Genetic and biochemical analysis of gp2 and its role in N4 RNAPII transcription initiation.

摘要

This thesis work describes two separate lines of study seeking to define the determinants of N4 RNA polymerase (RNAP) II specificity and transcriptional activation. Using genetic and biochemical approaches, I identified residues that contribute to the known functions of gp2, the single-stranded DNA binding protein essential to N4 middle transcription. I used a similar strategy to identify functional residues within the specificity loop of N4 RNAPII.;Site-directed mutagenesis of gp2, purification of loss-of-function mutants and subsequent in vitro characterization identified residues essential for its multiple activities, including ssDNA-binding and specific interaction with RNAPII. Mutants deficient in single-stranded DNA binding were generally found to retain affinity for RNAPII and the ability to activate in vitro transcription on single-stranded templates, clarifying that gp2's role in activation of transcription extends beyond simple recruitment of the polymerase to single-stranded DNA. Mutations that affected interaction with RNAPII were identified at both termini; only I3A-gp2 resulted in the concomitant loss of interaction with RNAPII and loss of activation of run-off transcription. The interaction between gp2 and RNAPII was further investigated through chemical crosslinking. I determined that gp2 contacts RNAPII through its N-terminal subunit, gp15, and that the loss-of-function gp2 mutant, I3A-gp2, failed to crosslink to gp15 to the same degree as wild-type gp2. Finally, I also identified a unique role for gp2 in enhancing first phosphodiester bond formation and demonstrated that the I3A-gp2 mutant was unable to accomplish this function.;The recent crystal structure of RNAPII identified an antiparallel beta-loop bearing structural homology to the specificity loop of single subunit RNAPs. To confirm that the structural homology extended to functional homology, I created a panel of specificity loop mutants by separately substituting each residue of the loop with alanine. Mutant proteins were purified to assay transcriptional activity in vitro. All mutants retained the ability to discriminate against a non-consensus initiating base, however several of the polymerases demonstrated significantly reduced activity on a wild-type promoter.;To contextualize these findings I will first provide an overview of transcription initiation by single-subunit polymerases and the present knowledge about their required cofactors, activating proteins and other determinants of specificity. I will also expound on the emerging diversity of transcription initiation strategies that exist within this family of polymerases and propose that T7 RNAP should be reclassified as an outlier in this family, rather than as its canonical representative.