Genetic analysis of poxvirus mRNA cap methyltransferase: suppression of conditional mutations in the stimulatory D12 subunit by second-site mutations in the catalytic D1 subunit.
The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme, composed of catalytic vD1(498-844) and stimulatory vD12 subunits, can function in vivo in Saccharomyces cerevisiae in lieu of the essential cellular cap methyltransferase Abd1. Coexpression of both poxvirus subunits is required to complement the growth of abd1Delta cells. A double-alanine scan of the vD12 protein identified lethal and temperature-sensitive vD12 alleles. We used this mutant collection to perform a forward genetic screen for compensatory changes in the catalytic subunit that suppressed the growth phenotypes of the vD12 mutants. The screen reiteratively defined a small ensemble of amino acids in vD1(498-844) at which mutations restored methyltransferase function in conjunction with defective vD12 proteins. Reference to the crystal structure of the microsporidian cap methyltransferase suggests that distinct functional classes of suppressors were selected, including: (i) those that map to surface-exposed loops, which likely comprise the physical subunit interface; (ii) those in or near the substrate binding sites, which presumably affect or mimic inter-subunit allostery.
Schwer
Beate
B
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA. bschwer@med.cornell.edu
Shuman
Stewart
S
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics*; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012315:RNA Caps; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013489:Suppression, Genetic; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014764:Viral Proteins
D009097:Multienzyme Complexes; D012315:RNA Caps; D012333:RNA, Messenger; D014764:Viral Proteins; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus