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Studies of initiation and elongation in T7 RNA polymerase

by Enuameh, Metewo Selase

Abstract (Summary)
During transcription initiation, competition between the initiation and elongation processes is thought to lead to the production of abortive transcripts (2-8 nucleotides of RNA) by the RNA polymerases. Our results show that A-16C and T-14G promoter mutations would lead to earlier promoter escape events relative to A-15C/G and wild type promoters as previously reported. This indicates that even for the A-15C/G promoter, interactions between the polymerase and bases at positions -16 and -14 are still important. The only physiological regulator of transcription known in the T7 phage is the phage protein T7 lysozyme. Several models have been proposed about its mechanism of action. By running transcription assays on crosslinked promoter-polymerase complexes in the presence and absence of lysozyme, in addition to partial tryptic digests on promoter complexes stalled at different translocation positions, we have shown that lysozyme largely inhibits the initiation complex to elongation complex transition by causing the increased dissociation of the already unstable initiation complex. In transcription, double stranded DNA is locally melted (forming a "bubble"), containing RNA annealed to the template strand. Thus, there is an energy cost to maintain the open bubble, but an energy gain from formation of the hybrid. We have independently varied the DNA-DNA and RNA-DNA duplex stabilities by employing 5-brominated pyrimidines, which stabilize duplexes by 4.3�° per substitution. Our results show that increasing hybrid stability stabilizes the elongation complex more than increasing DNA-DNA stability (favoring collapse of the bubble) destabilizes the complex. The recent solving of the crystal structures of T7 RNA polymerase initiation complexes involved in de novo RNA synthesis, has revealed a novel NTP binding site (the D-site), distinct from previously known P- and N-sites. The apparent inflexibility of the D- and P-sites in the initiation complex has inspired the hypothesis that initiation of transcription with GTP is by a lock-and-key mechanism. By using a 5-propynyl-dC base in place of dC at the +1 and +2 sites, which would still allow for initiation with GTP we observe a decrease in initiation efficiency. This indicates that initiation of transcription in the T7 RNA polymerase system is by a lock-and-key mechanism.
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School:University of Massachusetts Amherst

School Location:USA - Massachusetts

Source Type:Master's Thesis

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Date of Publication:01/01/2008

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