Fluorescence characterization of the interaction of various transfer RNA species with elongation factor Tu.cntdot.GTP: evidence for a new functional role for elongation factor Tu in protein biosynthesis

Janiak, F; Va, Dell; Jk, Abrahamson; Watson, Bonnie S.; Dl, Miller; Ae, Johnson

doi:10.1021/bi00470a002

Cited by 90 publications

(75 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We also show that the EF-Tu⅐GTP⅐Phe-tRNA Phe complex forms a high affinity (nM K D ) interaction (Fig. 1C) (17,19,20,23,25,26).…”

Section: Discussionsupporting

confidence: 56%

“…Likewise, the EF-Tu⅐GTP⅐EF-Ts species implicit in Reaction 2 is also transient in nature (k Ϫ ϭ 60 s Ϫ1 ) (28). The rates of ternary complex formation and dissociation (Reaction 3 in this reaction scheme) have been largely inferred from an array of steady state investigations (16,17,19,23,25,26). Direct presteady state information, however, is currently lacking.…”

Section: Resultsmentioning

confidence: 99%

“…Ensemble, steady state measurements of ternary complex (16,17,19,(23)(24)(25)(26)(27) together with estimated intracellular concentrations of EF-Tu, aa-tRNA (approximately tens of micromolar), and GTP (approximately millimolar) suggest that most (Ͼ90%) aa-tRNA is complexed with EF-Tu in actively growing bacteria (24,28). However, the steady state concentration of ternary complex is strongly dependent on numerous biochemical processes, including the rates of ribosome-catalyzed protein synthesis, amino acid availability, and the relative intracellular concentrations of GTP and GDP.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex

Burnett

Altman

Ferrao

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Aminoacyl-tRNA (aa-tRNA) enters the ribosome in a ternary complex with the G-protein elongation factor Tu (EF-Tu) and GTP. Results: EF-Tu⅐GTP⅐aa-tRNA ternary complex formation and decay rates are accelerated in the presence of the nucleotide exchange factor elongation factor Ts (EF-Ts). Conclusion: EF-Ts directly facilitates the formation and disassociation of ternary complex. Significance: This system demonstrates a novel function of EF-Ts.

show abstract

“…We also show that the EF-Tu⅐GTP⅐Phe-tRNA Phe complex forms a high affinity (nM K D ) interaction (Fig. 1C) (17,19,20,23,25,26).…”

Section: Discussionsupporting

confidence: 56%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex

Burnett

Altman

Ferrao

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…For example, both the initiator tRNA fMet esterified with either methionine or formylmethionine and the specialized elongator tRNA Sec esterified with either serine or selenocysteine (Sec) show very weak binding to EF-Tu⅐GTP in vitro (1,3,4,25). Because both these tRNAs enter translation by binding other proteins, this result is not unexpected.…”

Section: Discussionmentioning

confidence: 99%

The tRNA Specificity of Thermus thermophilus EF-Tu

Asahara

Uhlenbeck

2002

Proc. Natl. Acad. Sci. U.S.A.

100

112

View full text Add to dashboard Cite

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...

show abstract

“…These tight associations forming aminoacyl-tRNA⅐EF-Tu⅐GTP ternary complexes contrast strongly with the weak affinity of E. coli EF-Tu⅐GTP for uncharged tRNA (K d ϭ 2.6 -2.8 M; Ref. 3). The 2250-fold weaker affinity for tRNA Phe lacking the esterified phenylalanyl moiety (3) effectively prevents uncharged tRNA from reaching the ribosomal A site unless the normal aminoacylation status of cellular tRNAs is severely impaired.…”

mentioning

confidence: 99%

Quantitative Assessment of EF-1α·GTP Binding to Aminoacyl-tRNAs, Aminoacyl-viral RNA, and tRNA Shows Close Correspondence to the RNA Binding Properties of EF-Tu

Dreher

Uhlenbeck

Browning

1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

The translational elongation factors EF-1␣ (eEF1A) 1 and EF-Tu (EF1A) are GTP-binding proteins that serve similar roles in protein synthesis in prokaryotes and eukaryotes, respectively. Their normal role is to deliver aminoacylated tRNAs into the A site of the ribosome. While dispensing this function for tRNAs carrying all the standard 20 amino acids (including methionine) that are elongationally inserted into proteins, elongation factors must discriminate against non-aminoacylated tRNAs and against the methionylated initiator tRNA Met that is loaded into the ribosomal P site by initiation factors.The basis for these functions is well understood in bacterial systems, for which extensive ligand binding and structural studies have been performed. The reported equilibrium dissociation constants for the interactions of Escherichia coli EFTu⅐GTP with aminoacylated elongator tRNAs fall between 0.2 and 7 nM (1-3), varying over a 13.8-fold (4) or a 34-fold (2) range when determined in a single set of experiments. These tight associations forming aminoacyl-tRNA⅐EF-Tu⅐GTP ternary complexes contrast strongly with the weak affinity of E. coli EF-Tu⅐GTP for uncharged tRNA (K d ϭ 2.6 -2. Met from the A site thus relies only partially on EFTu⅐GTP discrimination, but also on A site competition by elongator methionyl-tRNA Met ⅐EF-Tu⅐GTP ternary complex, complex formation with IF2⅐GTP, and selective interaction of initiator tRNA with the ribosomal P site (5).The recently solved crystal structure of the phenylalanyltRNA Phe ⅐EF-Tu⅐GTP complex from Thermus aquaticus (6) has shown that the protein contacts the aminoacyl-tRNA only in the acceptor/T half of the tRNA molecule. Accordingly, aminoacylated tRNA half-molecules and tRNAs lacking the anticodon domain have been reported to bind Thermus thermophilus EF-Tu⅐GTP with affinities similar to those of the parental tRNAs (7,8).The overall similarity of EF-1␣ function to that of EF-Tu (see, e.g., Refs. 9 -11) has been convincingly established by the interchangeability of EF-Tu and EF-1␣ in binding to, although not in supporting protein synthesis by, bacterial and mammalian ribosomes (12). EF-1␣, like EF-Tu, has been shown to form ternary complexes with GTP and aminoacyl-tRNA (9, 10, 13). However, in contrast to the rich information available for EFTu, quantitative data on the interaction of EF-1␣ with tRNAs are almost non-existent. The only estimate for the stability of aminoacyl-tRNA⅐EF-1␣⅐GTP ternary complex we have found in the literature is one of "about 10 nM" for Phe-tRNA (11), and the extent of binding discrimination against uncharged tRNA is not known. With regard to the exclusion of initiator methionyltRNA Met from the A site of eukaryotic ribosomes, it has been shown that a 2Ј-phosphoribosyl modification of the purine at position 64 of the yeast and wheat germ initiator tRNAs is an important antideterminant of interaction with 15). After removal of this modification, both tRNAs could serve as elongators in in vitro protein synthesis (15); in the yeast case, the demodified met...

show abstract

Fluorescence characterization of the interaction of various transfer RNA species with elongation factor Tu.cntdot.GTP: evidence for a new functional role for elongation factor Tu in protein biosynthesis

Cited by 90 publications

References 56 publications

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex

The tRNA Specificity of Thermus thermophilus EF-Tu

Quantitative Assessment of EF-1α·GTP Binding to Aminoacyl-tRNAs, Aminoacyl-viral RNA, and tRNA Shows Close Correspondence to the RNA Binding Properties of EF-Tu

Contact Info

Product

Resources

About