Insights into the role of the non-template DNA within the transcription bubble of vRNAP and residues of NPH I that function in Vaccinia early gene transcription termination
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Poxviruses are large double stranded DNA viruses that conduct their entire infective cycle within the cytoplasm of their host cells. Due to their cellular location during infection, poxviruses must encode their own gene expression and replication machinery. Poxviruses encode their own multi-subunit DNA dependent RNA polymerase. Vaccinia virus, the prototypical member of the poxvirus family, provides a model to study transcription termination in eukaryotes. Vaccinia virus exhibits temporal control of its gene expression. The three classes of genes are named early, intermediate, and late genes. These distinct classes of genes are differentiated not only by the timing of expression after infection but also by associated regulatory factors and different sequences. Early gene transcription begins immediately upon infection and occurs within the viral core. Transcription termination of Vaccinia early genes requires the virion form of RNA polymerase (vRNAP), Nucleoside Triphosphate Phosphohydrolase I (NPH I), the Vaccinia termination factor (VTF), and the U5NU termination signal in the nascent RNA. The work of this dissertation focused on investigating the role of the non-template strand and NPH I in early gene transcription termination. Vaccinia virus NPH I can hydrolyze ATP or deoxy ATP (dATP) to release nascent RNA during transcription termination and it also acts as a positive elongation factor (L. M. Christen, Sanders, Wiler, & Niles, 1998; Deng, Hagler, & Shuman, 1996; S. Piacente, Christen, Dickerman, Mohamed, & Niles, 2008). NPH I accomplishes its function in transcription termination through a mechanism known as forward translocation (Tate & Gollnick, 2011). NPH I requires a single stranded (ss) DNA cofactor for ATPase activity (L. A. Christen, Sanders, & Niles, 1999; Martins, Gross, & Shuman, 1999). Also essential for transcription termination is the interaction between NPH I and the early gene specific subunit of vRNAP known as Rap94 (Ahn, Gershon, & Moss, 1994; S. C. Piacente, Christen, Mohamed, & Niles, 2003). These prior observations form the basis of the investigations presented in this dissertation. The first focus of the studies presented in this dissertation was to elucidate the role of the non-template strand in Vaccinia early gene transcription termination. The authors of a prior study had hypothesized that the non-template strand of DNA within the transcription bubble was the source of the required ssDNA cofactor of NPH I (L. A. Christen et al., 1999). Observations presented in this thesis demonstrate that the non-template strand of DNA within the transcription bubble is the source of the ssDNA cofactor required for NPH I to function in transcription termination. This thesis also demonstrates that transcription bubble collapse, or the re-annealing of the unpaired bases within the transcription bubble during transcript release, is important for Vaccinia early gene transcription termination as previously described for bacteria (J.-S. Park & Roberts, 2006; Ryder & Roberts, 2003). The second emphasis of the work presented in this dissertation was to identify residues of NPH I involved in the mechanism of transcription termination beyond those previously described to be involved in ATPase activity and for Rap94 interaction. To this end we employed DNA crosslinking and mass spectrometry analysis to identify residues involved in interaction with ssDNA. We also utilized in silico protein modeling identify residues of NPH I involved in forward translocation. We utilized ATPase activity assays, Rap94 pull down assays, and ssDNA binding assays to demonstrate NPH I mutants were specifically defective in transcript release activity.