Poster abstracts
Poster number 13 submitted by Rachel Boldt
The C-terminal domain of the vaccinia capping enzyme D12 subunit is important for its role in transcription termination
Rachel Boldt (Department of Biological Sciences, SUNY Buffalo), Dr. Paul Gollnick (Department of Biological Sciences, SUNY Buffalo)
Abstract:
Vaccinia virus, a member of the poxvirus family, is a double stranded, cytoplasmic DNA virus. Vaccinia genes are expressed in three different temporal classes; early, intermediate, and late. Early gene transcription requires a specific viral form of RNA polymerase. Termination of early gene transcription occurs in a site-specific, signal-dependent manner involving a U5NU signal in the nascent RNA together with several protein factors including, NPHI and the viral mRNA capping enzyme. The viral Capping Enzyme recognizes and binds a U5NU sequence which signals for termination to occur. In turn NPHI releases the terminated transcripts using a forward translocation method.
The Vaccinia mRNA capping enzyme is a heterodimeric protein composed of a large, 97kDa subunit (D1) and a small, 33kDa subunit (D12). The D1 subunit contains the active sites for capping activities. The N-terminal domain has both guanylyl-transerase and NTPase activities. The C-terminal domain contains the methyl-transferase active site; however dimerization with D12 is required for full enzymatic activity. Both subunits are required for its transcription termination activity.
Here we have made mutations in the C-terminal region of D12 and tested mutant D1 D12 dimers for early gene transcription termination activity. We have found that truncating D12 by as few as 3 amino acids from its C-terminal end inhibits transcription termination. These mutant proteins are fully active in capping activities suggesting that the C-terminal region of D12 is specifically involved in transcription termination function of the capping enzyme.
We have also detected an interaction between the C-terminal end of D12 and an as of yet unidentified viral factor using a Mts-Atf-Biotin label transfer reagent. Further work is being carried out to identify this factor and to determine whether this interaction is important for capping enzymes role in transcription termination.
Keywords: vaccinia , transcription termination