter  that  are  the  the  When t h e  A2/polymerase from  of  0.08  to  therefore  reflected  0.02  if  regarding  the  that  bound  0.06  to  G2.  0.2  ratio  is  with  were  rate the  at  These r e s u l t s stronger  does not at  least  complex  assay  the  to  from  (see  number  seem t o under  the  0.2  under  II)  from  the  A2 d e c r e a s e d  Table  of  were  II).  the  in  the  be  transcripts and  conditions  in  were which  prior  to  twice  as  same  incubated  the  A2 p r o m o t e r .  conditions  105  A2  was  would  G2 t r a n s c r i p t s  were  efficiently  low.  A2  It  s y n t h e s i z e d , yet  templates  <J>29 G2  pre-formed  RNA p o l y m e r a s e  suggested that  than  formation  (Table  s y n t h e s i z e d , under  e v e n when b o t h  considerably  the  However,  A2 t r a n s c r i p t s  G2 t r a n s c r i p t s  promoter,  A2  support  G2 t r a n s c r i p t s / e n z y m e  Similarly,  initiation  polymerase  further  had d i s s o c i a t e d f r o m  pre-incubated  together.  It  a dissociation  transcription  decrease in  synthesized.  conditions,  the  was a d d e d  transcripts/enzyme  A2 was n o t  many  complexes at  transcription  G2 t e m p l a t e  enzyme w h i c h  subsequently actually  II).  complexes formed  competition  complexes,  expected  synthesized  Table  heparin-resistant  The  information  promoter.  These data  (see  unstable.  G2 p r o m o t e r .  provided  that  non-initiated  Characteristics at  non-initiated  d i s s o c i a t e d so r a p i d l y  not  assay  c o m p l e x e s was  G2 p r o m o t e r T h e RNA  recognize which  is  the  A2  the enzyme:DNA  It  has been h y p o t h e s i z e d t h a t  transcription specific  from  sites  transcription 1982). the  on t h e from  therefore promoter  of  to  The n u m b e r  of  was  20).  Thus  formed that to  the  the  as were  with  readily  with  with  of  the  effect  assay  nonon a l . ,  indicated  that  A2 p r o m o t e r .  It  initiation  the  the  at  conditions  complex  the  was  of  formation  the  G2  our  at  the  G2.  A2 p r o m o t e r ,  binary  promoter  in  d i s s o c i a t e d and a r e  There  as is  (Figure the resistant  then,  a  complexes which  It  G2 p r o m o t e r  were  absence enzyme  the  G2 p r o m o t e r  B_. s u b t i l i s  the  GTP a n d ATP  G2 p r o m o t e r s . the  the  with  complexes. the  in  same a s when  unlike  the  between  of  that  G2 t e m p l a t e  GTP o r  initiated  A2 a n d  ability  from  c o m p l e x e s o b s e r v e d when t h e RNA  was e s s e n t i a l l y  strength  inhibit  (Achberger et  under  inhibit  complexes at  the  associate not  would  difference  at  if,  appeared t h a t ,  non-initiated  distinct  of  than  as  little  characterize  determine  incubated  it  heparin  to  have  competition  was s t r o n g e r  would  as w e l l  promoters  the  incubated  nucleotides  a n d DNA w e r e  to  of  system, delta  polymerase  DNA, but  interest  and  promoters,  strong  The r e s u l t s  G2 p r o m o t e r  assay  weak  delta  could  could  be  speculated  be d u e ,  RNA p o l y m e r a s e  binary  are  in  part,  to  complexes which  therefore  resistant  are  to  heparin . The e f f e c t the  A2 a n d  the  delta  G2 p r o m o t e r s .  was s h o w n t o round  of  be  inhibited  transcription  assay  subunit  on c o m p l e x  Transcription by d e l t a  in  (see F i g u r e 106  an  formation  from  the  in  vitro  multiple  There  is  13).  A2  at  promoter  no  round  transcription  trivial  assay  explanation  for  preparation  was shown t o  proteolytic  activity  experiment the is  did  not  transcription considerable  (Spiegelman therefore the  et  at  by  rather  and  the  than  productive  but  initiation directly  in  the  DNase,  Achberger delta  the  as  is  and  7).  to  no  delta  RNase  and F i g u r e  delta  This  which  step  inhibited,  however  acts  to  et  prior  a l . ,  affected  formation  in  complexes at  there  1982).  