Mutant species of EF‐Tu, altered at position 375, exhibit a reduced affinity for aminoacylated transfer‐RNAs

Sam, Tom; Pingoud, Alfred; Bosch, L.

doi:10.1016/0014-5793(85)80739-0

Cited by 9 publications

(6 citation statements)

References 17 publications

(12 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It can only be said that the functional changes observed in the three mutated factors isolated until now are probably all the results of long-range effects. This interpretation is supported by the finding that the two kirromycin-resistant EF-Tu mutants EF-TuAla375-*vai and EF-TuAla.375-Thr reveal specific changes in the interaction with GTP and aa-tRNA as well as in their basic GTPase activity independent of aa-tRNA, ribosomes, and kirromycin (intrinsic GTPase activity; Ivell et al, 1981;Swart et al, 1982;Sam et al, 1985).…”

Section: Discussionmentioning

confidence: 81%

Effects of the mutation Glycine-222 .fwdarw. Aspartic acid on the functions of elongation factor Tu

Swart¹,

Parmeggiani²,

Kraal³

et al. 1987

Biochemistry

Self Cite

View full text Add to dashboard Cite

We have studied the properties of a mutant elongation factor Tu, encoded by tufB (EF-TuBo), in which Gly-222 is replaced by Asp. For its purification from the kirromycin-resistant EF-Tu encoded by tufA (EF-TuAr), a method was developed by exploiting the different affinities to kirromycin of the two factors and the competition between kirromycin and elongation factor Ts (EF-Ts) for binding to EF-Tu. The resulting EF-TuBo kirromycin and EF-TuAr EF-Ts complexes are separated by chromatography on diethylaminoethyl-Sephadex A-50. For the first time we have succeeded in obtaining a tufB product in homogeneous form. Compared with wild-type EF-Tu, EF-TuBo displays essentially the same affinity for GDP and GTP, with only the dissociation rate of EF-Tu GTP being slightly faster. Protection of amino-acyl-tRNA (aa-tRNA) against nonenzymatic deacylation by different EF-Tu species indicates that conformational alterations occur in the ternary complex EF-TuBo GTP aa-tRNA. However, the most dramatic modification is found in the EF-TuBo interaction with the ribosome. Its activity in poly(Phe) synthesis as well as in the GTPase activity associated with the interaction of its ternary complex with the ribosome mRNA complex requires higher Mg2+ concentrations than wild-type EF-Tu (Mg2+ optimum at 10-14 vs. 6 mM), even if EF-TuBo can sustain enzymatic binding of aa-tRNA to ribosomes at low Mg2+. The anomalous behavior of EF-TuBo is reflected in a remarkable increase of the fidelity in poly(Phe) synthesis, especially at high Mg2+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

Section: Discussionmentioning

confidence: 81%

Effects of the mutation Glycine-222 .fwdarw. Aspartic acid on the functions of elongation factor Tu

Swart¹,

Parmeggiani²,

Kraal³

et al. 1987

Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The location of A375T in the cleft between domains I and III of EF-Tu suggests that it could do so by influencing the relative movement of these domains that occurs upon GTP hydrolysis (Berchtold et al 1993). Interestingly, A375T or other substitutions in the cleft lower the binding affinity of EF-Tu for aminoacyl-tRNA (Sam et al 1985;Vorstenbosch et al 2000), perhaps reflecting an enhanced ability to release aminoacyl-tRNA into the A site after GTP hydrolysis.…”

Section: Suppression Of Streptomycin-resistance Phenotypes Of R37c Anmentioning

confidence: 99%

A signal relay between ribosomal protein S12 and elongation factor EF-Tu during decoding of mRNA

