Determinants of vaccinia virus early gene transcription termination

Abstract

Vaccinia virus early gene transcription requires the vaccinia termination factor, VTF, nucleoside triphosphate phosphohydrolase I, NPH I, ATP, the virion RNA polymerase, and the motif, UUUUUNU, in the nascent RNA, found within 30 to 50 bases from the poly A addition site, in vivo. In this study, the relationships among the vaccinia early gene transcription termination efficiency, termination motif specificity, and the elongation rate were investigated. A low transcription elongation rate maximizes termination efficiency and minimizes specificity for the UUUUUNU motif. Positioning the termination motif over a 63 base area upstream from the RNA polymerase allowed efficient transcript release, demonstrating a remarkable plasticity in the transcription termination complex. Efficient transcript release was observed during ongoing transcription, independent of VTF or UUUUUNU, but requiring both NPH I and either ATP or dATP. This argues for a two step model: the specifying step, requiring both VTF and UUUUUNU, and the energy-dependent step employing NPH I and ATP. Evaluation of NPH I mutants for the ability to stimulate transcription elongation demonstrated that ATPase activity and a stable interaction between NPH I and the Rap94 subunit of the viral RNA polymerase are required. These observations demonstrate that NPH I is a component of the elongating RNA polymerase, which is catalytically active during transcription elongation.

Role of Forward Translocation in Nucleoside Triphosphate Phosphohydrolase I (NPH I)-mediated Transcription Termination of Vaccinia Virus Early Genes

“…2B). As seen previously (24,28), these results highlight the requirement for all termination factors (U 5 NU, VTF, (d)ATP, and NPH I) to achieve early gene transcription termination in a standard transcription reaction.…”

“…2B) (24,28). This observation also demonstrates that endogenous VTF and NPH I were efficiently removed from the TECs by the washing steps after Step 1 of the transcription assay.…”

“…Termination assays assess the production of released transcripts from actively elongating vRNAP, whereas transcript release assays evaluate transcript release from artificially stalled vRNAP. Prior studies have demonstrated the requirement of VTF and the termination signal for both termination and transcript release activity when NPH I is stimulated with ATP (16,24). In this study, we show that NPH I-mediated transcript release can occur independent of VTF and U 5 NU when NPH I is stimulated with dATP, as opposed to ATP.…”

“…The EcoRI sites flanking the promoter were first removed, and an EcoRI site was then created at the desired location on the template. Templates lacking the termination signal had the T9 termination sequence replaced with 5Ј-CGTGTGTTC-3Ј and were designed and provided by Dr. Edward Niles (24). Transcription templates were generated by PCR amplification of the appropriate plasmids using the M13 forward and reverse primers with the latter containing biotin at the 5Ј end (IDT).…”

“…ATPase activity is essential for NPH I-mediated transcript release of early genes (15,16). In addition to its function in early gene termination transcription, NPH I has been shown to be a positive transcription elongation factor (16,24). Evidence for this activity comes from the observation that NPH I allows vRNAP stalled at a run of A residues on the template strand to resume elongation (16).…”