Studies on the Peptidyl tRNA Translocase from Rat Liver

Moldave, Kivie; Galasiński, W; Rao, P. Nageswara; Siler, Jack

doi:10.1101/sqb.1969.034.01.041

Cited by 23 publications

(8 citation statements)

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“…The results thus raise the possibility that the binding site of factor G (the G site) and that of Complex II (which includes the A site) are near each other on the 50S subunit, or may even overlap. Such proximity or overlapping might make obligatory the release of factor G after translocation, before binding of Complex II (29), and the release of factor T. before binding of G. Since SOS subunits appear to be able to complemnent both the GTPase of G (4,5) and that of T. (23), it is tempting to suggest that a common, siomycin-sensitive site on this subunit might activate the GTPase of both factors. Accordingly, this site could be visualized as a region controlling both entry of acylated-tRNA into the A site mediated by Tu, and exit, mediated by G.…”

Section: Discussionmentioning

confidence: 99%

Inhibition by Siomycin and Thiostrepton of Both Aminoacyl-tRNA and Factor G Binding to Ribosomes

Ll¹,

Cabrer²,

Parmeggiani

et al. 1971

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

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Siomycin, a peptide antibiotic that interacts with the 50S ribosomal subunit and inhibits binding of factor G, is shown also to inhibit binding of aminoacyltRNA; however, it does not impair binding of fMet-tRNA and completion of the initiation complex. Moreover, unlike other inhibitors of aminoacyl-tRNA binding (tetracycline, sparsomycin, and streptogramin A), siomycin completely abolishes the GTPase activity associated with the binding of aminoacyl-tRNA catalyzed by factor Tu. A single-site interaction of siomycin appears to be responsible for its effect on both the binding of the aminoacyl-tRNA-Tu-GTP complex and that of factor G.Factor G, one of the supernatant proteins necessary for ribosomal activity in Escherichia coli, is involved in the coupling of GTP hydrolysis to polypeptide elongation. This hydrolysis is thought to provide the energy necessary for the rearrangements of tRNA, mRNA, and ribosomal subunits, in each round of peptide bond formation, that result in translocation of mRNA and peptidyl-tRNA (reviewed in ref. 1). GTP hydrolysis can also occur in the absence of mRNA and peptidyl-tRNA, the reaction requiring only factor G and ribosomes (2, 3). In recent work this reaction has been used to show that there is a binding site for factor G (the G-site) on the 50S subunit (4, 5). Our studies have further shown that a peptide antibiotic, siomycin, inactivates this site, preventing the interaction of this site with factor G. Moreover, the G-site is evidently distinct from the peptidyl transferase center on the 50S subunit (5).We have now shown that siomycin-treated ribosomes can carry out the initiation step in protein synthesis, with either viral messenger or triplet AUG, and the bound fMet-tRNA can reach the puromycin-reactive position. However, no peptide bond can be formed because the ribosomes do not bind aminoacyl-tRNA in the recognition site (the A-site). Moreover, the siomycin-treated ribosomes can complement neither the GTPase activity associated with factor T nor that associated with factor G. These effects appear to be the consequence of a single action of siomycin on the 50S subunit, thus suggesting a relationship between the binding site of factor G and that of the aminoacyl-tRNA-T.-GTP complex. MATERIALS AND METHODSRibosomes washed with ammonium chloride (1 M) and crude initiation factors from E. coli MRE600 were prepared as described (6, 7). Homogeneous initiation factor F1 and partially purified factor F2 were a gift of Dr. S. Ochoa. Elongation factors G and T (T. plus Tu) and ['y-82P]GTP were prepared as described (2, 8 Binding of aminoacyl-tRNA to ribosomes was assayed by filtration (9). Reaction mixtures for the binding of fMettRNA contained: 80 mM NH4Cl, 50 mM Tris HOl (pH 7.7), 5 mM Mg(acetate)2, 6 mM mercaptoethanol, 0.4 mM GTP, 0.3 mg/ml of crude initiation factors, 4.7 pmol f-[3H]MettRNA per A260 unit ribosomes, 1.2 mg/ml of R17-RNA, 35 A260 units/ml of ribosomes, and other additions as specified. Unless otherwise indicated, incubation was at 34°C for 10 min and was follo...

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Section: Discussionmentioning

confidence: 99%

Inhibition by Siomycin and Thiostrepton of Both Aminoacyl-tRNA and Factor G Binding to Ribosomes

Ll¹,

Cabrer²,

Parmeggiani

et al. 1971

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

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“…The obviously uncoupled reaction described above may therefore be somewhat different from the EF-2, ribosome-dependent GTPase previously reported for liver (12)(13)(14)(15) and reticulocytes (16). Experiments in progress have shown that the yeast GTPase as well as EF-2 are required for translocationdependent N-acetylphenylalanyl puromycin formation (22). Both proteins are therefore required for the translocation step and may interact simultaneously with ribosomes.…”

Section: Methodsmentioning

confidence: 99%

A ribosome-dependent GTPase from yeast distinct from elongation factor 2.

