TY - JOUR
T1 - The tRNA specificity of Thermus thermophilus EF-Tu
AU - Asahara, Haruichi
AU - Uhlenbeck, Olke C.
PY - 2002/3/19
Y1 - 2002/3/19
N2 - 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-tRNAGlu to -8.1 kcal/mol for Val-tRNATyr, clearly establishing EF-Tu·GTP as a sequence-specific RNA-binding protein. Because the ionic strength dependence of koff 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.
AB - 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-tRNAGlu to -8.1 kcal/mol for Val-tRNATyr, clearly establishing EF-Tu·GTP as a sequence-specific RNA-binding protein. Because the ionic strength dependence of koff 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.
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U2 - 10.1073/pnas.052028599
DO - 10.1073/pnas.052028599
M3 - Article
C2 - 11891293
AN - SCOPUS:0037133662
VL - 99
SP - 3499
EP - 3504
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 6
ER -