| Abstrakt: |
Prior phenotypic analysis of a vaccinia virus gene A18Rmutant, Cts23, showed the synthesis of longer than wild type (Wt) length viral transcripts during the intermediate stage of infection, indicating that the A18R protein may act as a negative transcription elongation factor. The purpose of the work described here was to determine a biochemical activity for the A18R protein. Pulse-labeled transcription complexes established from intermediate virus promoters on bead-bound DNA templates were assayed for transcript release during an elongation step that contained nucleotides and various proteins. Pulse-labeled transcription complexes elongated in the presence of only nucleotides were unable to release nascent RNA. The addition of Wt extract during the elongation phase resulted in release of the nascent transcript, indicating that additional factors present in the Wt extract are capable of inducing transcript release. Extract from Cts23 or mock-infected cells was unable to induce release. The lack of release upon addition of Cts23 extract suggests that A18R is involved in release of nascent RNA. By itself, purified polyhistidine-tagged A18R protein (His-A18R) was unable to induce release; however, release did occur in the presence of purified His-A18R protein plus extract from either Cts23 or mock-infected cells. These data taken together indicate that A18R is necessary but not sufficient for release of nascent transcripts. We have also demonstrated that the combination of A18R protein and mock extract induces transcript release in an ATP-dependent manner, consistent with the fact that the A18R protein is an ATP-dependent helicase. Further analysis revealed that the release activity is not restricted to a vaccinia intermediate promoter but is observed using pulse-labeled transcription complexes initiated from all three viral gene class promoters. Therefore, we conclude that A18R and an as yet unidentified cellular factor(s) are required for thein vitrorelease of nascent RNA from a vaccinia virus transcription elongation complex. |
