Conformational mechanisms in T7 RNA polymerase transcription

The ∼99 kDa single-subunit bacteriophage T7 RNA polymerase (T7RNAP) is the most extensively studied member of a widespread family of single-subunit RNA polymerases. Because of its structural simplicity and a remarkable degree of mechanistic similarity to multisubunit RNAPs, T7 RNAP serves as an excellent system to study transcription, a complex reaction involving initiation, elongation and termination phases, each of which is comprised of multiple steps. These different steps are regulated by conformational changes either in RNAP, DNA and/or RNA. In my Ph.D. dissertation I have studied (1) The nature of the conformational changes associated with T7RNAP transcription initiation, (2) The functional architecture of T7 RNAP elongation complexes, and (3) The conformational mechanisms of pausing/termination at the concatemer junction (CJ) of T7 DNA.;(1) T7 RNA polymerase undergoes a dramatic structural rearrangement in transitioning from initiation to elongation. An important question is how and when do these conformational changes occur? Do such changes occur incrementally as the RNA is extended during initial transcription or all at once in a step coincident with promoter release? Two approaches were used to address these questions. (I) Use of disulfide bonds to detect when conformational changes occur, (II) Site-specifically tethered chemical nucleases to monitor conformational changes. Results reveal that conformational changes during initial transcription are relatively limited but major conformational changes in the RNAP occur coincident with promoter release. (2) Crystal structures of T7 RNAP initiation and elongation complexes have provided a great deal of information about the possible interactions made by RNAP with the promoter and transcription bubble. However, the absence of DNA downstream of the melted region in the initiation complex structure and upstream of the transcription bubble in the elongation complex structure, has led to an incomplete picture of the functional architecture of T7 RNA polymerase transcription complex. To address this, I have characterized several RNAP mutations and analyzed cleavage patterns on labeled duplex DNA by site-specifically tethered chemical nuclease. Results indicate that downstream DNA interacts with a positive patch defined by polymerase residues K711/K713/K714 and these interactions are present during both initiation and elongation where they help stabilizing a bend in the downstream DNA that is important for promoter opening. The upstream DNA in the elongation complex is found to be sharply bent at the upstream edge of transcription bubble and thereby allowing formation of upstream duplex: polymerase interactions. (3) T7 RNAP elongation complexes (EC) pauses and are destabilized 7-8 b.p downstream of the concatemer junction (CJ) sequence (TATCTGTT), the sequence within which concatemerized T7 genomes are cleaved into single genomes for packaging. Pausing of the EC at CJ involves structural changes in both the RNAP and transcription bubble. However, these changes have not been fully defined and the mechanism by which the CJ sequence causes the EC to change its structure, pause and become less stable, is poorly understood. I have used approaches similar to those I used to study the normal EC architecture to define the conformational mechanisms of pausing/termination at the CJ. Results indicate that at the CJ, the RNA may form an unusual secondary structure similar to the HIV1 Tar-RNA hairpin bulge. This, in turn, leads to disruption of part of the RNA:DNA hybrid, collapse of the upstream half of the transcription bubble, unbending of the upstream DNA, establishment of a new set of DNA: polymerase interactions that retard EC translocation and, finally, conformational changes in the RNAP and termination. These results are relevant not just for our understanding of the mechanisms of transcriptional pausing and termination, but also for understanding phage DNA packaging as the unusual paused EC architecture that forms at the CJ appears to be what recruits the T7 DNA processing machinery to this sequence.