Stepwise synthesis of a tripeptide.

The supernatant initiation factor from Artemia salina embryos promotes, besides the AUGdependent binding of fMet-tRNAf, the poly(U)-dependent binding of N-acetylPhe-tRNA to 40S ribosomal subunits; the bound N-acylaminoacyl-tRNA reacts directly with puromycin upon addition of 60S subunits. Both the binding reaction and the synthesis of N-acylaminoacyl-puromycin occur in the absence of GTP or other ribonucleoside triphosphates. To a smaller extent, the factor also mediates the 40S ribosomal binding of Met-tRNAf and PhetRNA; in this case, the bound aminoacyl-tRNA is less reactive with puromycin. After the poly(U)-and supernatant factor-dependent binding of N-acetylPhe-tRNA to 40S subunits at low Mg2+ concentration, binding of a second aminoacyl-tRNA (Phe-tRNA), with ensuing formation of the first peptide bond, is dependent upon the addition of the 60S subunit, elongation factor EF-1, and GTP. Further growth of the polypeptide chain requires translocation and is, therefore, dependent upon the addition of elongation factor EF-2. As with the Escherichia coli system, once requirements for translation of the third codon have been met, no further additions are necessary for elongation of a peptide chain.In previous papers (1, 2), we reported on an initiation factor from postribosomal supernatants of embryos of the brine shrimp, Artemia salina, and its relation to factors from ratliver supernatant (3-6) and rabbit-reticulocyte ribosomal washes (7) described by other investigators. The Artemia factor and the Escherichia coli initiation factor IF-2 are not interchangeable (2) but, as previously noted (1), the reaction promoted by Artemia factor, namely the AUG-dependent binding of fMet-tRNAf to eukaryotic 40S ribosomal subunits, shares certain properties with the analogous reaction catalyzed by IF-2 in bacterial systems: (a) it occurs on the small ribosomal subunit, (b) it is sensitive to edeine and aurintricarboxylic acid, and (c) the bound fMet-tRNAf is converted directly to fMet-puromycin upon addition of the large subunit. However, the eukaryotic reaction is GTPindependent.In this paper, we report that, in further analogy to IF-2 (8), the Artemia factor also promotes the poly(U)-dependent binding of N-acetylPhe-tRNA to 40S subunits, and the bound N-acylaminoacyl-tRNA is directly converted to N-acetylPhe-puromycin after addition of 60S subunits. Furthermore, the Artemia factor catalyzes, at low Mg2+ concentrations, not only the reported (1, 3, 5, 6) binding of Phe-tRNA, but also that of Met-tRNAf to 40S subunits.However, the unacylated derivatives are bound to a lesser extent than the N-acylated ones, and the bound aminoacyltRNA is less reactive with puromycin. The poly(U)-directed synthesis of N-acetylPhe-(Phe)n by the Artemia system requires (a) supernatant factor for formation of the 40S initiation complex, (b) the 60S subunit, elongation factor EF-1, and GTP, for binding of the second aminoacyl-tRNA and ensuing synthesis of the first peptide bond, and (c) elongation factor EF-2 for further growth of the chain...

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“…As expected, the process is basically the same as in E. coli (12,14,15), except for the lack of GTP requirement in the initiation step.…”

Section: Lack Of Gtp Requirementsupporting

confidence: 71%

“…The reaction used was the poly(U)-directed synthesis of an oligopeptide, N-acetylPhe-(Phe),f. Similar studies have been done with bacterial systems (12)(13)(14)(15). Table 5 shows the simple, but stringent, requirements the experiment with Met-tRNAf (AUG as messenger).…”

Section: Lack Of Gtp Requirementmentioning

confidence: 89%

Polypeptide Chain Initiation and Stepwise Elongation with Artemia Ribosomes and Factors

McCroskey

Zasloff

Ochoa

1972

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

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“…It is the first time that such hydrolytic activity has been shown to be independent of a factor and that it can be directly attributed to ribosomal proteins. The necessity of GTP hydrolysis in protein synthesis has been well established (24), and increasing evidence has accumulated that EF-G (23,24,27) and EF-Tu (28)(29)(30)(31)(32) are involved when GTP is hydrolyzed.…”

Section: Discussionmentioning

confidence: 99%

ATPase and GTPase Activities Associated with a Specific 5S RNA-Protein Complex

Horne

Erdmann

1973

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

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An RNA-protein complex consisting of 5S RNA and two ribosomal proteins, B-L5 and B-L22, was isolated from Bacillus stearothermophilus ribosomes and found to be active in GTP hydrolysis. This activity was not influenced by elongation factor G. Further analysis of this complex showed that it was also able to hydrolyze ATP. Inhibition studies revealed that ATP was a noncompetitive inhibitor of GTP and that GTP was also a noncompetitive inhibitor of ATP, indicating that two different enzymatic sites were involved. Differences in pH optimum and optimal temperature also point to a twosite enzyme complex. Both enzymatic hydrolyses were inhibited by thiostrepton and fusidic acid, which are known inhibitors of protein synthesis.In order to accomplish protein synthesis, ribosomes perform an intricate pattern of enzymatic activities (for recent reviews on the structure and function of bacterial ribosomes see refs.

