Reaction of modified and unmodified tRNA(Tyr) substrates with tyrosyl-tRNA synthetase (Bacillus stearothermophilus).
Three species of tRNA(Tyr) have been examined as substrates for the transfer reaction of the tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus: Escherichia coli tRNA(Tyr), B. stearothermophilus tRNA(Tyr) expressed in E. coli, and B. stearothermophilus tRNA(Tyr) that has been transcribed in vitro. The binding of the first two substrates to TyrRS may be readily monitored by stopped-flow studies of tryptophan fluorescence to give the rate and equilibrium constants. The in vitro-transcribed tRNA(Tyr), which lacks the modified bases queuosine and 2-(methylthio)-N6-isopentenyladenosine in the anticodon loop, does not cause a significant change in tryptophan fluorescence upon binding. The three tRNA(Tyr) substrates exhibit very similar steady-state kinetics in the charging reaction. Pre-steady-state kinetics of the transfer reaction, monitored by stopped-flow measurements of the change in protein fluorescence on the addition of tRNA(Tyr) to the E.Tyr-AMP complex, show two exponential changes for the modified tRNA(Tyr) substrates. The first is that due to substrate binding. The second has an identical rate to the single change observed for the reaction with the in vitro-transcribed tRNA(Tyr) and to that monitored by quenched-flow measurements on the formation of Tyr-tRNA(Tyr). Hence, the transfer reaction can be observed by stopped-flow. The dissociation constants (KtRNA) of tRNA from the enzyme and rates of tyrosine transfer (k4) show that all three tRNA molecules are kinetically equivalent substrates for TyrRS. The value of k4 is also similar to that found for authentic tRNA(Tyr) from B. stearothermophilus.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Chemistry, University of Cambridge, United Kingdom.
J M
JM
Avis
A G
AG
Day
G A
GA
Garcia
A R
AR
Fersht
D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D012365:RNA, Transfer, Tyr / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014158:Transcription, Genetic; D014443:Tyrosine / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000378:metabolism*
D012365:RNA, Transfer, Tyr; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase