Structure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme Journal Article
| Authors: | Yu, L.; Martins, A.; Deng, L.; Shuman, S. |
| Article Title: | Structure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme |
| Abstract: | The N-terminal 60 kDa (amino acids 1 to 545) of the D1 subunit of vaccinia virus mRNA capping enzyme is an autonomous bifunctional domain with triphosphatase and guanylyltransferase activities. We previously described two alanine cluster mutations, R77 to A (R77A)-K79A and E192A-E194A, which selectively inactivated the triphosphatase component. Here, we characterize the activities of 11 single alanine mutants-E37A, E39A, Q60A, E61A, T67A, T69A, K75A, R77A, K79A, E192A, and E194A and a quadruple mutant in which four residues (R77, K79, E192, and E194) were replaced by alanine. We report that Glu-37, Glu-39, Arg-77, Glu-192, and Glu-194 are essential for γ-phosphate cleavage. The five essential residues are conserved in the capping enzymes of Shope fibroma virus, molluscum contagiosum virus, and African swine fever virus. Probing the structure of D1(1-545) by limited V8 proteolysis suggested a bipartite subdomain structure. The essential residue Glu-192 is the principal site of V8 cleavage. Secondary cleavage by V8 occurs at the essential residue Glu-39. The triphosphatase-defective quadruple mutant transferred GMP to the triphosphate end of poly(A) to form a tetraphosphate cap structure, GppppA. We report that GppppA-capped RNA is a poor substrate for cap methylation by the vaccinia virus and Saccharomyces cerevisiae RNA (guanine-7) methyltransferases. The transcription termination factor activity of the D1-D12 capping enzyme heterodimer was not affected by mutations that abrogated ATPase activity. Thus, the capping enzyme is not responsible for the requirement for ATP hydrolysis during transcription termination. |
| Keywords: | methylation; nonhuman; phosphatase; transcription, genetic; enzyme activity; acid anhydride hydrolases; structure activity relation; structure-activity relationship; methyltransferases; amino acid sequence; molecular sequence data; sequence homology, amino acid; amino terminal sequence; serine endopeptidases; messenger rna; rna caps; rna, messenger; vaccinia virus; binding sites; alanine; multienzyme complexes; glutamic acid; phosphoric monoester hydrolases; arginine; rna, viral; virus mutant; transcription termination; rna capping; nucleotidyltransferases; priority journal; article |
| Journal Title: | Journal of Virology |
| Volume: | 71 |
| Issue: | 12 |
| ISSN: | 0022-538X |
| Publisher: | American Society for Microbiology |
| Date Published: | 1997-12-01 |
| Start Page: | 9837 |
| End Page: | 9843 |
| Language: | English |
| PUBMED: | 9371657 |
| PROVIDER: | scopus |
| PMCID: | PMC230301 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 17 March 2017 -- Source: Scopus |
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