Interaction of elongation factor Tu from Escherichia coli with aminoacyl-tRNA carrying a fluorescent reporter group on the 3' terminus.
Transfer ribonucleic acids containing 2-thiocytidine in position 75 ([s2C]tRNAs) were prepared by incorporation of the corresponding cytidine analogue into 3'-shortened tRNA using ATP(CTP):tRNA nucleotidyltransferase. [s2C]tRNA was selectively alkylated with fluorescent N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS) on the 2-thiocytidine residue. The product [AEDANS-s2C]aminoacyl-tRNA, forms a ternary complex with Escherichia coli elongation factor Tu and GTP, leading to up to 130% fluorescence enhancement of the AEDANS chromophore. From fluorescence titration experiments, equilibrium dissociation constants of 0.24 nM, 0.22 nM and 0.60 nM were determined for yeast [AEDANS-s2C]Tyr-tRNATyr, yeast Tyr-tRNATyr, and the homologous E. coli Phe-tRNAPhe, respectively, interacting with E. coli elongation factor Tu.GTP. The measurement of the association and dissociation rates of the interaction of [AEDANS-s2C]Tyr-tRNATyr with EF-Tu.GTP and the temperature dependence of the resulting dissociation constants gave values of 55 J mol-1 K-1 for delta S degrees' and -34.7 kJ mol-1 for delta H degrees' of this reaction.
Ott
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Laboratorium für Biochemie and Bayreuther Institut für Makromolekulare Forschung der Universität Bayreuth.
Faulhammer
H G
HG
Sprinzl
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M
D003562:Cytidine / Q000031:analogs & derivatives; D004926:Escherichia coli / Q000378:metabolism*; D005456:Fluorescent Dyes; D006160:Guanosine Triphosphate / Q000378:metabolism; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008433:Mathematics; D009282:Naphthalenesulfonates; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D012346:RNA, Transfer, Amino Acyl; C002908:2-thiocytidine; D003562:Cytidine; C006421:1,5-I-AEDANS; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu; D007460:Iodoacetamide