(Verbatim from the application): A large body of evidence underscores the importance of transcription regulation in many complex eukaryotic processes. The long-term goal of this research is to elucidate the molecular mechanisms of transcription elongation by RNA polymerase II (RNAPII). The proposed work utilizes molecular, biochemical and immunological approaches to extend several related projects regarding the composition and regulation of RNAPII elongation complexes.
In Aim I we will determine the composition of yeast and human RNAPII elongation complexes. We have developed an approach for isolating native RNAPII elongation complexes from yeast or human cell nuclear extracts. We will scale-up and modify our purification scheme to obtain sufficient quantities of these complexes for molecular analyses. Immunoblotting and enzymatic assays will be used to determine the presence of specific components of the RNAPII transcription machinery or mRNA processing factors in these complexes.
In Aim 2 we will determine whether transcriptional regulatory proteins alter the composition of RNAPII elongation complexes. Promoter-bound transcriptional activators can enhance the processivity of RNAPII elongation complexes in vivo and in vitro. Modulation of elongation activity, imparted by the activator at the promoter, suggests that activators may alter the composition of RNAPII elongation complexes. Upon establishing conditions that allow for the identification of components of yeast and human RNAPII elongation complexes (Aim 1), we will test the hypothesis that promoter-bound transcriptional activators can alter the subunit composition of elongation complexes.
In Aim 3 we will expand the analyses of Aims 1 and 2 and determine whether variations in core promoter sequence confer alterations in the activity and/or composition of RNAPII elongation complexes.
In Aim 4 we will identify/purify the factor(s) that restores elongation competence to blocked human RNAPII elongation complexes. Human RNAPII elongation complexes blocked by the incorporation of 3'-O-methyl-GTP into the nascent transcript require a nuclear factor(s) to resume elongation. To identify the factor or factors that confer this activity, HeLa cell nuclear extracts will be fractionated and the fractions will be assayed for their ability to restore elongation to blocked complexes. We will determine whether multiple factors confer this activity and whether any of these factors correspond to known elongation-associated factors. If it is determined that resumption of elongation is conferred by an apparently novel factor, it will be purified to enable cloning and an investigation into its mechanism of action. The experiments described in this proposal will provide novel and important information regarding the molecular composition of RNAPII elongation complexes and the mechanism of activator-dependent regulation of transcriptional elongation.
| Yang, Chen; Ponticelli, Alfred S (2012) Evidence that RNA polymerase II and not TFIIB is responsible for the difference in transcription initiation patterns between Saccharomyces cerevisiae and Schizosaccharomyces pombe. Nucleic Acids Res 40:6495-507 |
| Yang, Chen; Khaperskyy, Denys A; Hou, Min et al. (2010) Improved methods for expression and purification of Saccharomyces cerevisiae TFIIF and TFIIH; identification of a functional Escherichia coli promoter and internal translation initiation within the N-terminal coding region of the TFIIF TFG1 subunit. Protein Expr Purif 70:172-8 |
