Photo‐Induced Crosslinking between Phenylalanine Transfer RNA and Messenger RNA on the <i>Escherichia coli</i> Ribosome

Matzke, A. J. M.; Barta, Andrea; Kuechler, Ernst

doi:10.1111/j.1432-1033.1980.tb04998.x

Cited by 6 publications

(5 citation statements)

References 35 publications

(7 reference statements)

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“…The data on the Ywye photocrosslinking (3,4) as well as fluorescence (15,16) and chemical modification studies (17,18) are consistent with the notion that the anticodon-loop of tRNA on the ribosome occurs in the 3'-stacked conformation both in the P-and in the A-site. Nevertheless, some differences in the arrangements of the nucleotides of the anticodon-loop might exist between P-site and A-site bound tRNA.…”

Section: Resultssupporting

confidence: 76%

“…500 pnol ribosomes (8) were incubated for 10 min at 200 C in buffer B (50 mM NH4Cl, 50 mM N-2-hydroxyethylpiperazine-N'-2ethanesulfonate-sodium salt (HEPES), 10 mM MgCl2, 1 mM dithioerythritol, pH 7.4) containing 0. (3,4). After irradiation the incubation mixture was layered on a Sepharose SB column (10 x 0.4 cm) and eluted with buffer B in order to separate the ribosomes from unbound [32P]-oligonucleotides.…”

Section: Materials and Methods Materialsmentioning

confidence: 99%

“…Upon irradiation at 320 nm Ywye undergoes a photoreaction (1). This photoreactivity has been utilized in several photocrosslinking studies (2,3). Using ribosomal complexes Phe-tRNA from yeast was found to be photocrosslinked with poly(U) at the Ywye residue (3,4).…”

Section: Introductionmentioning

confidence: 99%

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Crosslinking transfer RNA and messenger RNA at the ribosomal decoding region: identification of the site of reaction on the messenger RNA

Steiner¹,

Lührmann²,

Kuechler³

1984

Nucl Acids Res

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Wybutine (Ywye), situated next to the 3'-side of the anticodon of tRNAPhe from Saccharomyces cerevisiae, can be photo-crosslinked to mRNA when bound to Escherichia coli ribosomes. Crosslinking can be obtained with poly(U) as well as with oligonucleotides such as pAUGUUU or p(U)6. In order to identify the site of reaction on the mRNA, 5'-[32P]-labelled pAUGUUU was crosslinked by irradiation at 320 nm with Phe-tRNAPhe from yeast bound to the acceptor-site. The photoproduct was subsequently digested with P1-nuclease and analyzed by electrophoresis followed by homochromatography in the second dimension. As a result of the photoreaction the wybutine was found to be crosslinked to the U at the 5'-position of the corresponding UUU-codon.

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Section: Resultssupporting

confidence: 76%

Section: Materials and Methods Materialsmentioning

confidence: 99%

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Crosslinking transfer RNA and messenger RNA at the ribosomal decoding region: identification of the site of reaction on the messenger RNA

Steiner¹,

Lührmann²,

Kuechler³

1984

Nucl Acids Res

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“…The ribosome editor hypothesis (19) proposes that peptidyl-tRNA dissociates because its structure is inappropriate for the mRNA codon. However, decoding interactions can occur at both the A and the P sites (10,14,16,17,28,32). Cabafias and Modolell (2) have recently shown that inappropriate peptidyltRNA can dissociate, with high probability, from the A site, but in their experiments, translocation was blocked, and a less common route of dissociation may thereby have been revealed.…”

Section: Methodsmentioning

confidence: 99%

Erythromycin, carbomycin, and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes

Menninger¹,

Otto²

1982

Antimicrob Agents Chemother

144

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In mutant Escherichia coli with temperature-sensitive peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase; EC 3.1.1.29), peptidyl-tRNA accumulates at the nonpermissive temperature (40°C), and the cells die. These consequences of high temperature were enhanced if the cells were first treated with erythromycin, carbomycin, or spiramycin at doses sufficient to inhibit protein synthesis in wildtype cells but not sufficient to kill either mutant or wild-type cells at the permissive temperature (30°C). Since peptidyl-tRNA hydrolase in the mutant cells is inactivated rapidly and irreversibly at 40°C, the enhanced accumulation of peptidyl-tRNA and killing were the result of enhanced dissociation, stimulated by the antibiotics, of peptidyl-tRNA from ribosomes. The implications of these findings for inhibition of cell growth and protein synthesis are discussed. Certain alternative interpretations are shown to be inconsistent with the relevant data. Previous conflicting observations on the effects of macrolide antibiotics are explained in terms of our observations. We conclude that erythromycin, carbomycin, and spiramycin (and probably all macrolides) have as a primary mechanism of action the stimulation of dissociation of peptidyl-tRNA from ribosomes, probably during translocation.The mechanism of action of the macrolide antibiotics has been a matter of controversy for some time (7,31). A few years ago, we reported data concerning a novel effect of erythromycin on protein synthesis in vivo, namely, the enhancement of the dissociation of peptidyl-tRNA from ribosomes (J. R. Menninger, Fed. Proc. 33:1335. This phenomenon was subsequently verified in vitro (24). Our in vivo studies have now been extended to two other antibiotics of the macrolide group, carbomycin and spiramycin.We used a strain of Escherichia coli (ts8) with a mutation in the structural gene (pth) for peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase, EC 3.1.1.29) which renders that enzyme activity temperature sensitive. Peptidyl-tRNA hydrolase normally acts in the cell to catalyze the hydrolysis of intact peptides from peptidyltRNA that has dissociated from ribosomes during protein synthesis. A temperature-sensitive hydrolase activity allows measurement of the dissociation of peptidyl-tRNA from ribosomes at 40°C since the first step in the scavenging pathway for dissociated peptidyl-tRNA is blocked by the high temperature (18). Shortly after cultures growing at 30°C are shifted to the nonpermissive temperature (40°C), protein synthesis is inhibited (1, 18), and the cells die; i.e., they are unable to form a colony at permissive temperatures (21). All of these effects were assayed in the presence of erythromycin, carbomycin, and spiramycin at doses chosen, after the evaluation of several concentrations, so that no lethal effects of the drugs were observed at permissive temperatures.The results presented below show that all three of these antibiotics stimulated the dissociation of peptidyl-tRNA from ribosomes of E. coli when given at doses that inhibited the gr...

