The objective of this proposal is to elucidate molecular mechanisms and control of transcription initiation of RNA polymerase III-mediated genes in human KB cells. Since most of the RNA products of these genes are directly involved in regulation of protein synthesis, the studies in this proposal will further our understanding of control mechanisms in two of the key life processes: transcription and translation. Therefore, the knowledge gained for this project should be related to health problems, such as control of cancer growth, control of virus infections in target tissues, host defense mechanism against infectious agents and control of human cell metabolism. Early studies have delimited the outer boundaries of the transcriptional control region of Ad2 specific VARNAL gene as from +10 to +69 using an in vitro cell-free transcription system to study effects of deletion mutations on overall transcription of this gene. Our studies have further defined the organization of functional domains in this control region and gained insight into the mechanisms of transcription initiation of this gene. The studies outlined in this proposal represent continuing investigation of this aspect. The major objective of this proposal, therefore, is to define precisely the function of each base pair in every domain and to study mechanisms of interactions of transcriptional regulatory factors and RNA polymerase III with each nucleotide in these functional domains leading to initiation. Our studies will focus on: (a) effect of deletion mutations in the control region on formation of stable preinitiation and initiation complexes; (b) construction of linkerscanning mutants for transcription studies to define further the function and boundaries of each domain; (c) construction of point mutants for transcription studies to define the function of each base pair in every functional domain; (d) purification of regulatory factors for reconstitution of transcription machinery and for studying mechanisms of interactions of proteins-proteins and proteins-DNA sequence in each functional domains; and, (e) comparisons of mechanisms of transcription initiation in this gene with human tRNA genes and other RNA polymerase III-mediated genes.
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