It  was  transcription  of  in  initiation  from  AG-initiated  that if  required A2,  manner. rate  complex it  12 s h o w e d t h a t  it  to  at  the  affects  some s t e p  in  explanation  enzyme w h i c h  elongates  synthesis  of  the  is  less  complete  core/sigma+delta  during  This  seems i m p l a u s i b l e  explanation  the  the  data  by  that  delta  pathway. in  transcripts  for  14  after  Figure  delta  efficiently,  10 m i n u t e  107  quantitative,  indicating  the  form  delta  A2 was i n h i b i t e d  initiation  for  if  Figure  associated with  RNA c h a i n s fewer  a  reaction  c o m p l e x e s had been f o r m e d ,  GTP t o  that  from  shown i n  formation  was a d d e d  was i n  The d a t a  assay  both  ATP a n d  suggesting  initiation  transcription  not  Figure  core/sigma+delta  qualitative  be t h a t  initiation  that  inhibiting  An a l t e r n a t i v e could  I  There  since  information  that  process of  demonstrated delta  of  was b e i n g  1978;  presented  heparin-resistant affected  inhibition  Table  pathway  13).  A2.  The d a t a core/sigma  (see  likely  Figure  be f r e e  evidence  A2 p r o m o t e r  complexes  the  provide  a_l. ,  most  (see  during  resulting  by  elongation several  14  reaction.  reasons.  First,  the  A2 t r a n s c r i p t  easily  be c o m p l e t e d  during  was  a l s o no d e t e c t a b l e  237  bases  that  (on  or  It  synthesizes DNA t h a n latter  enhanced  delta  either  s e l e c t i o n of  transcripts evidence  directly  to  inhibit  Although decreased the  the  the  that  the  rate  in  the  possibility  delta  initiated  in  Results,  complexes  was t e s t e d  no e v i d e n c e t h a t  or  the  delta  possible enzyme  from  the  phage  1978).  elongation  SP82  The  sites  directly  from  There that  or  which is  full-  thus  delta  no  might  reaction.  delta  to  15),  it  were  inhibited  core/sigma  stability  was n o t  the  which  One  initiation  rate  complexes.  As  the  (see F i g u r e  formed  obvious  altered.  the  of  clearly  c o m p l e x e s were  non-initiated  altered  than  inhibiting  hypothesis  pathway  directly  is the  a_l. ,  than  initiation  Figure  initiation  described  of  which  (see  was t h a t  destabilizing  the  smaller  it  if  vitro  promoter  elongation  at  There  core/sigma/delta  be s y n t h e s i z e d .  addition  interval.  random a l o n g  in  rather  strong  support  A2 p r o m o t e r  step(s)  at  stimulated  could  should  be n o t i c e a b l e  transcripts  suggested that,  and  although  been r e p o r t e d  large  long  RNA p r o d u c t s  does c o r e / s i g m a (Spiegelman et  elongation,  prior  not  bases  elongation  transcription  has a l s o  more  data  length  the  237  polyacrylamide gels),  terminated  template.  only  increase in  such a change might  paused  at  is  by  initiation 17)  stability  and t h e r e of  was  those  complexes. Spiegelman composition  of  and W h i t e l e y  the  (1979)  Bacillus subtilis  108  studied  the  subunit  RNA p o l y m e r a s e  during  transcription, analysis, enzyme  using  and  prior  observed to  non-initiated SP82  early  which  the  These  formed  are  initiated  transcription  Figure  of  and of  the  that and  saturated  promoter.  the  A2 p r o m o t e r  has  concentration, of  Figure this  It  9).  work  of  acts  The  of  overall  et to  a l . , the  that  1982).  formation  delta  at  did  the  lowered at of  enzyme  approaches  assay  the  from  109  not  A2  14  for  which  by  enzyme  the  from  value the  holoenzyme,  course from  attained In at  the  (see  A2 t r a n s c r i p t s rarely  the  enzyme  concentrations  throughout  only  the  in  of  transcription  a plateau  of  curves  level  transcript/promoter.  transcription  of  of  Figure  be a f f e c t e d  observed  at  initiated  level  (see  level  high  from  core/sigma/delta  prior  point  to  an  hypothesis.  delta the  the  sites  The enzyme c o n c e n t r a t i o n  at  one  number  complexes  that  at  different  (Achberger  maximum y i e l d  of  the  promoter.  