The products of many genes that are transcribed by eukaryotic RNA polymerase III have roles in the regulation and execution of protein synthesis. Transfer RNA and 5S RNA are required for the translation of all mRNAs; 7S RNA, with tRNAs and 5S RNA, is required for translation of secretory protein mRNA. Because these gene products are vital to the health and well-being of the organism, it is important to understand how these RNA polymerase III transcription units are expressed. We have used human HeLa cell extracts as the starting material for the isolation of RNA polymerase III transcription factors. We have developed, using partially purified transcription factor fractions, assays that not only allow the identification of those protein factors but also allow their functional characterization. We propose to use this information to develop the means to obtain large quantities of factors. We will first use conventional biochemical methods in efforts to improve our current fractionation scheme and to explore alternative mammalian sources for the factors. We will develop DNA filter binding assays for the factors which bind to their DNA template; these assays will allow us to study the kinetic parameters of factor-DNA interaction. This information will be used to design DNA affinity purification methods. Protein microsequencing will be used to determine (partial) amino acid sequence of transcription factors isolated using conventional and/or DNA affinity purification methods. We will raise antibodies to a synthetic peptide derived from the protein sequence; anti-peptide IgGs will be used to characterize putative transcription factor clones. Mixed oligodeoxynucleotide probes, whose sequences are derived from the protein sequence, will be used to probe cDNA libraries for recombinants carrying coding sequence for the factor. Finally isolation of the factor gene from genomic libraries will allow the reconstruction of the entire gene for insertion into E. coli expression vectors. This gives final assurance of the availability of that factor. The ability to isolate large quantities of RNA polymerase III transcription factors, whether achieved through conventional biochemical methods, DNA affinity methods, or recombinant DNA technology, will enable us to undertake detailed studies of how these proteins interact with each other and with their DNA template to allow transcription of genes whose products are essential to protein synthesis.
