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Updated: Mar 31, 2026
In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection
Published on: March 26, 2012
Biochemical analysis of the multifunctional vaccinia mRNA capping enzyme encoded by a temperature sensitive virus
Jessica Tate1, Rachel L Boldt2, Baron D McFadden1
1Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, P.O. Box 100266, Gainesville, FL 32610, United States.
Abstract:
Prior biochemical analysis of the heterodimeric vaccinia virus mRNA capping enzyme suggests roles not only in mRNA capping but also in early viral gene transcription termination and intermediate viral gene transcription initiation. Prior phenotypic characterization of Dts36, a temperature sensitive virus mutant affecting the large subunit of the capping enzyme was consistent with the multifunctional roles of the capping enzyme in vivo. We report a biochemical analysis of the capping enzyme encoded by Dts36. Of the three enzymatic activities required for mRNA capping, the guanylyltransferase and methyltransferase activities are compromised while the triphosphatase activity and the D12 subunit interaction are unaffected. The mutant enzyme is also defective in stimulating early gene transcription termination and intermediate gene transcription initiation in vitro. These results confirm that the vaccinia virus mRNA capping enzyme functions not only in mRNA capping but also early gene transcription termination and intermediate gene transcription initiation in vivo.