on t h e  the  been shown t h a t  observation  The  although  value  to  A2 p r o m o t e r  transcription  theoretical level  with  been shown  the  also  from  core/sigma/delta  initiated  was a l s o  that  productive  the  the  changed  transcription  by  same  adding  transcription  level  the  was u n e x p e c t e d .  16 s h o w  has  significantly  delta  delta  from  was r e l e a s e d  respect  the  consistent  It  sedimentation  c o r e / s i g m a and  that  stability  The e f f e c t  example)  the  complexes,  is  delta  are  with  virtually suggest  the  promoter  by  gradient  formed  promoter  complexes  data  affect  complexes  enzyme c o n t a c t s  complexes enzymes  that  initiation.  gene  core/sigma  glycerol  of a  the  general,  high  concentrations  of  enzyme,  transcripts/promoter, was  somewhat  whereas  higher,  usually  transcript/promoter. change that the  the  is,  shown  reaction of  in  could  decreasing the  of  the  three  is  the  rate  of of  the  that  delta  complex  equilibrium  tau  0.8  from  core/sigma  that  delta  1.0  in  the  consistent delta  lowering  the  of  might  reaction,  synthesizing  adding  RNA  with  to  from  the  the  concentration  complex  but  core/sigma/delta  extent  1)  formation  that  delta  reaction,  plot  did  at  at  the  was n o t  able  mechanism would  110  act  to  by  interfering  presented the  K^  rate  as  S )  content.  initiate to  could  be u n a b l e  A2 p r o m o t e r  constant,  to  complex  complexes.  core/sigma/delta  was u n a b l e  could  thereby  affect  and d e l t a  of  transcription,  might  analysis  not  rate  initiate  delta  3)  and  core/sigma/delta  not  or  dissociation  unlikely  the  but  formation  between  either  is  AG-initiated  suggested  Although  of  rate  ways:  promoter,  the  A2 p r o m o t e r  to  therefore  associated with  from  appeared  as  both  The d a t a  correlation  18;  the  promoter  formation  initiation the  inhibit  one  the  0.7  and  decreases.  enzyme w h i c h  with  15 a n d  reaction;  with  from  possibility  the  with  transcription  capable  in  in  interact  III  Figures  is  same e f f e c t  formation interact  that  the  Delta  0.5  enzyme c o n c e n t r a t i o n  This  A2 p r o m o t e r  between  appeared  has  enzyme  the  2)  enzyme  A2 p r o m o t e r .  data  It  effective  the  ranged  by  altering  there  the  was  no  therefore with  transcription with  Table  of  interacted  interact explain  It  in  the  or  the that  promoter.  observation  that  delta and  alters  the  would  the  number  expect  a manner,  have  a higher  K  values  the  definitive. which  slows  of  the  the  of  had  in  the  promoter,  one  complex  formation  in  delta  had III  content  in for  been d e p l e t e d are  of  therefore  between  although of  the  not  those for  data  to  the  were  not  other  enzymes which the  therefore  possible  complex  formation  to  #3,  enzymes,  had  enzymes w h i c h  is  is  the  enzyme p r e p a r a t i o n  comparison  than  transcription  would  interpretation.  exception  It  complexes  A2  a high  Table  content,  delta  delta.  at  be a c o r r e l a t i o n  lower  60%  rate  productive  to  the  AG-initiated  inhibited  above  a low  was  approximately depleted  formed  of  enzyme w h i c h  presented  delta  had  value  delta  than  With  also  kg  if  appeared  and  formation  complexes  A #  with  There  of  enzyme w h i c h  The d a t a  consistent  the  of  that  such  delta.  rate  that  had delta  s u c h an e x t e n t  interfered  with  at  been  the  that  A2  promoters. The by  interpretation  altering  converted The  tau  the  to  plot  that delta  the  at  previously  into  has  the  sigma  content of  the  would  must  promoters.  It  sigma/delta  different affect  of  the  111  be made  the is  kinetic  are  cautiously.  only  for  E_. c o l i  RNA  quite  ratio,  B_. s u b t i l i s  formation  complexes  been used  interactions  or  complex  non-initiated  analysis  different  preparations,  which  affects  complexes  at  content  delta  AG-initiated  investigations polymerase  rate  that  possible  as w e l l  as  the  RNA p o l y m e r a s e  parameters.  As a  consequence, comparisons It the  for  plot  analysis  for  terms,  presented  in  non-A2  to  RNA w h i c h  migrates  gels.  reaction  of  could  a n  b show  above  K  that  the  In  of  the  this affect  concentration-  Q n  .  The  there  data  is  indeed  A2 t r a n s c r i p t  The c a l c u l a t e d  with  available  therefore  have d  all  is  interest.  which  for  problem  assumed t h a t  the  specifically 12a and  potential  is  promoter  parameters  Figure  polyacrylamide  added  it  inadequate  preparations.  another  that  the  may be  enzyme  transcription  kinetic  dependent  that  is  is  with  non-specific  analysis  different  enzyme w h i c h  values  plot  be n o t e d  interaction  assay  tau  between  should  tau  active  the  values  for  some  on  K  #  a n (  j  £ on  A  may t h e r e f o r e  be o v e r e s t i m a t e d  and  underestimated,  respectively. Delta  is  obviously  specific  transcription.  promoter  was  A2,  to  21). not et  result  inhibit a l . ,  complex  inhibitor  Complex f o r m a t i o n contrast the  supports  with  presence the  formation  The  chromosomal  A+T  content  and  which  could  1982).  by  a general  complex of  strong  promoter-  the  G2  formation  delta  hypothesis at  at  of  (see  that  Figure  delta  promoters  at  would  (Achberger  1 982 ).  1982)  the  in  be u n a f f e c t e d This  high  shown,  not  also  of  of  been p r o p o s e d the  51%  a  relatively  regions  to  play  a role  and  and  non-specifically  B_. s u b t i l i s 112  has  (Geiduschek  single-stranded  RNA p o l y m e r a s e  has  specificity  _B. s u b t i l i s  approximately  contains  bind  Delta  DNA o f  in  RNA p o l y m e r a s e  Ito,  nicks  (Doi, enhancing by  preventing stable  transcription,  complexes, at  or  of  interactions in  this  with  some way w i t h  expected  that  transcribe  the  in  promoter  more be  delta  this  that  probably  delta  template  decreased.  specific  interactions  not  was a l s o  that  This  interfere  therefore delta  while  be  would  non-specific  transcription  work.  Although  the  from  the  some from  was  most  of  A2  non-specific the  plasmid  when c o r e / s i g m a w a s overall  Thus d e l t a  level  from  the  appears  of  A2 to  transcription, promoter  inhibit  a n d A2 p r o m o t e r - s p e c i f i c  observed  c o m p l e x e s any  core/sigma.  of  result  the  important enzyme t o  does n o t ,  that more  This  complexes formed  kinetically  ability  sequences  non-  interactions  well.  initiation  specific  The  1982).  would  originated  and n o n - s p e c i f i c ,  did  It  as d i s c u s s e d a b o v e ,  both  than  A-T r i c h  on n o n - s p e c i f i c  with  It  Doi,  non-specific  efficiently,  assay used i n  was,  Bacillus  associated with  reconstituted  equally  that  the  of  reduced.  was o b s e r v e d  specific  on  1982;  location. is  formation  was s y n t h e s i z e d o r i g i n a t e d  there  It  of  the  transcription vector.  promoter  would  RNA w h i c h  DNA o r  enzyme w h i c h  The e f f e c t observed  nicked  is  the  sites  et . a l . ,  hypothesis  promoters  transcription  least  non-specific  c h r o m o s o m a l DNA ( A c h b e r g e r implication  at  core/sigma+delta rapidly  result with at  would  the  least  locate  however,  113  at  the  suggest  plasmid with  out  by  form  promoter that  to  promoter  the  not  non-  core/sigma  respect  available rule  G2  did  are  the sites.  possibility  that  alter  the  template  kinetics were  that and  and D o i  sigma  had  a greater  also  observed  that  concomitant  release  that  the  and  delta  regarding promoter with  the  are  effect  complex  sigma  Based  on  the  core  to  the  initiation Some o f  delta with site  model.  effectively  at  A2.  