Vaccinia virus mRNAs are synthesized by a multisubunit RNA polymerase homologous to cellular RNA polymerase II. The 5' m7GpppN mRNA cap is formed on nascent transcripts by a virus-encoded capping enzyme that possesses triphosphatase, guanylyltransferase and methyltransferase activities. The 3' ends of early mRNAs are formed by transcription termination downstream of a cis-acting signal UUUUUNU in the nascent RNA. Termination requires a trans-acting vaccinia termination factor (VTF), which is identical to the capping enzyme. The termination reaction requires the hydrolysis of ATP to ADP. The energy coupling step is catalyzed by NPH-I, a DNA-dependent ATPase tightly associated with the elongation complex. NPH-I serves two functions: (i) it acts as a termination factor in concert with VTF to catalyze release of the nascent UUUUUNU-containing RNA from the polymerase and (ii) it acts as an elongation factor to suppress intrinsic pausing by vaccinia RNA polymerase. This proposal outlines a biochemical and molecular genetic analysis of NPH-I and VTF/capping enzyme. The applicant is focusing on the following questions. What structural features of NPH I are responsible for nucleic acid binding and ATP hydrolysis? How does NPH-I interact with the transcription elongation complex? Does NPH-I play a role in recruiting VTF/capping enzyme to the elongation complex? How are distinct functional domains organized within the VTF/CE molecule? How does ATP hydrolysis trigger transcript release: the experimental plan includes a comprehensive structure-function analysis of NPH-I and VTF/capping enzyme via mutagenesis and structure determination. Footprinting and crosslinking methods will be applied to dissect protein- nucleic acid and protein-protein interactions that contribute to the formation of a termination-competent transcription complex. Template signals that influence the elongation properties of RNA polymerase will be characterized.
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