Importance of structural features for tRNA(Met) identity.
We showed previously that the tRNA tertiary structure makes an important contribution to the identity of yeast tRNA(Met) (Senger B, Aphasizhev R, Walter P, Fasiolo F, 1995, J Mol Biol 249:45-58). To learn more about the role played by the tRNA framework, we analyzed the effect of some phosphodiester cleavages and 2'OH groups in tRNA binding and aminoacylation. The tRNA is inactivated provided the break occurs in the central core region responsible for the tertiary fold or in the anticodon stem/loop region. We also show that, for tRNA(Met) to bind, the anticodon loop, but not the anticodon stem, requires a ribosephosphate backbone. A tertiary mutant of yeast tRNA(Met) involving interactions from the D- and T-loop unique to the initiator species fails to be aminoacylated, but still binds to yeast methionyl-tRNA synthetase. In the presence of 10 mM MgCl2, the mutant transcript has a 3D fold significantly stabilized by about 30 degrees C over a wild-type transcript as deduced from the measure of their T(m) values. The k(cat) defect of the tRNA(Met) mutant may arise from a failure to overcome an increase of the free energetic cost of distorting the more stable tRNA structure and/or a tRNA based MetRS conformational change required for formation of transition state of aminoacylation.
Aphasizhev
R
R
CNRS, UPR 9002 Structure des Macromolécules Biologiques et Mécanismesde Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.
Senger
B
B
Fasiolo
F
F
D000926:Anticodon; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D007700:Kinetics; D015636:Magnesium Chloride; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012358:RNA, Transfer, Met / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D000926:Anticodon; D012358:RNA, Transfer, Met; D015636:Magnesium Chloride