G2 p r o m o t e r ,  a mechanism would  be  that  core/delta promoter  delta  complexes  complexes.  therefore  determine  core/promoter  synthesis.  This  The d a t a  mechanism to is at  not  A2 b u t  is  rate  complexes particular  at to  to  allow  of  it  or  near  after  formation If  at  delta  does  the  delta  here  the  A2  competes binds  core/sigma, the  for  rate  suggest  of  delta  from from  release  sigma could  be  that  promoters.  effect  initiation  of  complex  released  mechanism would  114  to  efficiently  which  data  presented  certain  rapidly  The e f f i c i e n c y the  data  would  explain  released  with  RNA s y n t h e s i s  obtained  only  complex.  DNA  these  inhibit  however,  pertain  sigma  and c o r e / d e l t a  than  formation  to  enzyme  of  the  on c o r e  did  complex,  on c o m p l e x that  demonstrate  a core/sigma  of  delta.  interactions  than  bind  c o r e / s i g m a would  the  to  could  1978).  less  able core  from  to  at  another  the  core/delta  allow  of  An a l t e r n a t i v e  the  for  delta  formation  could  were  s i g m a bound  a binding  A2 p r o m o t e r  and  directed  consistent  for  adding  such  that  and D o i ,  the  sigma  then  delta  was r e l e a s e d , t o  (Williamson  if  investigated  affinity  displace  was p r o p o s e d  to  and d e l t a  actually  promoter  association  (1978)  could  They the  core,  delta  promoter  used.  Williamson between  of  of  G2  would  bind  to  RNA  consistent  synthesis.  This  particular  mechanism would  with  the  observed e f f e c t  both  the  A2 a n d G2 p r o m o t e r s .  to  reconcile  different the  with  sigma f a c t o r s  composition  uncertain  and w i l l  structure  Model  of  for  Based  on  about  delta,  the  a modulating  postulated (1982).  the  which  For  polymerase  function  to  aid  between  the  core/sigma  and  the  1981).  the  A2 a n d G2  core/sigma/delta  promoters.  to  include  in  as has  been  ( 1978 ) and A c h b e r g e r  it  delta is  is  of  information  RNA p o l y m e r a s e  of  simplicity  The  promoters.  promoters,  effect  on  therefore  and p r e v i o u s  and D o i  is  an i n v e s t i g a t i o n  the  A2 a n d G2 p r o m o t e r s  sake of  Pero,  c a n be c o n s i d e r e d  specific  the  it  selectivity  enzyme i s  by  at  above  delta  for  at  formation  of  the  and  at  difficult  that  promoter  be r e s o l v e d  presented  formation  observations  (Losick  consistent  nevertheless  only  by W i l l i a m s o n  at  is  confer  role  A model  formation  It  promoter-bound  complex  the  on c o m p l e x  of  the  data  discriminating  22.  model  delta  B a c i l l u s RNA p o l y m e r a s e  subunit  the  the  of  be  on  al.  complex  presented  assumed  interact  e_t  in  Figure  that  with  the  A2 a n d G2  promoters. In  the  initiation the  model  associates  case  of  the  G2 p r o m o t e r  the  c o m p l e x e s c a n be i n t e r p r e t e d presented with  conformational intermediates. open complex  the  by M c C l u r e promoter  (1985).  one  The c l o s e d c o m p l e x does not  to  readily .1-15  is  of  be a n a l o g o u s  to  RNA p o l y m e r a s e  and u n d e r g o e s  changes i n v o l v i n g  that  formation  or  a series  more  then  closed  of complex  converted  dissociate.  Delta  to is  an  G2  1)  E/cr/i  +  p ^ ^ E / c r / ^  - P  c  _  ^E/cr / J - P  >E/CT-P  Q  0  Figure 22. M o d e l f o r t h e e f f e c t o f d e l t a on i n i t i a t i o n a t t h e A2 a n d G2 p r o m o t e r s . The p r o m o t e r s and c o r e enzyme a r e d e s i g n a t e d P and E , r e s p e c t i v e l y . P P and P represent the c l o s e d , open and i n i t i a t e d c o m p l e x e s . The s t e p s i n t h e r e a c t i o n s w h i c h a r e h y p o t h e s i z e d t o be e s s e n t i a l l y i r r e v e r s i b l e are i n d i c a t e d ( >) a n d t h e potential p o i n t s at which d e l t a c o u l d act to slow the r e a c t i o n at the A2 p r o m o t e r a r e i n d i c a t e d b y a n a s t e r i s k . C >  116  q  I  released of  from  events  the  and  enzyme It  the  is  has  complexes  in  to  that  is  that  the with  be  for  at  delta  is  the to  for  the  open  open c o m p l e x e s  has  that  of  that  an SP82 p r o m o t e r  in  bound  account  postulated  (1982)  pathway  the  with DNA.  enzyme  promoter.  