Studies of human RNA polymerase II holoenzyme

grantor: University of TorontoThe holoenzyme form of RNA polymerase II, which contains RNA polymerase II, most or all of the general transcription factors, and many other factors involved in transcription, is able to bind to promoters and to initiate transcription. Using in vitro transcription assays, I have shown that the addition of TBP and GAL4-VP16 to reactions increases transcription by holoenzyme. In an effort to examine the components present in complexes of the holoenzyme form of RNA polymerase II engaged in elongation, I have investigated a system for isolation of such complexes by using the interaction between RNA containing the bacteriophage lambda nut site and the lambda N protein fused to GST. I found that the binding of GST-N to nut site RNA in transcripts made by holoenzyme in in vitro transcription reactions was too inefficient to allow purification of elongation complexes using this system. I discuss alternative methods for purification of elongating holoenzyme complexes.M.Sc