Skogerson¹,

Wakatama²

1976

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

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Three proteins required for poly(U)directed polyphenylalanine synthesis have been separated from yeast. Two of the factors correspond to the elongation factors 1 and 2 described for other eukaryotic systems, according to the criteria of phenylalanyl-tRNA binding and diphtheria toxin-. catalyzed ADP-ribosylation. The third protein, while absolutely required for polyphenylalanine synthesis, was a more active ribosome-dependent GTPase than elongation factor 2. Ribosomal functions in vitro require the participation of specific soluble proteins that are readily removed by standard isolation procedures (1). Two such proteins, the elongation factors (EF), are required for the poly(U)-directed formation of polyphenylalanine. One factor participates in codon recognition by binding the correct aminoacyl-tRNA to ribosomes while the other factor is required for translocation. Binding factor from eukaryotic cells, EF-1, is functionally very similar to that from bacteria, EF-Tu. The bacterial EF-G appears analogous to EF-2 in that both are required for translocation (2, 3).In the course of purifying elongation factors from the yeast, Saccharomyces ceretsiae, we have identified and partially purified a third protein with elongation factor activity. Our studies show that the third factor is required, in addition to EF-1 and EF-2, for poly(U)-directed polyphenylalanine synthesis. In examining possible functions for this new factor we found that it promoted the ribosome-dependent hydrolysis of GTP. On the other hand EF-2 had only a small effect on ribosome-dependent GTP hydrolysis, in either the presence or absence of the third factor. T-500, 339 ml of 30% (weight/weight) polyethylene glycol 6000, and sufficient NH4Cl to give a final concentration of 0.1 M. The suspension was stirred for 30 min and the phases were separated by centrifugation at 5000 X g for 10 mmin. The upper polyethylene glycol phase contained about 30-409k of the protein, but negligible elongation factor activity and was discarded. To each 100 ml of the lower Dextran phase was added 247 ml of buffer A, 82 ml of the 30% polyethylene glycol solution, and sufficient NH4Cl to give a final concentration of 2.0 M. The polyethylene glycol phase was recovered as before (2 M polyethylene glycol) and was treated with ammonium sulfate as previously described (4) to remove the bulk of the polyethylene glycol. To each 1000 ml of the 2 M polyethylene glycol fraction 144 g of solid ammonium sulfate was added (25% saturation). When the salt had dissolved, the phases were centrifuged at 5000 X g for 10 min and the lower phase was removed by aspiration. Proteins were precipitated from this solution by adding 267 g of ammonium sulfate per 1000 ml (65% saturation) of solution. The precipitate was collected by centrifugation, dissolved in buffer B (0.1 M potassium phosphate, pH 6.8, 10% glycerol, and 10 mM 2-mercaptoethanol), and desalted on a Sephadex G-25 column equilibrated with the same buffer.Hydroxylapatite Chromatography. Hydroxylapatite (Bio-Rad, HT) was packed in a...

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“…It was thought that a variable contamination of protein factors (Miller & Schweet, 1968;Heywood, 1970;Moldave et al, 1969;Smulson & Rideau, 1970) in preparations of crude ribosomes from the adequate-protein and lowprotein groups may account for the above findings. This possibility is supported by the observation that the enhanced protein-synthetic activity of ribosomes prepared from spleens of immunized rats is due to higher contamination of these ribosomes with bound transfer factors as compared with ribosomes obtained from control spleens (Willis & Starr, 1971).…”

Section: Resultsmentioning

confidence: 99%

“…It is known that various protein factors are associated with crude ribosome preparations, and these include the initiation (Miller & Schweet, 1968;Pritchard et al, 1970;; Heywood, 1970) and elongation (Moldave et al, 1969;Smulson & Rideau, 1970) factors. Hence, a variable concentration of these factors in crude ribosome preparations may account in part for the differences observed by Young & Alexis (1968) in Vol.…”

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confidence: 99%

Dietary protein intake and skeletal-muscle protein metabolism in rats. Studies with salt-washed ribosomes and transfer factors

Alexis

Basta

Young

1972

Biochemical Journal

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1. Aspects of skeletal muscle protein synthesis in vitro were studied in young rats given a low-protein diet for up to 10 days and during re-feeding with an adequate diet. 2. Partially purified muscle transfer factors (transferases I and II), crude and purified (NH(4)Cl-washed) ribosomes and a pH5 enzyme fraction were prepared for this purpose. 3. A marked decrease in the capacity of crude ribosomes to carry out cell-free polypeptide synthesis occurred within 4 days of feeding the low-protein diet. 4. The capacity of salt-washed ribosomes to promote amino acid polymerization, in the presence of added transfer factors and aminoacyl-tRNA, was only slightly decreased by the dietary treatment. 5. However, the capacity of salt-washed ribosomes to bind (14)C-labelled aminoacyl-tRNA was decreased by feeding the low-protein diet. 6. The capacity of the pH5 enzyme fraction to promote amino acid incorporation in a complete cell-free system was decreased within 2 days of feeding the low-protein diet. There is no evidence that the change is associated with aminoacyl-tRNA synthetase or binding enzyme activities of the pH5 fractions. 7. These changes are discussed in relation to the diminished rate of protein synthesis in the intact muscle cell when rats are given a low-protein diet.

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Studies on the Peptidyl tRNA Translocase from Rat Liver

Cited by 23 publications

References 0 publications

Inhibition by Siomycin and Thiostrepton of Both Aminoacyl-tRNA and Factor G Binding to Ribosomes

Inhibition by Siomycin and Thiostrepton of Both Aminoacyl-tRNA and Factor G Binding to Ribosomes

A ribosome-dependent GTPase from yeast distinct from elongation factor 2.

Dietary protein intake and skeletal-muscle protein metabolism in rats. Studies with salt-washed ribosomes and transfer factors

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