therefore  those  the  the  are  An  been  observations.  release  transition  of  delta,  steps,  is  initiation.  proceed  is to  by  not the  associated with "strong"  pathway  2)  possible  to  define  G2 p r o m o t e r  or  how t h e y  E_. c o l i  e_t j a l .  some p o i n t  one  sequence  nucleotides  enzyme c o n t a c t s  to  efficiently  unlike  the  pathway  Enzyme w h i c h  not  at  associated with  necessary  is  which  the  further  would  at  this  appropriate  core/sigma  intermediate  It  is  Achberger  from  during  transcription.  complex  in  could  the  open  included  bind  by  associated with  additional  of  core/sigma/delta  sites  shows  some p o i n t  initiate  different  the  result  to  by  at  presence  been shown  formed  This  which  the  able  significantly respect  complex  it  complex  does  appear  formation  at  G2 p r o m o t e r  shown  the  in  Figure  precise  might that the  delta  and 22.  to  basic  lacUV5  Although events  the  it at  situation  process  promoter,  also  initiation  sequence of  compare the  would  is  not  for  example. The also  proposed  shown  enzyme complex  in  Figure  interacts which  The c l o s e d  and  sequence of 22.  with  the  undergoes open  In  events the  at  the  A2 p r o m o t e r  case  of  core/sigma  A2 p r o m o t e r  to  form  a transition  complexes  are  11 7  to  a  an open  kinetically  is  the  closed complex.  unstable  but  the  final  open  initiation  complex  complex  in  the  Core/sigma/delta the  rate  at  which  transcription core/sigma  is  is  is  with  the  that  promoter  There alter  the  unstable  able  initiation  enzyme w h i c h  that  presence of  two  rate  of  are  the  in  (1980,  1982),  postulated  The  would  thus  transition  that  that  therefore  kinetic  not  be  from  than  A2 p r o m o t e r  the  easily  to  In  the  at  which  postulated  forms  to  a  complexes to  open  d  case  k ,  to  step  for  2  could  the  transcription  an u n s t a b l e  core/cr / J  core/  first  delta  and  open  in  rate  Gralla  be a c l o s e d c o m p l e x .  to  n  which  Stefano  rate-limiting t t  rate  converted  the  p r o p o s e d by  K^  is  delta  but  initiate  the  It  productive  complex  parameters refer  the  transcription.  the  to  would  slower  reaction.  analogous  from  to  p o s s i b l e m e c h a n i s m s by  assay,  transition  bind  associated with  intermediate  is  (heparin-resistant)  ATP a n d G T P .  complexes.  can i n i t i a t e  are  to  considerably  forms  a stable  c o r e / s i g m a / d e l t a complexes  that  complexes  can form  The  complex  the  reaction.  core/sigma/delta  - P _ complex core/cr/d  c  and  the  -P„, o  respectively. The  other  associated this the  case  possibility  with the  the  in  parameters  and  c o n v e r s i o n of  the step  intermediate  the  kinetic the  that  rate-limiting  unstable  intermediate  is  r e l e a s e of in  might  the  £  would  core/cr/c5-P  11 8  refer to  to  is  reaction.  be d i f f e r e n t  core/sigma i n i t i a t i o n and k  delta  pathway.  In from The  core/cr/J - P  core/C3~-P  r t  Q  Comparison There  were  two  of  the  sequences of  differences  polymerase  interacted  nucleotide  requirements  formation  in  with  the  for  and 2)  the  effect  The s e q u e n c e s o f  the  two  differences for  the  two  with  in  and  delta  promoters of  way  the  the  1983)  are  differs  base a l t e r a t i o n s  at  position  (E.  coli)  promoter  the  G residue  of  3 in  -35  1979;  from is  the  -35  of  and M c C l u r e ,  sequences  of  B a c i l l u s promoters,  region.  