The goal of this project is to understand the mechanism of transcription termination during eukaryotic mRNA synthesis. The problem is being approached in a viral model system, vaccinia, where biochemical and genetic complexity are reduced relative to that of the cell. Vaccinia early mRNAs are synthesized by a multisubunit virus-encoded RNA polymerase. Transcription of early genes terminates downstream of a cis- acting termination signal, UUUUUNU, in the nascent RNA. A separate factor, termed VTF (vaccina termination factor), is required for termination in vitro by purified vaccinia RNA polymerase. Extensive purification of VTF, and the reconstitution of VTF from its constituent polypeptides, has established that the termination factor is identical to the vaccinia mRNA capping enzyme, a virus-encoded protein of 95 kDa and 31 kDa subunits. This proposal outlines a combined biochemical and molecular genetic analysis of the role of the multifunctional capping enzyme in mRNA biogenesis, focusing on the following questions. How are distinct functional domains organized within the heterodimeric VTF/capping enzyme molecule? How does the factor interact with other constituents of the transcriptional apparatus? What is the structure of the polymerase elongation complex? What subunit interactions contribute to the assembly and function of the vaccinia RNA polymerase? To answer these questions, the vaccinia genes encoding the two subunits of VTF/capping enzyme have been expressed in bacteria. By assessing the effects of amino acid sequence alterations on enzyme activity in vitro, we will fine-map the three individual catalytic domains for RNA modification (triphosphatase, guanylyltransferase, and methyltransferase) and will delineate the (as yet) uncharted requirements for factor-dependent termination. Probing the structure of the ternary complex will entail purification of active complexes of RNA polymerase halted in vitro at unique template positions. UV crosslinking will be used to determine which polypeptide subunits of the elongation complex make contact with nascent RNA and with the DNA template. Analysis of transcription complexes halted at various intervals on the template will reveal whether, and how, the complex changes as polymerase moves away from the promoter and as the termination signal is transcribed. The """"""""two-hybrid system"""""""" in yeast will be exploited to define the spectrum of binary protein-protein interactions among the vaccinia gene products involved in early transcription. The proposed studies of viral transcription termination should provide insights into analogous aspects of cellular mRNA synthesis. Indeed, termination has been implicated as a regulatory step in the expression of cellular proto-oncogenes (e.g. c-myc) during tumorigenesis, and in the genetic program of human immunodeficiency virus (HIV).
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