Gregory¹,

Carr²,

Dahĺberg³

2008

RNA

View full text Add to dashboard Cite

Codon recognition by aminoacyl-tRNA on the ribosome triggers a process leading to GTP hydrolysis by elongation factor Tu (EF-Tu) and release of aminoacyl-tRNA into the A site of the ribosome. The nature of this signal is largely unknown. Here, we present genetic evidence that a specific set of direct interactions between ribosomal protein S12 and aminoacyl-tRNA, together with contacts between S12 and 16S rRNA, provide a pathway for the signaling of codon recognition to EF-Tu. Three novel amino acid substitutions, H76R, R37C, and K53E in Thermus thermophilus ribosomal protein S12, confer resistance to streptomycin. The streptomycin-resistance phenotypes of H76R, R37C, and K53E are all abolished by the mutation A375T in EF-Tu. A375T confers resistance to kirromycin, an antibiotic freezing EF-Tu in a GTPase activated state. H76 contacts aminoacyl-tRNA in ternary complex with EF-Tu and GTP, while R37 and K53 are involved in the conformational transition of the 30S subunit occurring upon codon recognition. We propose that codon recognition and domain closure of the 30S subunit are signaled through aminoacyl-tRNA to EF-Tu via these S12 residues.

show abstract

“…Kirromycin-resistant factors have a higher intrinsic GTPase activity (Fasano & Parmeggiani, 1981;Swart et al, 1982), and GTP hydrolysis is more readily stimulated by ribosomes (Figure 3C). Since Phe-tRNAPhe is not added in saturating amounts and the kirromycin-resistant EF-TuAla.375_Thr has lower affinity for aa-tRNA than the wild-type factor (Sam et al, 1985), slightly less GTP is hydrolyzed when the ternary complex is added (Figure 5C). As expected, the GTPase activity of EF-TuGiy.222-Asp is hardly stimulated by ribosomes (Figure 3B; Swart et al, 1987); nevertheless, its ternary complex can somewhat distinguish the different ribosomal complexes (Figure 5B).…”

Section: Resultsmentioning

confidence: 99%

TRNA and the guanosine triphosphatase activity of elongation factor Tu

Swart¹,

Parmeggiani²

1989

Biochemistry

View full text Add to dashboard Cite

Different sites of the tRNA molecule influence the activity of the elongation factor Tu (EF-Tu) center for GTP hydrolysis [Parlato, G., Pizzano, R., Picone, D., Guesnet, J., Fasano, O., & Parmeggiani, A. (1983) J. Biol. Chem. 258, 995-1000]. Continuing these studies, we have investigated some aspects of (a) the effect of different tRNA(Phe) species, including Ac-Phe-tRNA(Phe) and 3'-truncated tRNA-CCA in the presence and absence of codon-anticodon interaction, and (b) the effect of occupation of the ribosomal P-site by different tRNA(Phe) species. Surprisingly, we have found that 3'-truncated tRNA can enhance the GTPase activity in the presence of poly(U), in contrast to its inhibitory effect in the absence of codon-anticodon interaction. Moreover, Ac-Phe-tRNA(Phe) was found to have some stimulatory effect on the ribosome EF-Tu GTPase in the presence of poly(U). These results indicate that under specific conditions the 3'-terminal end and a free terminal alpha-NH2 group are not essential for the stimulation of the catalytic center of EF-Tu; therefore, the same structure of the tRNA molecule can act as a stimulator or an inhibitor of EF-Tu functions, depending on the presence of codon-anticodon interaction and on the concentration of monovalent and divalent cations. EF-Tu-GTP does not recognize a free ribosomal P-site from a P-site occupied by the different tRNA(Phe) species. When EF-Tu acts as a component of the ternary complex formed with GTP and aa-tRNA, the presence of tRNA in the P-site strongly increases the GTPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

Mutant species of EF‐Tu, altered at position 375, exhibit a reduced affinity for aminoacylated transfer‐RNAs

Cited by 9 publications

References 17 publications

Effects of the mutation Glycine-222 .fwdarw. Aspartic acid on the functions of elongation factor Tu

Effects of the mutation Glycine-222 .fwdarw. Aspartic acid on the functions of elongation factor Tu

A signal relay between ribosomal protein S12 and elongation factor EF-Tu during decoding of mRNA

TRNA and the guanosine triphosphatase activity of elongation factor Tu

Contact Info

Product

Resources

About