): The study of basal transcription has proven central to our understanding of a number of diseases including malignancy, as the products of several oncogenes and anti-oncogenes turn out to represent transcriptional regulators that modulate directly the basal transcription machinery. In eukaryotes, transcription is carried out by three types of DNA-dependent RNA polymerases, each of which must be recruited specifically to its target promoters. The promoters of the human snRNA genes are very similar in structure yet some of these are recognized by RNA polymerase II while others are recognized by RNA polymerase III. SnRNA gene promoters recruit at least two common factors, the transcriptional activator Oct-1 and a TBP-containing complex termed the snRNA RNA activating protein complex (SNAP complex or SNAPc). Because RNA polymerase II and III snRNA promoters are so similar yet are transcribed by different RNA polymerases, they constitute an ideal model system to identify the key components that determine RNA polymerase specificity. The broad goal of this application is to pursue the characterization of the basal transcription factors involved in transcription of the human snRNA genes by RNA polymerases II and III.
The specific aims are to obtain cDNAs encoding all of the subunits of SNAPc as well as antibodies directed against each of these subunits, to use these reagents to overexpress each of the SNAPc components, reconstitute SNAPc from recombinant subunits, and thereby understand how SNAPc is assembled and how it recognizes DNA, and to identify other factors involved in basal RNA polymerase II and III transcription of snRNA genes. The experiments will take advantage of the availability of in vitro transcription systems capable of reproducing accurately both RNA polymerase II and III snRNA gene transcription. Together, the proposed experiments will characterize the transcription initiation complexes formed on RNA polymerase II and III snRNA promoters. The comparison of these complexes with each other and with more conventional RNA polymerase II and III initiation complexes will allow the characterization of the key features that determine RNA polymerase specificity.
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