Studies on the catalytic rate constant of ribosomal peptidyltransferase

Synetos, Dennis; Coutsogeorgopoulos, C.

doi:10.1016/0304-4165(87)90014-6

Cited by 41 publications

(41 citation statements)

References 25 publications

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“…2). The rate of the faster reaction component agrees with previous literature values (19,47,48). The origin of the slow reacting component has yet to be delineated.…”

Section: Surface-immobilized Ribosomes Are Highly Active In Peptide Bondsupporting

confidence: 89%

tRNA dynamics on the ribosome during translation

Blanchard

Kim

Gonzalez

et al. 2004

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

449

575

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Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by Ϸ6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance energy transfer was also used to observe aa-tRNA accommodation coupled with elongation factor G-mediated translocation. Dynamic rearrangements in tRNA configuration are also observed subsequent to the translocation reaction. This work underscores the importance of dynamics in ribosome function and demonstrates single-particle enzymology in a system of more than two components. P rotein synthesis, catalyzed by the ribosome, is rapid, processive, and highly regulated. In bacteria, two RNA-protein subunits consisting of a small 30S subunit and a large 50S subunit assemble around a mRNA template into a roughly spherical 70S particle that translates the nucleotide sequence into a polypeptide chain through repetitive, codon-dependent binding of aminoacylated tRNA.The landmark structures of the 30S (1, 2), 50S (3, 4), and 70S (5) ribosomal particles provide a molecular basis for understanding ribosome function. tRNA molecules bind to the ribosome in a solvent-accessible channel at the subunit interface. Three binding sites for tRNA, called the aminoacyl site (A site), peptidyl site (P site), and exit site (E site), have been identified on both the large and small subunit (Fig. 1). Each tRNA is separated from its neighbor at the elbow region (the site of Ϸ90°b ending) by 25-45 Å (5, 6). The anticodon stem loops of A-and P-site tRNA form Watson-Crick base pairs with adjacent mRNA codons on the 30S subunit (5, 7), whereas the 3Ј CCA terminal residues of A-and P-site tRNAs base-pair with conserved ribosomal RNA (rRNA) loops (8-10) within the 50S subunit peptidyltransferase center, the site of peptide bond formation (11). Additional interactions with rRNA and ribosomal proteins position tRNA molecules on the ribosome (5).The essential components and principal steps of translation have been delineated through genetic, biochemical, and kinetic methods (12). The process of polypeptide elongation has been most extensively characterized (13). Binding of aminoacyl-tRNA (aa-tRNA) to the A site occurs as a ''ternary complex'' with the GTPase elongation ...

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“…2). The rate of the faster reaction component agrees with previous literature values (19,47,48). The origin of the slow reacting component has yet to be delineated.…”

Section: Surface-immobilized Ribosomes Are Highly Active In Peptide Bondsupporting

confidence: 89%

tRNA dynamics on the ribosome during translation

Blanchard

Kim

Gonzalez

et al. 2004

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

449

575

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“…Among the several species formed during complex C preparation, only poly(U)-programmed 70 S ribosome complexes with AcPhe-tRNA bound at the P-site could react with puromycin. The radioactive noise of other species, except for complex C, was corrected by dividing the x values by the extent of puromycin reaction (23), before fitting these values into the integrated rate law. Thus, complex C was normalized to the same baseline at which 100% of the bound AcPhe-tRNA reacts quantitatively with puromycin.…”

Section: Methodsmentioning

confidence: 99%

Polyamines Affect Diversely the Antibiotic Potency

Πετρόπουλος

Xaplanteri

Dinos

et al. 2004

Journal of Biological Chemistry

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The effects of spermine on peptidyltransferase inhibition by an aminohexosylcytosine nucleoside, blasticidin S, and by a macrolide, spiramycin, were investigated in a model system derived from Escherichia coli, in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes. Kinetics revealed that blasticidin S, after a transient phase of interference with the A-site, is slowly accommodated near to the P-site so that peptide bond is still formed but with a lower catalytic rate constant. At high concentrations of blasticidin S (>10 ؋ K i ), a second drug molecule binds to a weaker binding site on ribosomes, and this may account for the onset of a subsequent mixed-noncompetitive inhibition phase. Spermine enhances the blasticidin S inhibitory effect by facilitating the drug accommodation to both sites. On the other hand, spiramycin (A) was found competing with puromycin for the A-site of AcPhe-tRNA⅐poly(U)⅐70 S ribosomal complex (C) via a two-step mechanism, according to which the fast formation of the encounter complex CA is followed by a slow isomerization to a tighter complex, termed C*A. In contrast to that observed with blasticidin S, spermine reduced spiramycin potency by decreasing the formation and stability of complex C*A. Polyamine effects on drug binding were more pronounced when a mixture of spermine and spermidine was used, instead of spermine alone. Our kinetic results correlate well with cross-linking and crystallographic data and suggest that polyamines bound at the vicinity of the antibiotic binding pockets modulate diversely the interaction of these drugs with ribosomes.Blasticidin S and spiramycin are representatives of two distinct antibiotic families, the aminohexosylcytosine nucleosides and the 16-membered lactone ring macrolides, respectively, both of which have been proposed to inhibit protein synthesis by binding to the large ribosomal subunit. Blasticidin S consists of a cytosine bonded to a pyranose ring, to which an amino acid-like substituent is attached (see Fig. 1). Therefore, it is not surprising that blasticidin S and puromycin have been considered as iso-structural, both with one another and with the 3Ј-end of aminoacyl-tRNA (1, 2). Consistently, blasticidin S has been found to inhibit the binding of CACCA (Phe) to the A-site of ribosomes (3) and to compete with designated A-site inhibitors (4). In addition, the inhibition of peptidyl-or AcPhe 1 -puromycin synthesis by this antibiotic shows a competitive phase in concert with noncompetitive or mixed-noncompetitive phases (5, 6), a fact supporting the notion that blasticidin S influences, at least transiently, the affinity of the A-site. Further support derives from chemical probing studies in Escherichia coli ribosomes (7), suggesting that blasticidin S protects A2439, one of the three nucleosides in domain V of 23 S rRNA, which show altered chemical reactivity on removal of the aminoacyl group from A-site-bound tRNAs (8). Moreover, A2439 is one of the preferable cross-linking...