The  -35 JL*  could  a G residue  therefore  G2 p r o m o t e r  these  region. coli  had  The b a s e a l t e r a t i o n  promoter  One o f  all  base i s  and B a c i l l u s  and M c C l u r e ,  has  1983)  in  the  it  23. three  conserved G  Of  the  known  promoters  in  are  Furthermore, compiled  position -35  sites.  by  of  "down" the  Moran  3 in  region  which  the the  e_t  -35 A2  promoter  only  changes from consensus.  two  at  very  promoters  but  at  1982;  diminish  changes occurs This  1983).  of  consensus  has been a l t e r e d  (Hawley  (1982),  highly  the  if  account  sequences  Figure  region.  see  could  Moran et _ a l . ,  the  all  region  The  consensus at  at  to  RNA p o l y m e r a s e  subtilis  mutations  al. ,  the  the  formation.  compared  which  1)  complex  promoters  shown i n  mutations,  the  nine  G2 p r o m o t e r s :  were  E_. c o l i / B .  promoters.  t h e RNA  on c o m p l e x  the  in  ( R o s e n b e r g and C o u r t ,  and M c C l u r e ,  residue  A2 a n d  of  the  that  A2 a n d G2 p r o m o t e r s .  T h e A2 p r o m o t e r One o f  way  A2 a n d G2  heparin-resistant  structures  the  promoters  sequence Hawley  the  differences  interacts the  in  the  the  is  the  119  second T r e s i d u e  highly  conserved in  (Moran et  not  activity.  clear  al. ,  1982;  in  the  both  the  Hawley  how a b a s e c h a n g e  at  A2  T T T A C A  17 b . p .  G2  T A G A C A  18 b . p .  Consensus  [T {^G  (kjc^k  -16  cfi  T A T A C T  + 1 b.p.-iT^A  -35 Position  A A V  T(T)A{T -10  1 2 3 4 5 6  1 2 3 4 5 6  Figure 23. P r o m o t e r s e q u e n c e s o f t h e A2 a n d G2 p r o m o t e r s ( t a k e n f r o m D o b i n s o n and S p i e g e l m a n , 1985 and G a r v e y e t a l . , 1985). The s e q u e n c e s a r e c o m p a r e d w i t h t h e c o n s e n s u s s e q u e n c e ( R o s e n b e r g and C o u r t , 1 9 7 9 ; Moran e t a l . , 1 9 8 2 ; H a w l e y a n d M c C l u r e , 19 8 3 ) f o r t h e p r o m o t e r s r e c o g n i z e d b y t h e B a c i l l u s s u b t i l i s s i g m a - 4 3 a n d E . c o l i RNA p o l y m e r a s e . The b a s e s w h i c h a r e u n d e r l i n e d i n t h e c o n s e n s u s s e q u e n c e a r e t h o s e w h i c h a p p e a r t o be h i g h l y c o n s e r v e d i n B_. s u b t i l i s ( M o r a n e_t a_^. , 1 9 8 2 ) , a n d t h e b a s e s i n t h e A2 a n d G2 promoters which are changed from consensus are boxed.  120  this  site  would  alter  promoter,  which  has an A r e s i d u e  fairly  strong  promoters (Hawley  except in  promoter  may e i t h e r  for  E_. c o l i  but  the  two  This  3. of  the  site nine  T h e A2 p r o m o t e r  has an A r e s i d u e  pair  could  The length  of  regions. the  two  relative  also differ  of  alter  profoundly.  It  possible that  efficiently  longer  spacer region,  on t h e  b a s i s of  be  the  of  a E.  coli  activity  but two  identical  weakly  that  conserved  it  the  This  factor two  -10  to  and  is  not  clear  function  the  DNA w h i c h  important  for  promoter  surrounds function.  121  the -35  how  and such  so  B a c i l l u s RNA p o l y m e r a s e which  have  such a c o n c l u s i o n cannot  of  in  17 b a s e p a i r s l o n g  promoters  promoters.  base  promoters.  respect  the  analyzed  position.  site.  the  with  promoter  with  are  at  that  A2 i s  would  structure  at  18 b a s e p a i r s b u t ,  only  in  promoter  be a n o t h e r  a difference  more  site  is  B a c i l l u s promoters  strengths  The s p a c e r r e g i o n  is  this  only  spacer sequence between  G2 s p a c e r i s  interacts  is  had a T r e s i d u e  therefore  promoters  the  at  T h e G2  position,  promoters  (1982)  the  this  diminish  by M o r a n e t _ a l .  determining  at  and m u t a t i o n s  of  eight  alteration  strength.  1983).  regions  position  promoter  enhance or  and M c C l u r e ,  The - 1 0  the  Furthermore, the  promoter  the  be made the may  also  REFERENCES  A c h b e r g e r . 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