Speed-accuracy relationships during in vitro and in vivo protein biosynthesis

Laughrea, Michael

doi:10.1016/s0300-9084(81)80189-7

Cited by 35 publications

(18 citation statements)

References 193 publications

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“…Unfortunately, in only one case have both these effects been shown to be due to a single mutation (23) and there are many "accuracy" mutants that do not show any change in chain elongation rate. In addition, in vitro experiments have generally shown a correlation between speed and accuracy that is the opposite of that predicted by the kinetic theory (24), and so the concern of most investigations has been with structural problems of distinguishing AA-tRNAs on the basis of the anticodons (3,4,(25)(26)(27). The experiments reported here provide unambiguous evidence that kinetic rather than structural factors limit the accuracy of AA-tRNA selection when it operates at a speed close to those observed in vivo.…”

mentioning

confidence: 90%

The accuracy of protein biosynthesis is limited by its speed: high fidelity selection by ribosomes of aminoacyl-tRNA ternary complexes containing GTP[gamma S]

Thompson¹,

Karim²

1982

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

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Guanosine 5'-[y-thioltriphosphate (GTP[yS])forms a stable ternary complex with polypeptide chain elongation factor Tu (EF-Tu) and aminoacyl-tRNA, and this complex binds rapidly and tightly to a properly programed ribosome. However, the rate constant for the subsequent hydrolysis of the f-y pyrophosphate bond (3.9 x 10-3 sl at 50C) is less than 1/2,500th of that for the analogous reaction of GTP. We have taken advantage ofthis low rate to determine the rate constant for dissociation of the complex of poly(U)-programed ribosomes, EF-Tu, PhetRNAPhe, and GTP['yS] (2.7 x 10-3 s-') and the second-order rate constant for formation ofthis complex (3.3 x 106 M-' s-1). Therefore, the Kd of the complex may be calculated to be 8.2 x 10-10M. An analogous near-cognate complex-with Leu-tRNAI" in place of Phe-tRNA~Ie has been determined by equilibrium methods to have a Kd >1.7 x 10-6 M. These results indicate that under equilibrium conditions the ribosome can distinguish cognate and nearcognate ternary complexes with great accuracy. Therefore, its failure to showthis high specificitywith the physiological ternary complexes containing GTP isdue to the speed of GTP hydrolysis being similar to the speed of dissociation of the near-cognate complex. The low specificity of the physiological reaction is corrected by subsequent proofreading. The results reported here suggest that proofreading is necessary not simply for high accuracy but for the combination ofspeed and accuracy required in protein biosynthesis.The selection of an aminoacyl-tRNA (AA-tRNA) by a mRNAprogramed ribosome is one ofthe key reactions determining the fidelity of protein biosynthesis which, by current estimates (1, 2), exceeds 99.95%. The mechanism of this selection process has attracted a good deal ofinterest since Hopfield (3) proposed that it exemplified a form ofsubstrate selection known as kinetic proofreading. The selection is thought to occur according to the following scheme: the mRNA-programed ribosome binds a ternary complex ofEFTu, GTP, and the AA-tRNA with rate constant ki, and it either dissociates this ternary complex with rate constant k-1 or hydrolyzes its GTP with rate constant k2. Second, the products EF-Tu-GDP and Pi dissociate from the ribosome, and the AAtRNA may either enter the ribosomal A (acceptor) site with rate constant k3 or dissociate from the ribosome with rate constant k4, depending on the strength of the codon-anticodon interaction (4-6). We have shown previously (6) that errors are made quite frequently in the first recognition step but that they are usually corrected by the second recognition step, which is therefore known as proofreading (3). This paper addresses the question ofwhy the first recognition step-selection and hydrolysis of a ternary complex-is insufficiently accurate to achieve the required fidelity by itself and hence requires subsequent proofreading. Failure to obtain high specificity in the GTPase reaction has generally been attributed to there being only a small difference in free energy between the cog...

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mentioning

confidence: 90%

The accuracy of protein biosynthesis is limited by its speed: high fidelity selection by ribosomes of aminoacyl-tRNA ternary complexes containing GTP[gamma S]

Thompson¹,

Karim²

1982

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

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“…In contrast, HM, a monosubstituted 2-deoxystreptamine-containing aminoglycoside antibiotic, is a poor misreader (6) and preferentially inhibited chain elongation. These results are the inverse of the relationship between misreading and elongation inhibition found in E. coli (9,19).…”

Section: Discussionmentioning

confidence: 79%

Mechanisms of action of aminoglycoside antibiotics in eucaryotic protein synthesis

Eustice¹,

Wilhelm²

1984

Antimicrob Agents Chemother

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Tetrahymena thermophila is a eucaryotic organism that is highly susceptible to growth inhibition by aminoglycoside antibiotics. Concentrations of paromomycin, gentamicin G418, and hygromycin B at 22, 10, and 17 ,uM, respectively, inhibited growth by 50%. A combination of in vitro and in vivo methods was used to determine the mechanisms of action of these aminoglycoside antibiotics on protein synthesis in T. thermophila. Analysis of polysome profiles from paromomycin-and gentamicin G418-treated cells showed clear, progressive depletions of polysomes concomitant with an inhibition of in vivo [14C]lysine incorporation. In vitro, paromomycin and gentamicin G418, which are disubstituted 2-deoxystreptamine-containing molecules, were not very effective inhibitors of either the translocation of peptidyl-tRNA or the elongation of nascent polypeptide chains on polysomes. In contrast, we found that the translocation of phe-tRNA on polyuridylate programmed ribosomes was susceptible to inhibition by paromomycin. We conclude that the primary inhibitory action of paromomycin and gentamicin G418 was at (i) an early stage of elongation after initiation, (ii) the initiation stage of translation, or (iii) a stage of translation before initiation. Hygromycin B, which is a monosubstituted 2-deoxystreptamine-containing aminoglycoside, potently inhibited the elongation of nascent chains during the translation of polysomes. In addition, the in vitro translation of polysomes from two hygromycin B-resistant mutants was resistant to the inhibition of elongation caused by hygromycin B.Aminoglycoside antibiotics are well-established inhibitors of growth, stimulators of misreading, and inhibitors of protein synthesis in procaryotes and eucaryotes (18). Vazquez and co-workers have studied the in vitro activities of many aminoglycoside antibiotics in eucaryotic model systems derived from yeasts and mammalian cells (20 and references therein). Of the aminoglycoside antibiotics tested, those active in inhibiting protein synthesis were usually found to inhibit the polypeptide chain elongation phase (20). Model systems to study the interaction of antibiotics with the ribosome at separate stages of the elongation cycle have proven to be powerful tools in the elucidation of antibiotic action on ribosomes. Furthermore, these studies have greatly enhanced the understanding of the structure-function relationships of ribosomes (8,15).Eucaryotic protein synthesis has not, however, received as much attention as have procaryotic systems, and the majority of current concepts about the structure of eucaryotic ribosomes have been derived by analogy with the extensive information available from Escherichia coli ribosomes. Model systems derived from different eucaryotic sources do not have uniform responses to a particular aminoglycoside, as is exemplified by the failure of paromomycin (PM) to inhibit the cytoplasmic protein synthesis of yeasts (14), mammalian cells (21), and wheat germ (22). In contrast, cytoplasmic protein synthesis in the lower eucaryote Tet...

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“…Elongation rates are higher in E.coli and yeast and vary from 12 to 18 residues/s at 37 o C [65,69]. The maximum elongation rate observed is 20.4 residues/s for the E.coli EF-Tu polypeptide chain [ 66 ].…”

Section: Protein Folding At the Ribosomementioning

confidence: 99%

“…Taking into account this value, it can be supposed that the duration of the ribosome elongation cycle is no shorter than 0.01s on the time scale. [64,[68][69][70]. Thus, it should be outlined from the above data that the residues of a synthesized polypeptide are imported into the cytosol from the ribosome in a definite way: sequentially with the N-terminal residue and gradually with the periodicity of no less than 10 -2 s on the time scale.…”

Section: Protein Folding At the Ribosomementioning

confidence: 99%

Protein folding

Basharov

2003

J Cellular Molecular Medi

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The problem of protein folding is that how proteins acquire their native unique three-dimensional structure in the physiological milieu. To solve the problem, the following key questions should be answered: do proteins fold co-or post-translationally, i.e. during or after biosynthesis, what is the mechanism of protein folding, and what is the explanation for fast folding of proteins? The two first questions are discussed in the current review. The general lines are to show that the opinion, that proteins fold after they are synthesized is hardly substantiated and suitable for solving the problem of protein folding and why proteins should fold cotranslationally. A possible tentative model for the mechanism of protein folding is also suggested. To this end, a thorough analysis is made of the biosynthesis, delivery to the folding compartments, and the rates of the biosynthesis, translocation and folding of proteins. A cursory attention is assigned to the role of GroEL/ES-like chaperonins in protein folding.

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Speed-accuracy relationships during in vitro and in vivo protein biosynthesis

Cited by 35 publications

References 193 publications

The accuracy of protein biosynthesis is limited by its speed: high fidelity selection by ribosomes of aminoacyl-tRNA ternary complexes containing GTP[gamma S]

The accuracy of protein biosynthesis is limited by its speed: high fidelity selection by ribosomes of aminoacyl-tRNA ternary complexes containing GTP[gamma S]

Mechanisms of action of aminoglycoside antibiotics in eucaryotic protein synthesis

Protein folding

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