Structure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme.
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.
Yu
L
L
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Martins
A
A
Deng
L
L
Shuman
S
S
D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000378:metabolism*; D000409:Alanine; D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D018698:Glutamic Acid; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D012315:RNA Caps; D012333:RNA, Messenger; D012367:RNA, Viral / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000201:enzymology*; D014764:Viral Proteins
D009097:Multienzyme Complexes; D012315:RNA Caps; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D018698:Glutamic Acid; D001120:Arginine; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase; D000409:Alanine