The long term objective of this study is to determine how RNA polymerase III (pol III) is recruited to structurally distinct promoters. It is likely that while the basal transcription complex is assembled differently on different promoters, one or more essential factors that recruit the polymerase may be shared by all the promotes. A likely candidate is transcription factor IIIB (TFIIIB) because in the yeast, Saccharmomyces cerevisiae, this complex is the only essential RNA pol III factor and contacts the enzyme directly. The mammalian TFIIIB complex appears to have more subunits than the yeast factor and its role in transcription is not clearly defined.
The specific aims of this project are: 1) -- to purify human TFIIIB and clone cDNAs encoding the subunits; 2) -- The clones will be used to overexpress the TFIIIB components for raising monoclonal antibodies and for functional studies; 3) -- The TFIIIB complex will be assembled in vitro from its components and the functions of the holo- and partial complexes evaluated in transcription from the human 5S, U6 and 7SL genes, the Adenovirus VA1 and the Epstein-Barr Virus EBER2 genes. The promoters of these genes represent the spectrum of structurally distinct RNA pol III promoters. Mutagenesis, biochemical assays and footprinting techniques will be used to define i) the interactions among the components of TFIIIB ii) the interactions with other known RNA polymerase III factors, RNA polymerase and DNA; 4) -- Transcription complexes will be assembled on the biochemically less well characterized 7SL and EBER2 promoters to determine which of the know RNA pol III factors are required. Potentially these studies will identify novel pol III factors. A knowledge of how RNA pol III recognises disparate promoters is important in understanding how transcription is regulated, and may ultimately provide a means for selectively inhibiting viral gene expression in human cells.
| Zhou, D; Lobo-Ruppert, S M (2001) Transcription of the Schizosaccharomyces pombe U2 gene in vivo and in vitro is directed by two essential promoter elements. Nucleic Acids Res 29:2003-11 |
| McCulloch, V; Hardin, P; Peng, W et al. (2000) Alternatively spliced hBRF variants function at different RNA polymerase III promoters. EMBO J 19:4134-43 |
| Zhou, D; Frendewey, D; Lobo Ruppert, S M (1999) Pac1p, an RNase III homolog, is required for formation of the 3' end of U2 snRNA in Schizosaccharomyces pombe. RNA 5:1083-98 |
| Ruppert, S M; McCulloch, V; Meyer, M et al. (1996) Monoclonal antibodies directed against the amino-terminal domain of human TBP cross-react with TBP from other species. Hybridoma 15:55-68 |
