GTPase center of elongation factor Tu is activated by occupation of the second tRNA binding site.

Noort, Johannes M. van; Kraal, Barend; Bosch, L.

doi:10.1073/pnas.83.13.4617

Cited by 19 publications

(14 citation statements)

References 41 publications

(38 reference statements)

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“…How can the results reported in this paper be correlated with the recently proposed model (Van Noort et al, 1986) that on the ribosome the GTPase of EF-Tu may be triggered by an interaction with peptidyl-tRNA located in the P-site? In this regard, we have examined whether EF-Tu can recognize a free from an occupied P-site in poly(U)-programmed ribosomes.…”

Section: Discussionsupporting

confidence: 56%

“…Here, there are also some differences in the extent of the evoked GTPase activity, depending on whether Ac-Phe-tRNAphe or Phe-tRNAPhe or tRNA0H had been located in the ribosomal P-site. Therefore, in this condition peptidyl-tRNA or in general tRNA in the P-site may interact directly with EF-Tu, as proposed by Van Noort et al (1986). Alternatively, these effects may be indirect and mediated by the ribosome.…”

Section: Discussionmentioning

confidence: 91%

“…Recently, EF-Tu has been suggested to harbor an additional binding site for a second tRNA molecule (Van Noort et al, 1982. In this context an interaction between EF-Tu and peptidyl-tRNA on the ribosome has been proposed to be involved in the stimulation of the EF-Tu• GTPase center (Van Noort et al, 1986).…”

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confidence: 99%

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TRNA and the guanosine triphosphatase activity of elongation factor Tu

Swart¹,

Parmeggiani²

1989

Biochemistry

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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)

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Section: Discussionsupporting

confidence: 56%

Section: Discussionmentioning

confidence: 91%

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TRNA and the guanosine triphosphatase activity of elongation factor Tu

Swart¹,

Parmeggiani²

1989

Biochemistry

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“…The EF-Tu complex has two distinct binding for tRNAs: one the classical binding site for aminoacyl-tRNA, and a second binding site for peptidyl-tRNA which is induced by binding to the ribosome (49). The ribosome-bound interaction with peptidyl-tRNA activates the EF-Tu GTPase, the first step in acceptance of cognate aminoacyl-tRNA into the A site (50 (51). The structure of the frameshift-prone peptidyl-tRNA, then, by binding to the incoming EF-Tu complex could misposition the aminoacyltRNA in the A site leading to out-of-frame binding and frameshifting.…”

Section: Methodsmentioning

confidence: 99%

Special peptidyl-tRNA molecules can promote translational frameshifting without slippage.

Vimaladithan¹,

Farabaugh²

1994

Mol. Cell. Biol.

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Recently we described an unusual programmed +1 frameshift event in yeast retrotransposon Ty3.Frameshifting depends on the presence of peptidyl-tRNA " on the GCG codon in the ribosomal P site and on a translational pause stimulated by the slowly decoded AGU codon. Frameshifting occurs on the sequence GCG-AGU-U by out-of-frame binding of a valyl-tRNA to GUU without slippage of peptidyl-tRNAV&. This mechanism challenges the conventional understanding that frameshift efficiency must correlate with the ability of mRNA-bound tRNA to slip between cognate or near-cognate codons. Though frameshifting does not require slippery tRNAs, it does require special peptidyl-tRNAs. We show that overproducing a second isoacceptor whose anticodon had been changed to CGC eliminated frameshifting; peptidyl-tRNAVZ' must have a special capacity to induce +1 frameshifting in the adjacent ribosomal A site. In order to identify other special peptidyl-tRNAs, we tested the ability of each of the other 63 codons to replace GCG in the P site. We found no correlation between the ability to stimulate + 1 frameshifting and the ability of the cognate tRNA to slip on the mRNA-several codons predicted to slip efficiently do not stimulate frameshifting, while several predicted not to slip do stimulate frameshifting. By inducing a severe translational pause, we identified eight tRNAs capable of inducing measurable +1 frameshifting, only four of which are predicted to slip on the mRNA. We conclude that in Saccharomyces cerevisiae, special peptidyl-tRNAs can induce frameshifting dependent on some characteristic(s) other than the ability to slip on the mRNA.Protein synthesis produces a low frequency of errors: both missense errors and processivity errors (those leading to premature termination). However, spontaneous frameshift errors are extremely infrequent (34). This means that the translational machinery must much more efficiently avoid changes in frame than it does other errors. Some genes have evolved sequences which manipulate this machinery to allow very high levels of frameshifting, with efficiencies from a few to nearly 100% (2, 22, 28). All such sequences, termed programmed frameshift sites, consist of two elements: the actual site at which the frame shifts, termed the recoding site (22), and sequences which increase the probability that the ribosome will slip, termed stimulators (2).In all but one case, the recoding site is a sequence which allows slippage of one or more tRNAs between cognate or near-cognate codons and thus is termed a slippery site. Frameshifts occur in either the upstream, or leftward, direction (e.g., -1 frameshift) or in the downstream, or rightward direction (e.g., +1 frameshifting). In -1 simultaneous slippage frameshifting, common in retroviruses but found in other viruses and one chromosomal gene, the slippery site is a heptamer of the form X-XXY-YYZ. Frameshifting occurs by the simultaneous slippage of two tRNAs from XXY-YYZ to 46). Mutational changes which decrease the likelihood of the slip tend to decrease or elimi...

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“…All preparations of EF-Tu were homogeneous and intact as judged by SDS polyacrylamide gel electrophoresis and all were active for at least 90% as judged by the GDP exchange assay via nitrocellulose membrane filtration. The aminoacylation of bulk tRNA and complex formation with EF-Tu were according to (14), with the exception that the aminoacylated tRNA was not dialysed against reaction buffer, but was dissolved in 2 mM potassium acetate pH 5. Aminoacylated tRNA was stored at -70°C.…”

Section: Methodsmentioning

confidence: 99%

Isolation and stability of ternary complexes of elongation factor Tu, GTP and aminoacyl-tRNA

Abrahams¹,

Kraal²,

Clark³

et al. 1991

Nucl Acids Res

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Intact, native EF-Tu, isolated using previously described methods and fully active in binding GTP, was never found to be fully active in binding aminoacyl-tRNA as judged by high performance liquid chromatography (HPLC) gel filtration and zone-interference gel-electrophoresis. In the presence of kirromycin, however, all these EF-Tu.GTP molecules bind aminoacyl-tRNA, although with a drastically reduced affinity. For the first time, the purification of milligram quantities of ternary complexes of EF-Tu.GTP and aminoacyl-tRNA, free of deacylated tRNA and inactive EF-Tu, has become possible using HPLC gel filtration. We also describe an alternative new method for the isolation of the ternary complexes by means of fractional extraction in the presence of polyethylene glycol. In the latter procedure, the solubility characteristics of the ternary complexes are highly reminiscent to those of free tRNA. Concentrated samples of EF-Tu.GMPPNP.aminoacyl-tRNA complexes show a high stability.

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GTPase center of elongation factor Tu is activated by occupation of the second tRNA binding site.

Cited by 19 publications

References 41 publications

TRNA and the guanosine triphosphatase activity of elongation factor Tu

TRNA and the guanosine triphosphatase activity of elongation factor Tu

Special peptidyl-tRNA molecules can promote translational frameshifting without slippage.

Isolation and stability of ternary complexes of elongation factor Tu, GTP and aminoacyl-tRNA

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