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“…After all, it is a well known and frequently used fact that yeast tRNAs, in particular tRNAphe, behave normally with E. coli ribosomes (see, e.g. [24,25]). …”

Section: Comparison Qf Yeast and E Coli Trnasmentioning

confidence: 99%

The Properties of the tRNA · protein Complex of the Escherichia coli Ribosome

Matspalu¹,

Ustav²,

Villems³

1982

European Journal of Biochemistry

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A complex between immobilized tRNA and Escherichiu coli 50-S ribosomal subunit proteins provides binding sites for several other components of the protein biosynthesis system. We found that this complex binds 5-S RNA, tRNA and 30-S ribosomal subunits. The binding of 5-S RNA and tRNA takes place at equilibrium; in 10 mM Tris/HCl buffer, pH 7.5, containing 10 mM MgC12 and 100 mM KCI at 4 "C it is characterized by the dissociation constants 1.7 x M and 4 x lo-' M respectively. We show that the perfornicd complex between tRNA and 5 0 3 ribosomal subunit protein can bind these two RNAs simultaneously by forining a subribosoinal domain consisting of two molecules of tRNA, 5-S R N A and the ribOSomd1 proteins L2. L15, L16, L17, L18, L22, L33 and L34. The preformed t R N A . protein complex interacts also with the 30-S ribosomal subunit, suggesting that it is located at the ribosomal interface.A number of recent reviews deal with various aspects of the interaction between tRNA and the ribosomes [I -51. In spite of its comparatively long history, the mechanism of the tRNAjribosome interaction is still largely an open question. What we know about tRNA interaction with the components of the ribosome (ribosomal proteins and rRNA) comes from results obtained by indirect methods and can be, therefore, considered only as the first approximation when we ask a question about the direct contacts between tRNA and ribosomes. Four laboratories have published results showing interactions between ribosomal proteins and immobilized tRNA [6-131. These results demonstrate the possibility, but only the possibility, of direct interactions between certain ribosomal proteins and tRNA.We argued earlier [8-111 that the proteins forming a complex with the immobilized tRNA are those located at the ribosomal decoding site or in the vicinity of the peptidyl transferase center. In a more recent study [12] we showed that the complex of 5-S RNA with 50-S ribosomal subunit proteins binds one molecule of tRNA. Here we extend this observation and describe subribosomal complex containing ribosomal proteins and two molecules of tRNA as well as 5-S RNA. MATERIALS A N D METHODS Isolation OfrRNA, 5-S R N A 50-S Ribosomal Subunit Proteins und 30-S SubunitsAll these components have been isolated from Escherichiu coli MRE600 strain according to published procedures [8 -10, 12,141. The purity of tRNA and 5-S RNA was controlled byAbbreviations. TP50, total protein fraction of E. co/i 5 0 4 ribosomal subunit; bisoxyrane, 1,4-bis(2,3-epoxypropoxy)butane.Dcjinition. 1 AZ60 unit, the quantity of RNA in 1 ml solution which has an absorbance of 1 at 260 nm, when measured in a I-cm pathlength cell. urea/polyacrylamide gel electrophoresi\ according to Maxam and Gilbert [I 51. Ribosomes and their subunits were isolated as described in [I61 and 50-S subunit proteins were extracted by acetic acid [17]. We also isolated tKNA from baker's yeast by the phenol/sodium dodecyl sulphate method followed by further purification on DEAF.-Sephadex A-50 chromatography and Sephadex G-1...

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Photo‐Induced Crosslinking between Phenylalanine Transfer RNA and Messenger RNA on the Escherichia coli Ribosome

Cited by 6 publications

References 35 publications

Crosslinking transfer RNA and messenger RNA at the ribosomal decoding region: identification of the site of reaction on the messenger RNA

Crosslinking transfer RNA and messenger RNA at the ribosomal decoding region: identification of the site of reaction on the messenger RNA

Erythromycin, carbomycin, and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes

The Properties of the tRNA · protein Complex of the Escherichia coli Ribosome

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