A growing body of evidence underscores the central importance of transcription regulation in many complex eukaryotic processes including human disease. It has been proposed that the general transcription factor TFIIB plays a crucial role in the regulation of transcription by RNA polymerase II. We have cloned the gene encoding Saccharomyces cerevisiae TFIIB and have isolated a temperature-sensitive TFIIB mutant. In the experiments proposed here, we will utilize genetic, molecular, and biochemical approaches to continue to investigate the function of TFIIB in both basal and activated transcription. Specifically, we will 1) Identify and characterize gene products that interact with TFIIB or affect its function. We have isolated both dominant and recessive suppressor mutations in at least 2 distinct genes that suppress the growth defect of our temperature-sensitive TFIIB strain. The suppressor genes will be cloned using standard genetic and molecular techniques. Their encoded proteins will be analyzed by affinity chromatography and by gel mobility-shift assays in order to characterize their interactions with TFIIB and with other transcription factors. 2) Determine the requirements for TFIIB in basal and activated transcription. We will utilize purified yeast general transcription factors and activator proteins, yeast nuclear extracts depleted of TFIIB, and in vitro transcription and gel mobility-shift assays to study the concentration dependence for TFIIB as a function of promoter activity. Specifically, we will determine the concentrations of TFIIB required to achieve defined levels of transcription or transcription complex formation under conditions of variable basal promoter strength, activator strength, and under conditions of synergistic activation. 3) Identify and characterize mutants of TFIIB with altered properties in basal or activated transcription. We will analyze the various functions of TFIIB by employing several genetic screens and selections to identify TFIIB mutants that: a) suppress mutations in the acidic activation region of the Herpes virus transcriptional activator VP16 in an allele-specific manner; b) support basal transcription but show a decreased response to acidic activators; c) cause increased levels of basal transcription; and d) are transcription-deficient. The mutant forms of TFIIB will be analyzed biochemically using in vitro transcription and by characterizing their interactions with other general transcription factors and activator proteins.
| 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 |
