Structure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme. Academic Article uri icon

Overview

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 gamma-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.

publication date

  • December 1, 1997

Research

keywords

  • Acid Anhydride Hydrolases
  • Alanine
  • Arginine
  • Glutamic Acid
  • Methyltransferases
  • Multienzyme Complexes
  • Nucleotidyltransferases
  • Phosphoric Monoester Hydrolases
  • RNA Caps
  • RNA, Viral
  • Vaccinia virus

Identity

PubMed Central ID

  • PMC230301

Scopus Document Identifier

  • 0030728958

Digital Object Identifier (DOI)

  • 10.1128/JVI.71.12.9837-9843.1997

PubMed ID

  • 9371657

Additional Document Info

volume

  • 71

issue

  • 12