Enormous advances have been made in the past decade toward understanding transcriptional control mechanisms in eukaryotic cells. Biochemical studies have identified different classes of transcription factors, defined as (i) """"""""general"""""""" factors, required for basal expression of protein-encoding genes; (ii) """"""""promoter-specific"""""""" factors that control the rate of initiation; and (iii) the """"""""coactivators"""""""" that mediate, at least in some cases, the interaction between the general and specific factors. In addition, the chromatin structure of the DNA template can affect the rate of initiation. One of the most remarkable outcomes of this research is the extent to which transcription factors and their known functions are conserved throughout the eukaryotic kingdom. This expands the repertoire of experimental approaches available to investigate transcriptional mechanisms. The yeast Saccharomyces cerevisiae is especially valuable for such studies because of its ability to be manipulated by extraordinarily powerful genetic methods. Accordingly, genetic approaches can be used to investigate the mechanisms of transcription initiation with the outcome being informative with respect to transcriptional control in higher organisms. The yeast gene (SUA7) encoding TFIIB has been cloned and sequenced. SUA7 is an essential gene that is required for normal transcription start site selection in vivo. In this application experiments are proposed to take advantage of the sua7 genetic system to define the functional role of TFIIB.
Four Specific Aims are proposed.
Aim #1 is to generate mutant forms of both TFIIB and the newly discovered Ssu71 protein, and to define their effects on transcription initiation.
Aim #2 is to uncover genes whose products functionally interact with TFIIB during initiation.
Aim #3 proposes to identify the products of the genes uncovered in Aim #2.
Aim #4 is to characterize the altered forms of TFIIB in vitro and to determine the relationship between TFIIB and the products of the genes defined in Aim #3. The cloned SUA7 gene and existing sua7 mutants, combined with the power of classical yeast genetics and modern biochemical methods, offer a unique opportunity to define the role of TFIIB, and the factors that interact with TFIIB, to control gene expression and cell proliferation. Although the flow of genetic information can be regulated at many different levels, transcription initiation is generally the focal point in this process. The discovery that several proto-oncogenes are transcription factors underscores the importance of transcriptional control with respect to disease processes and cancer in particular. The successful outcome of the proposed experiments should contribute significantly to our understanding of the factors and mechanisms that regulate gene expression in eukaryotic organisms.
| Lamas-Maceiras, Mónica; Singh, Badri Nath; Hampsey, Michael et al. (2016) Promoter-Terminator Gene Loops Affect Alternative 3'-End Processing in Yeast. J Biol Chem 291:8960-8 |
| Olayanju, Bola; Hampsey, James Jensen; Hampsey, Michael (2015) Genetic analysis of the Warburg effect in yeast. Adv Biol Regul 57:185-92 |
| Singh, Badri Nath; Hampsey, Michael (2014) Detection of short-range chromatin interactions by chromosome conformation capture (3C) in yeast. Methods Mol Biol 1205:209-18 |
| Rosado-Lugo, Jesús D; Hampsey, Michael (2014) The Ssu72 phosphatase mediates the RNA polymerase II initiation-elongation transition. J Biol Chem 289:33916-26 |
| Yadon, Adam N; Singh, Badri Nath; Hampsey, Michael et al. (2013) DNA looping facilitates targeting of a chromatin remodeling enzyme. Mol Cell 50:93-103 |
| Hampsey, Michael (2012) Molecular biology. A new direction for gene loops. Science 338:624-5 |
| Goel, Shivani; Krishnamurthy, Shankarling; Hampsey, Michael (2012) Mechanism of start site selection by RNA polymerase II: interplay between TFIIB and Ssl2/XPB helicase subunit of TFIIH. J Biol Chem 287:557-67 |
| Hampsey, Michael; Singh, Badri Nath; Ansari, Athar et al. (2011) Control of eukaryotic gene expression: gene loops and transcriptional memory. Adv Enzyme Regul 51:118-25 |
| Seibold, Steve A; Singh, Badri Nath; Zhang, Chunfen et al. (2010) Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase. Biochim Biophys Acta 1799:575-87 |
| Laine, Jean-Philippe; Singh, Badri Nath; Krishnamurthy, Shankarling et al. (2009) A physiological role for gene loops in yeast. Genes Dev 23:2604-9 |
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