The tRNA Specificity of Thermus thermophilus EF-Tu

Abstract

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)⅐GTP 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 kcal͞mol for Val-tRNA Glu to ؊8.1 kcal͞mol for Val-tRNA Tyr , clearly establishing EF-Tu⅐GTP 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. E longation factor Tu (EF-Tu) binds GTP and aminoacyl tRNA (aa-tRNA) to form a ternary complex that subsequently binds ribosome and participates in codon-directed binding of the aa-tRNA to the ribosomal A site. Although EFTu⅐GTP binds poorly to tRNAs lacking the esterified amino acid (1), the protein is generally considered to lack specificity because it binds all elongator aa-tRNAs with a similar affinity (2-4). However, recent experiments have shown that EF-Tu⅐GTP exhibits substantial specificity for both the esterified amino acid and the tRNA body of aa-tRNAs (5). This specificity was previously unappreciated because the contributions of the amino acid and the tRNA body to the overall affinity are arranged in a compensatory manner such that the cognate aa-tRNAs all bind with similar affinities. However, misacylated tRNAs were found to bind EF-Tu⅐GTP with a wide range of affinities that were either tighter or weaker than the cognate aa-tRNAs (5). The four different tRNA bodies that were tested displayed about a 100-fold range of K D values with Thermus thermophilus EFTu⅐GTP when each was esterified with the same amino acid. The same range of K D values was observed when the four tRNAs were esterified with a different common amino acid, clearly establishing that EF-Tu shows specificity toward the tRNA body. However, the four tRNAs used in these experiments (Escherichia coli tRNA Ala , tRNA Gln , tRNA Val , and yeast tRNA Phe ) have very similar overall architectures, raising the possibility that EFTu⅐GTP could exhibit a much larger range of affinities with tRNAs of different architectures.Experiments presented here take advantage of the important role of the anticodon as a identity element for valyl-tRNA synthetase (ValRS) and phenylalanyl-tRNA synthetase (PheRS) (6-10) to prepare 21 different E. coli tRNAs that were misacylated with either valine or phenylalanine. By comparing the affinities of the different tRNAs esterified with a common amino acid, the specificity of EF-Tu⅐GTP for the different tRNA bodies was established. Materials and MethodsE. coli...

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

“…We made use of the observation that tRNA Val can be misacylated by high concentrations of yeast-PheRS, and the resulting Phe-tRNA Val binds EF-Tu twofold to threefold tighter than Val-tRNA Val (10). Table 5 shows that the k pep values of WT and T1 are 4.3-and 2.2-fold slower when phenylalanylated than when valylated, which agrees reasonably well with the twofold to threefold difference reported in k off (10).…”

“…As previously observed with other tRNAs, (12,21,22) the unmodified WT tRNA Val GAC bound EF-Tu very similarly to native, fully modified tRNA Val GAC consistent with the absence of modifications in the EF-Tu binding site. The seven mutations showed the desired broad range of binding affinity to E. coli EF-Tu that is similar to the range in affinities observed for different tRNA bodies with T. thermophilus EF-Tu (10,16). T1 to T3 bound from 0.5 to 1.0 kcal∕mol tighter, W1 to W3 bound from 1.0 to 2.0 kcal∕mol weaker, and Ψ bound nearly the same as the WT tRNA Val .…”

“…Although all elongator aa-tRNAs bind EF-Tu•GTP with similar affinities (7)(8)(9), studies with misacylated tRNAs reveal that the protein shows substantial specificity for both the esterified amino acid and the tRNA body (10)(11)(12). The nearly uniform EF-Tu binding affinity observed for tRNAs acylated with their correct (cognate) amino acid occurs because the sequence of each tRNA has evolved to compensate for the variable thermodynamic contribution of the esterified amino acid.…”

Biogenesis of glutaminyl-mt tRNA Gln in human mitochondria

“…These observations suggest that the rejection of misaminoacylated tRNAs appears to be a common feature of EF-Tu. Molecular recognition of the amino acid moiety of an aa-tRNA by EF-Tu has been studied by Uhlenbeck and coworkers (39,40). tRNA Glu binds more tightly to EF-Tu than tRNA Gln does.…”

Aminoacyl-tRNAs: setting the limits of the genetic code

“…The noncognate aa-tRNA species, Asp-tRNA Asn and Glu-tRNA Gln , are formed by a nondiscriminatingtype aaRS (see below) as precursors for subsequent conversion to Asn-tRNA Asn and Gln-tRNA Gln by Asp/GlutRNA amidotransferase . The noncognate species do not present a threat to translational fidelity, as they are effectively discriminated against by EF-Tu (Stanzel et al 1994;Becker and Kern 1998;Asahara and Uhlenbeck 2002). Genome sequence and biochemical analyses provided a major surprise in revealing the lack of conservation in Gln-tRNA formation.…”