Structure and function studies of vaccinia virus early transcription factor

Jing Li, Purdue University

Abstract

VETF is a transcription activator specific for vaccinia early gene transcription. Gel shift assays showed that VETF activated transcription by direct interaction with RNA polymerase at its promoter-binding site to form an initiation complex. DNaseI footprint and methylation interference analyses revealed a conformation change in the promoter area induced by the complex formation. The formation of the complex on the promoter sequence did not require any nucleotide, but the synthesis of a short RNA chain absolutely required ATP. Vaccinia D6 and A8 genes were expressed in human cells using the hybrid vaccinia-T7 expression system. Both A8 and D6 subunits are required for all the biochemical activities associated with VETF. Mutations of an A-type ATP binding motif $\rm (K\sb{51}I)$ and a DEAH box $\rm (E\sb{136}Q)$ resulted in VETF with no ATPase activity and were unable to activate transcription. These results indicate that the DNA-dependent ATPase activity is essential for VETF to activate early transcription. VETF versions from two temperature sensitive vaccinia viruses were shown to be defective for promoter binding and transcription activation, indicating that the small subunit of VETF is essential for its promoter-binding activity. The mutant proteins retained DNA-dependent ATPase activity, but required higher DNA concentration to activate the ATPase. The two mutant virion particles contained about half the VETF of the wild type virion. RNA polymerase, mRNA capping enzyme, and nucleoside triphosphate phosphohydrolase I were found at similarly reduced levels in the virion. This suggested that promoter binding may contribute to packaging of VETF into the virion particle, and that packaging of some virion core enzyme is interdependent. Site-directed mutations in the sequence of the D6 subunit revealed two areas important for DNA binding in D6 sequence. One is located in the N-terminus of D6, the other in the middle of the C-terminal part of D6. Both of them are conserved regions among the VETF family members. The same regions were also important for the DNA-dependent ATPase activity of VETF.

Degree

Ph.D.

Advisors

Broyles, Purdue University.

Subject Area

Biochemistry|Molecular biology|Microbiology

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