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“…Cells from wild-type or mutant strains of S. cerevisiae were grown to OD 600 ϭ 0+9 in YPD+ S30 extracts were prepared as described (Hussain & Leibowitz, 1986;Synetos & Coutsogeorgopoulos, 1987;Leibowitz et al+, 1991) with some modifications+ These include the exposure of the S30 extracts to 0+1 mM of the antibiotic puromycin in the presence of 0+1 mM GTP at 30 8C for 20 min to release the nascent polypeptide chains+ The puromycin-treated crude S30 extract was then applied to a Sephadex G-10 column+ Excluded fractions with the highest A 260 were pooled and processed+ They contained about 50 A 260 ribosomes/mL and 6-10 mg protein/mL+…”

Section: Preparation Of a Yeast Cell-free System For Translation In Vmentioning

confidence: 99%

“…Translation of poly(U) templates was carried out as described recently (Synetos et al+, 1996) cpm/A 260 unit), essentially as described previously for the preparation of [ 3 H]Phe-tRNA from E. coli (Synetos & Coutsogeorgopoulos, 1987)+ Reaction mixtures were incubated at 30 8C for the appropriate time intervals and the reaction was stopped by adding an equal volume of 1 N KOH+ Binding of tRNA to the P-and A-sites of the ribosome The P-site binding was carried out in 0+1 mL of a binding buffer (80 mM Tris-HCl, pH 7+4, 160 mM NH 4 Cl, 11 mM Mg(CH 3 COO) 2 , 6 mM b-mercaptoethanol, 0+4 mM GTP, 2 mM spermidine) containing 2+5 A 260 units of ribosomes, 40 mg poly(U), and 1+3 A 260 units of yeast Ac-[…”

Section: Translation Of Poly(u) Templates In Vitromentioning

confidence: 99%

“…Phe to bind to the P-site of the ribosome and form Ac-Phe-puromycin product in a titration assay (data not shown)+ Thus, the occupation of the P-sites with tRNA Phe was performed in 0+1 mL of binding buffer containing 15 mM Mg(CH 3 COO) 2 by adding 40 mg poly(U), 2+5 A 260 units of ribosomes, 0+1 mg of soluble protein factors, and tRNA Phe at a molar ratio of 4:1 to ribosomes+ The reaction mixtures were incubated at 30 8C for 30 min to fill all P-sites, after which 1+3 A 260 units of yeast (Synetos & Coutsogeorgopoulos, 1987)+ By using this method, Ac-[ 3 H]Phe-tRNA Phe was charged with 14+9 pmol of [ 3 H]Phe (170,000 cpm total)/A 260 unit+ The puromycin reaction in solution was carried out essentially as described previously for E. coli ribosomes (Coutsogeorgopoulos et al+, 1975)+…”

Section: Translation Of Poly(u) Templates In Vitromentioning

confidence: 99%

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Mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA affect the function of the decoding center of the ribosome

Velichutina

Dresios

Hong³

et al. 2000

RNA

Self Cite

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A dynamic structural rearrangement in the phylogenetically conserved helix 27 of Escherichia coli 16S rRNA has been proposed to directly affect the accuracy of translational decoding by switching between "accurate" and "error-prone" conformations. To examine the function of helix 27 in eukaryotes, random and site-specific mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA have been characterized. Mutations at positions of yeast 18S rRNA corresponding to E. coli 886 (rdn8), 888 (rdn6 ), and 912 (rdn4 ) increased translational accuracy in vivo and in vitro, and caused a reduction in tRNA binding to the A-site of mutant ribosomes. The double rdn4rdn6 mutation separated the killing and stop-codon readthrough effects of the aminoglycoside antibiotic, paromomycin, implicating a direct involvement of yeast helix 27 in accurate recognition of codons by tRNA or release factor eRF1. Although our data in yeast does not support a conformational switch model analogous to that proposed for helix 27 of E. coli 16S rRNA, it strongly suggests a functional conservation of this region in tRNA selection.

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Studies on the catalytic rate constant of ribosomal peptidyltransferase

Cited by 41 publications

References 25 publications

tRNA dynamics on the ribosome during translation

tRNA dynamics on the ribosome during translation

Polyamines Affect Diversely the Antibiotic Potency

Mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA affect the function of the decoding center of the ribosome

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