UBC Theses and Dissertations
Studies on the regulation of RNA polymerase in Escherichia coli Little, Robert
The regulation of RNA polymerase subunit synthesis and its relationship to the expression of ribosome component genes have been investigated in strains of Escherichia coli having a mutation in one of the genes specifying either the β or β' subunit of the core enzyme.' Particular attention has been focused on the L10 transcriptional unit (organization: P[sub= L10] – rp1J(LlO) - rplL(L7/L12) - attenuator - rpoB(β) -rpoC(β ') ). The mutant strain XH56 produces a defective β ' subunit which renders the RNA polymerase inactive in transcription initiation at 42°C; at somewhat lower temperatures, the RNA polymerase activity is only partially restricted. A temperature shift of this strain from 30°C to 39°C resulted in a rapid 5-fold increase in the transcription of the rpo β and CI genes and in the synthesis rates of the β and β' subunits, indicating that β and β' synthesis is regulated primarily at the transcriptional level. Transcription of the α subunit gene, located in the spc-str region of the chromosome, was also enhanced. Transcription of the lacZ gene (coding for β -galactosidase) was decreased to undetectable levels, indicating that the dramatic increase of rpo β and C. transcription occurred at the expense of transcription of other operons. The mutant strains Ts4 and A2R7 produce defective β' and β subunits respectively which are unable to assemble into core RNA polymerase at the nonpermissive temperature. In these strains RNA polymerase assembled prior to a temperature shift from 30°C to 42°C retains its activity but little or no enzyme is assembled after the shift. Prolonged incubation of these strains after such a shift produced a gradual 1.5- to 2-fold increase in the transcription of the rpoβ and C genes and in the synthesis rates of the β and β' subunits. During the restrictions, transcription of ribosome component genes was essentially unchanged. RNA polymerase assembly was also inhibited in strains carrying both a temperature-sensitive amber suppressor mutation and an amber mutation in the rpoβ gene. Under permissive conditions these amber mutations are suppressed by insertion of serine into the β protein at the UAG codon. After a temperature shift to 42°C, core RNA polymerase synthesis is restricted due to the failure to produce 3 in the non-polar amber strain MX515 and both β and β' in the polar amber strain MX515. Core enzyme synthesized prior to the shift retains its activity. Inhibition of core enzyme synthesis in this manner resulted in a gradual stimulation of rpoβ and C transcription; in the polar strain this was accompanied by a concomitant increase in the synthesis rate of the β' subunit protein. The increase of rpoβ and C transcription involved both increased initiation at P[sub L10] and relaxed termination in the rp1L-rpoβ intergenic space. It was also observed that transcription of the a subunit gene was specifically stimulated during the restriction, suggesting that the regulatory mechanisms are specific for genetic units containing core RNA polymerase genes. These results therefore indicate that the mechanisms which govern the transcriptional frequency of operons containing RNA polymerase genes are coupled to the demand for active RNA polymerase; a sudden restriction of enzyme activity produces a rapid and dramatic increase of rpoβ and C transcription whereas a slow restriction results in only a gradual and less extensive induction. The regulatory mechanisms operating within the L10 transcription unit were accentuated by introducing the composite colEl plasmid pJC701 into the RNA polymerase activity mutant strain XH56.. All of the genes in the L10 transcription unit except the distally located rpoC are present on this plasmid and therefore were amplified in the transformed bacteria. The partial temperature inactivation of RNA polymerase activity in this strain allowed us to modulate the transcription of the proximal rp1J-rp1L genes and the distal rpoβ gene over a 10-fold and 30-fold range respectively. The observed imbalance in transcription between the proximal and distal portions of the L10 transcription unit strongly. suggest that the restriction has :two distinct effects: (i) it stimulates initiation at the major L10 promotor and (ii) it reduces termination at the attenuator located within or near the rp1L-rpoβ intergenic space. The synthesis rates of L7/L12 and β subunit proteins were also measured and compared to their respective mRNA levels under these conditions. The synthesis rate of L7/L12 protein and β protein varied by less than 2-fold and by 15-fold respectively. These measurements clearly indicate that translation of excess L7/L12 ribosomal protein mRNA is severely restricted and contributes to maintaining the balanced synthesis of ribosome components. The translational efficiency of 3 mRNA was also reduced by about 50%. Under the above conditions, β protein is produced in large excess relative to β' subunit protein.
Item Citations and Data