RRM
2011 Rustbelt RNA Meeting
RRM
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Talk abstracts
Abstract:
Vaccinia is a member of the Pox virus family containing a double-stranded DNA genome. Early genes, the first of three transcriptional classes, are transcribed by enzymes packaged within the Vaccinia virion. Nucleoside Triphosphate Phosphohydrolase I (NPH I) is an integral component of Vaccinia early gene transcription termination complex. NPH I releases paused transcripts by hydrolyzing ATP or deoxyATP. NPH I ATPase activity requires ssDNA as a cofactor, which is hypothesized to originate from the non-template strand of the DNA being transcribed. To test this hypothesis we constructed bead-bound transcription DNA templates lacking portions of the non-template strand. Transcription was paused using nucleotide starvation and NPH I mediated transcript release was measured. Lengthening the non-template strand restored NPH I mediated transcript release activity. Hence the non-template strand may be the source of the ssDNA cofactor. Another possibility is that transcription bubble collapse may be restored when the non-template strand is lengthened. Transcription bubble collapse is an important component of terminator hairpin function, rho mediated termination, and MFD mediated transcript release in bacteria. To test whether bubble collapse is an important component of NPH I mediated transcript release, templates were constructed with sequences of non-complementarity within the transcription bubble and assayed for NPH I mediated transcript release and oligonucleotide mediated transcript release. Preliminary results suggest that bubble collapse is an important component of NPH I mediated transcript release. Future directions will include attempting to crosslink NPH I to regions of single stranded DNA within the transcription bubble.
Keywords: Vaccinia, Early Gene Transcription
