Poster abstracts
Poster number 15 submitted by Rachel Boldt
Specific Early Gene Transcription Termination Signal Binding by Vaccinia Capping Enzyme
Rachel Boldt (Dept. of Biological Sciences, SUNY Buffalo), Paul Gollnick (Dept. 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 termination occurs in a site-specific manner and depends on U5NU signal in the nascent RNA. A specific interaction between the viral mRNA capping enzyme and the U5NU sequence is required for proper transcription termination.
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.
The N-terminal domain of the D1 capping enzyme subunit has been shown to crosslink to the U5NU termination signal in RNA. Here, we differentiate specific U5NU sequence binding from non-specific RNA 5’end binding associated with mRNA capping. Chimeric oligonucleotides with 10 bases of DNA at the 5’ends followed by 26 bases of RNA were used in binding studies. Capping enzyme was found to have a higher affinity for U5NU containing oligos compared to oligos without the termination signal. Furthermore, chimeric oligos containing the U5NU were able to stimulate transcription termination in vitro. These chimeric oligos will be used in future studies to identify the specific residues within D1 that interact with the U5NU sequence. Both alanine scanning and a crosslinking followed by mass spec approach will be used.
Keywords: Vaccinia, Transcription