The Role of an Aminoacyl-tRNA-GTP-Protein Complex in Polypeptide Synthesis

Ravel, Joanne M.; Shorey, RoseAnn L.; Garner, Charles W.; Dawkins, Ross C.; Shive, William

doi:10.1101/sqb.1969.034.01.039

Cited by 32 publications

(14 citation statements)

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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: 55%

“…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: 81%

“…For determination of radioactivity insoluble in hot trichloroacetic acid, the samples were treated with 3 ml of 5% acid, heated to 900 for 15 min, cooled, and filtered through Millipore filters. The filters were thoroughly washed with 5% trichloroacetic acid, dried, and counted in Omnifluor as above.…”

Section: Methodsmentioning

confidence: 99%

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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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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: 55%

Section: Lack Of Gtp Requirementmentioning

confidence: 81%

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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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“…Elongation factor Tu (EF-Tu) delivers the charged tRNA to the A-site of the ribosome in a ternary complex with GTP and an aminoacylated tRNA, hydrolyzing the GTP to GDP in the process (14,39). Elongation factor Ts (EF-Ts) then interacts with EF-Tu to regenerate EF-Tu to an active form, facilitating the replacement of bound GDP with GTP (50).…”

mentioning

confidence: 99%

Discovery and Analysis of 4 H -Pyridopyrimidines, a Class of Selective Bacterial Protein Synthesis Inhibitors

Ribble

Hill

Ochsner

et al. 2010

Antimicrob Agents Chemother

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Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation protein synthesis system composed of phenyltRNA synthetases, ribosomes, and ribosomal factors from Escherichia coli. This system, utilizing purified components, has been used for high-throughput screening of a small-molecule chemical library. We have identified a series of compounds that inhibit protein synthesis with 50% inhibitory concentrations (IC 50 s) ranging from 3 to 14 M. This series of compounds all contained the same central scaffold composed of tetrahydropyrido[4,3-d]pyrimidin-4-ol (e.g., 4H-pyridopyrimidine). All analogs contained an ortho pyridine ring attached to the central scaffold in the 2 position and either a five-or a six-member ring tethered to the 6-methylene nitrogen atom of the central scaffold. These compounds inhibited the growth of E. coli, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, with MICs ranging from 0.25 to 32 g/ml. Macromolecular synthesis (MMS) assays with E. coli and S. aureus confirmed that antibacterial activity resulted from specific inhibition of protein synthesis. Assays were developed for the steps performed by each component of the system in order to ascertain the target of the compounds, and the ribosome was found to be the site of inhibition.

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“…Elongation factor Ts (EF-Ts) and EF-Tu are interacting proteins involved in extending the nascent polypeptide chain during the elongation stage of bacterial translation (18). During polypeptide chain synthesis, the complex of GTP and EF-Tu (EF-Tu‫ء‬GTP) triggers the binding of an aminoacyl-tRNA to the acceptor site of the ribosome (3,15). EF-Ts subsequently binds to EF-Tu, which promotes the release of GDP from EF-Tu after GTP hydrolysis, resulting in regeneration of the active form of EF-Tu (10).…”

mentioning

confidence: 99%