Early vaccinia virus gene transcription takes place in the virion core and is the only class that demonstrates signal dependent termination. Early gene transcription termination requires the signal UUUUUNU in the nascent RNA, VTF, the vaccinia termination factor that is also the viral mRNA capping enzyme, NPH I a single stranded DNA dependent ATPase to provide energy and the virion form of the multi-subunit RNA polymerase containing the additional H4L subunit. The goal of this research is to define the mechanism of early gene transcription termination. In this submission, we propose three specific aims.
In Aim 1, we will focus on the role of NPH I in termination. We will determine whether NPH I binds to the nontemplate strand of the DNA to activate the ATPase activity. In addition, we will investigate the conformational change in NPH I induced upon ssDNA binding. Mutants in NPH I known to affect termination, in vitro, will be constructed in virus and their phenotypes investigated in vivo. Finally, the role of NPH I in transcription elongation will be evaluated.
In Aim 2, our attention switches to VTF, the vaccinia termination factor. We will determine whether VTF is the factor that senses UUUUUNU in the nascent RNA. In addition, we will identify additional VTF interacting partners. Mutagenesis of VTF will permit identification of the termination specific functions. Finally, our novel observation of UUUUUNU specific activation of premature termination in trans will be fully explored.
In Aim 3, we will address the role of the H4L subunit of the virion RNA polymerase in both early gene transcription initiation and termination. We propose to locate the NPH I binding site on H4L. We will map the region of H4L that is required for pre-initiation complex formation. We will search for H4L interacting proteins to both identify the factors required for PIC formation and additional proteins that may function in termination. Finally, we will construct mutations in virus that exhibit altered NPH I binding and evaluate their phenotypes in vivo. Through these studies we will define aspects of the mechanism of early gene transcription. These results will provide important insights into this essential step in gene expression and define new sites for the development of specific poxvirus antiviral agents.
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