Activation of RNA Polymerase II Mediated Transcription

grantor: University of TorontoI have developed a sensitive and highly selective in vitro crosslinking strategy to characterize the protein-protein interactions mediated by a sequence-specific activator of transcription with components of the RNA polymerase II transcriptional machinery. The basis of this approach involved the selective modification of the chimeric transactivator LexA-E2F-1 with the photoreactive crosslinking reagent maleimide-4-benzophenone at a single cysteine residue located within its activation domain. Using this approach, I have demonstrated that LexA-E2F-1 can interact in a direct and binding-site-dependent manner with the TATA-binding protein TBP. I provided evidence that this interaction is biologically relevant by showing that mutations within the E2F-1 activation domain which impair activation by LexA-E2F-1 also reduce crosslinking of LexA-E2F-1 to TBP. I have refined my original crosslinking methodology in order to identify additional protein targets of LexA-E2F-1 in an in vitro transcription system derived from a yeast cell extract. Using this approach, I have shown that the activation domain of LexA-E2F-1 interacts in a promoter-dependent manner with a novel component of the yeast RNA polymerase II transcriptional machinery, XTC1. The XTC1 gene product also interacts directly with the activation domains of the herpes virion protein VP16 and the yeast activator GAL4, suggesting it is a common target of activators. Yeast strains deleted for the XTC1 gene exhibit growth defects and altered responses of the RNA polymerase II transcriptional machinery to activators in vivo consistent with XTC1 being a physiologically relevant target of activators in yeast. Finally, I have performed affinity chromatography experiments aimed at identifying human proteins which interact with the evolutionarily conserved carboxy-terminal domain (CTD) of the largest polypeptide subunit of the RNA polymerase II. I have purified and identified two such CTD-binding proteins as the essential splicing factor PSF and the putative splicing factor p54$\rm\sp{nrb}.$ Since splicing of messenger RNA is intimately coupled to the process of transcriptional elongation in vivo, this observation suggests that the CTD may be directly involved in the processing of nascent RNA transcripts in addition to its role in regulating transcription by Pol II.Ph.D