Prevention of mis-aminoacylation of a dual-specificity aminoacyl-tRNA synthetase.
Accurate aminoacylation of tRNAs by aminoacyl-tRNA synthetase is essential for the fidelity of protein synthesis. For Methanococcus jannaschii tRNA(Pro), accuracy is difficult because the cognate prolyl-tRNA synthetase also recognizes and aminoacylates tRNA(Cys) with cysteine. We show here that the unmodified transcript of M. jannaschii tRNA(Pro) is indeed mis-acylated with cysteine. However, the origin of mis-charging is not at the anticodon or acceptor stem, the two hotspots for tRNA(Pro) and tRNA(Cys) identity determinants. Instead, replacement of the D loop in the tRNA core with that of tRNA(Cys) suppresses mis-charging with cysteine without compromising the activity of aminoacylation with proline. The reduced level of cysteine activity of the chimera is not due an editing response of the synthetase and is consistent with a relaxed sensitivity of the tRNA to the analog thiaproline in aminoacylation with cysteine. We suggest that mis-acylation is not due to the presence of cysteine determinants, but to a mis-placed 3' end into the cysteine catalytic site that activates and transfers cysteine to the tRNA. Prevention of mis-placement by alteration of the core structure or by nucleotide modifications in the tRNA illustrates a novel strategy of the dual-specificity synthetase.
Lipman
Richard S A
RS
Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.
Wang
Jinling
J
Sowers
Kevin R
KR
Hou
Ya-Ming
YM
D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000926:Anticodon / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D007700:Kinetics; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011392:Proline / Q000235:genetics / Q000378:metabolism; D017393:RNA Editing; D012350:RNA, Transfer, Cys / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012361:RNA, Transfer, Pro / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D014158:Transcription, Genetic
D000926:Anticodon; D012350:RNA, Transfer, Cys; D012361:RNA, Transfer, Pro; D000255:Adenosine Triphosphate; D011392:Proline; D000604:Amino Acyl-tRNA Synthetases; D003545:Cysteine