Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding.
In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNA(Met)i The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNA(Met)i, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present.
Ozanick
Sarah G
SG
Department of Biological Sciences, Marquette University, P.O. Box 1881, Milwaukee, WI 53201, USA.
Bujnicki
Janusz M
JM
Sem
Daniel S
DS
Anderson
James T
JT
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D039621:Eukaryotic Initiation Factor-3 / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012331:RNA, Fungal / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D012359:tRNA Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D039621:Eukaryotic Initiation Factor-3; D012331:RNA, Fungal; D012358:RNA, Transfer, Met; D029701:Saccharomyces cerevisiae Proteins; C409023:Gcd10 protein, S cerevisiae; D012359:tRNA Methyltransferases; C119384:GCD14 protein, S cerevisiae