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“…It should be noted that any comparison between the puromycin reactions of Phe-tRNA and AcPhe-tRNA has to take into account the different rates of product formation (Phe-puromycin and AcPhe-puromycin, respectively [24,251). The initial rate of the formation of AcPhe-puromycin is larger by a factor of 3.6 than that of the formation of Phe-Puromycin (Fig.…”

Section: Ef-g Is Directly Invohed In Translocation and Is Not Only A mentioning

confidence: 99%

The Ribosomal Elongation Cycle: tRNA Binding, Translocation and tRNA Release

Rheinberger

Schilling

Nierhaus

1983

European Journal of Biochemistry

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Experiments concerning the ribosomal elongation cycle have been performed; the following results were obtained.1. A measurement of the percentage of tightly coupled ribosomes participating in a tRNA binding reaction is obtained by determining both the stoichiometry of binding and the site location. In the case of AcPhe-tRNA it is sufficient to determine the maximal binding since at moderate Mg2+ concentration ( 5 15 mM) one ribosome cannot bind more than one AcPhe-tRNA molecule, which can be present at either the A or P site (exclusion principle).2. tRNA binding to poly(U)-programmed ribosomes does not follow the predictions of simple models with rigid tRNA binding sites as postulated by the classical A/P site model. For example, 2.7 molecules of tRNAPh' are found per ribosome at a ribosome concentration of 0.33 pM and a [14C]tRNAPh' excess over ribosomes of about 40-fold, in agreement with our previous report. At a 70s ribosome concentration of 3.3 pM and a sevenfold excess of tRNAPh' a binding of 2.5 tRNA molecules/ribosome would be expected according to the binding constants reported by us, but in fact a binding of only 1.8 molecules/70S ribosome is found. These findings explain the current discrepancies concerning the number of tRNA sites on the ribosome and confirm previous reports that three tRNA molecules can be bound simultaneously to one ribosome.3. The ternary complex EF-Tu . GTP . Phe-tRNAPhe binds to the A site at 0°C even if the P site is not occupied. Only if the P site is free does the addition of EF-G provoke a translocation to the P site at 0 "C, whereas at 37 "C the Phe-tRNAPh' 'slides' from the A to the P site without EF-G. This EF-G-independent sliding is not observed if the P site is filled by a deacylated tRNAPhc. Thus, EF-G is directly involved in the translocation reaction, and its role is not restricted to tRNA release.4. Kinetics of the puromycin reaction with Phe-tRNA and AcPhe-tRNA, respectively, were compared. The initial rate of AcPhe-puromycin formation exceeds that of Phe-puromycin formation by a factor of 3.6 (0 "C, 15 mM Mg2+).5. Poly(U)-programmed ribosomes carrying only [14C]tRNA release tRNA upon addition of EF-G. tRNA release is observed only if at least two tRNAPh" molecules are present per ribosome. The tRNA bound first is released with a higher probability than that bound second. Such a complex is not related to a classical pretranslocation complex carrying a peptidyl-tRNA at the A site. Therefore, when tRNA release during the course of an EF-G-induced translocation reaction is studied, care must bc taken that no significant amounts of the complex 70s . mRNA . (deacylated tRNA) are present.Our recent observation that E,ycherichiu coli ribosomes have, in addition to the classical A and P sites, a third tRNA binding site, the E site, which codon-specifically binds deacylated tRNA only [1,2], has revived the discussion of the number of tRNA binding sites. These findings, which were obtained with the standard nitrocellulose-filter technique, have been questioned by Gassen and...

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Stepwise synthesis of a tripeptide.

Cited by 117 publications

References 24 publications

Polypeptide Chain Initiation and Stepwise Elongation with Artemia Ribosomes and Factors

Polypeptide Chain Initiation and Stepwise Elongation with Artemia Ribosomes and Factors

ATPase and GTPase Activities Associated with a Specific 5S RNA-Protein Complex

The Ribosomal Elongation Cycle: tRNA Binding, Translocation and tRNA Release

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