Effect of activators on promoter clearance by RNA polymerase II

Abstract

Eukaryotic transcription is a complex and highly regulated process. The adenovirus major late (AdML) promoter, a model promoter for transcription by RNA polymerase II (pol II), contains a binding site for the transcriptional activator upstream stimulatory factor (USF). Previous experiments showed that deletions in the USF site in AdML promoter decrease the stability of ATP-activated transcription complexes. As a result we wanted to look at the effect USF had on the rate of promoter clearance by pol II in vitro. To develop an assay for promoter clearance, special DNA templates were constructed such that a G at position +11 is the only G incorporated into the growing RNA in the first 36 nucleotides of RNA synthesis. Our assay for promoter clearance is designed to measure whether pol II has passed position +11 at a given time, t, and depends on the ability of the chain terminating nucleotide 3'-O-methyl GTh (added at t) to quantitatively stop transcription if it is incorporated into the RNA at +11. Controls for the promoter clearance assay are in progress. So far controls have shown the presence of promoter specific transcripts from our templates that are distinguishable from the background. In addition, 3'-O-methyl GTP at high concentrations has been shown to terminate transcription almost completely. However, it is still not quite clear if it is efficient enough for the promoter clearance assay.