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The organization, expression, function and evolution of some essential genes from the hyperthermophilic eubacterium thermotoga maritima

University of British Columbia

The hyperthermophilic eubacterium Thermotoga maritima grows optimally at 80°C near marine geothermal locales. Phylogenetic analyses based on various molecular sequences indicate that T. maritima and other hyperthermophilic prokaryotes have very deep phylogenetic placements; i.e., that they have diverged early from the ancestor of living organisms. Thus, studies on the biochemistry and molecular biology of hyperthermophilic organisms such as T. maritima may shed light on the early evolution of life, as well as enhance our understanding of life at high temperature. In this study, a 5,800-base-pair DNA fragment from the chromosome of T. maritima was cloned and sequenced. This fragment encodes five tRNAs, the ribosomal protein L33, an integral membrane protein, SecE, which is probably involved in protein translocation, the transcription factor NusG, four large subunit ribosomal proteins (L11, L1, L10 and L12), and the N-terminus of the RNA polymerase 0 subunit. The transcriptional patterns of this gene cluster were analyzed using S1 nuclease protection and primer extension techniques. The tRNAgenes and the protein-encoding genes are co transcribed, except the 13 gene, which is transcribed separately. The following regulatory sequence elements were identified in this cloned fragment: five promoters (Pi and P2 in front of the first and second methionine tRNAs, respectively, PLio in the L1-L10 intergenic space, PL12 at the end of the L10 gene, and PR in the L12-(3 intergenic region), a transcription attenuator upstream of the L10 gene, a transcription terminator located between theL12 and the (3 subunit gene of the RNA polymerase, and an autogenous translational regulation site (the Ll binding site) located upstream of the L11 gene. The transcription factor NusG encoded in this cluster exhibits 43% amino acid sequence identity when aligned to its E. coli counterpart; the alignment is interrupted by a 171-amino-acid-long insertion into the T. maritima protein. The T. maritima NusG was over expressed in E. coli, and the recombinant NusG protein was purified. The NusG protein binds to DNA cooperatively, but nonspecifically. Two types of NusG-DNA complexes have been observed. The first type forms instantly and can be stained with ethidium bromide ("loose" complex); the second type forms more slowly, and is probably converted from the loose structure(s). The second type is probably more compact, as it can not be stained with ethidium bromide ("tight" complex). This protein binds to both ds- and ssDNA, but preferentially to dsDNA in a mixture of both DNA molecules. About 40 and 60NusG monomers per kilobases (pairs) of ds- and ssDNA, respectively, are required to form cooperative NusG-DNA complexes. When a relatively large amount ofNusG was added to an in vitro transcription assay, it appears to selectively suppress aberrant transcription initiation and termination, and at the same time, the production of specific transcripts is, at most, only marginally reduced. Available sequences that correspond to the E. coli ribosomal proteins L11, L1,L10 and L12 from eubacteria, archaebacteria and eukaryotes have been aligned, and the alignments were subjected to quantitative phylogenetic analysis. Eubacteria andeukaryotes each form a well-defined, coherent and non-overlapping group. Archaebacteria also form a coherent phylogenetic group by themselves, but the relationships between the major groups of archaebacteria and out groups (eubacteriaand eukaryotes) can not unambiguously be established. On the other hand, T.maritima does not appear as the deepest branch within the eubacterial kingdom; however, this placement is less definitive.

Thesis/Dissertation

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2008-09-05

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http://hdl.handle.net/2429/1701

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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