isatin-beta-thiosemicarbazone and sarkosyl

The vaccinia virus RNA polymerase is a multi-subunit enzyme that contains eight subunits in the postreplicative form. A prior study of a virus called IBT(r90), which contains a mutation in the A24 gene encoding the RPO132 subunit of the RNA polymerase, demonstrated that the mutation results in resistance to the anti-poxvirus drug isatin-beta-thiosemicarbazone (IBT). In this study, we utilized an in vitro transcription elongation assay to determine the effect of this mutation on transcription elongation. Both wild type and IBT(r90) polymerase complexes were studied with regard to their ability to pause during elongation, their stability in a paused state, their ability to release transcripts, and their elongation rate. We have determined that the IBT(r90) complex is specifically defective in elongation compared with the WT complex, pausing longer and more frequently than the WT complex. We have built a homology model of the RPO132 subunit with the yeast pol II rpb2 subunit to propose a structural mechanism for this elongation defect.

Topics: Biotinylation; Cell Line, Tumor; Cytoplasm; DNA; DNA-Directed RNA Polymerases; Dose-Response Relationship, Drug; Drug Resistance, Viral; HeLa Cells; Humans; Isatin; Models, Genetic; Models, Molecular; Mutation; Plasmids; Polymerase Chain Reaction; RNA, Viral; Salts; Sarcosine; Software; Time Factors; Transcription, Genetic; Uridine Triphosphate; Vaccinia virus