The goal of this laboratory is to understand at the biochemical level the role of transcription termination in 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. Synthesis of vaccinia early mRNAs by the virus-encoded RNA polymerase terminates some 50 nucleotides downstream of a cis- acting termination signal, TTTTTNT, in the non-transcribed template strand. A novel trans-acting factor, termed VTF (vaccinia termination factor), is required for termination in vitro by purified vaccinia RNA polymerase. Extensive purification of VTF has established that the termination factor is identical to the vaccinia virus mRNA capping enzyme. This proposal outlines a combined biochemical and genetic analysis of the role of this multifunctional 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? How might the multifunctional VTF/capping enzyme act at both 5' and 3' ends of the mRNA synthetic process? What are the physiologic roles of RNA capping and transcription termination in vaccinia RNA metabolism? To answer these questions, the vaccinia genes encoding the two subunits of VTF/capping enzyme will be expressed in active form and at high level in E. coli. Directed mutagenesis will then be used to delineate the functional domains of VTF/capping enzyme, and to correlate enzyme structure and function in vitro. Assays are described to study the physical interaction of VTF/capping enzyme with other components of the transcriptional apparatus. The physiologic roles of termination and capping will be addressed through the isolation of conditional virus mutants specifically affecting these reactions. The proposed studies of viral transcription may provide insights into analogous aspects of cellular mRNA synthesis. Indeed, termination has been implicated as a major 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), the causative agent of AIDS.
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