The tRNA specificity of Thermus thermophilus EF-Tu.
By introducing a GAC anticodon, 21 different Escherichia coli tRNAs were misacylated with either phenylalanine or valine and assayed for their affinity to Thermus thermophilus elongation factor Tu (EF-Tu)*GTP by using a ribonuclease protection assay. The presence of a common esterified amino acid permits the thermodynamic contribution of each tRNA body to the overall affinity to be evaluated. The E. coli elongator tRNAs exhibit a wide range of binding affinities that varied from -11.7 kcal/mol for Val-tRNA(Glu) to -8.1 kcal/mol for Val-tRNA(Tyr), clearly establishing EF-Tu*GTP as a sequence-specific RNA-binding protein. Because the ionic strength dependence of k(off) varied among tRNAs, some of the affinity differences are the results of a different number of phosphate contacts formed between tRNA and protein. Because EF-Tu is known to contact only the phosphodiester backbone of tRNA, the observed specificity must be a consequence of an indirect readout mechanism.
Asahara
Haruichi
H
Department of Chemistry and Biochemistry, University of Colorado, 215 UCB, Boulder, CO 80309-0215, USA.
Uhlenbeck
Olke C
OC
D000215:Acylation; D000926:Anticodon / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering; D009154:Mutation / Q000235:genetics; D023282:Nuclease Protection Assays; D009994:Osmolar Concentration; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D011485:Protein Binding; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology*
D000926:Anticodon; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D016601:RNA-Binding Proteins; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu