Photoaffinity modification of E-site of Escherichia coli ribosomes

Vladimirov, S.N.; Graifer, D. M.; Зенкова, М. А.; Karpova, G. Y.; Olenina, Ludmila V.; Kirillov, S.V.; Makarov, Evgeny M.; Makhno, V.I.; Semenkov, Yu.P.

doi:10.7124/bc.000044

Cited by 4 publications

(7 citation statements)

References 13 publications

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“…Free 70 S ribosomes and the initiation complex were treated with 1 mM Pt2amCI4 at 20°C for 50 min. Treated ribosomes were divided into subunits according to [2,4]. rRNA-protein cross-links were isolated as in [2,4] and reversed by treatment with 2 M thiourea for 1 h at 37°C and pH 3.5.…”

Section: Methodsmentioning

confidence: 99%

“…Treated ribosomes were divided into subunits according to [2,4]. rRNA-protein cross-links were isolated as in [2,4] and reversed by treatment with 2 M thiourea for 1 h at 37°C and pH 3.5. Ribosomal proteins were analysed by two-dimensional polyacrylamide gel electrophoresis as described in [4].…”

Section: Methodsmentioning

confidence: 99%

“…In the study of ribosomal topography, Pt(II) compounds are rather promising [1][2][3][4]. The reagent { dichloro [N,N,N ',N '-tetrakis(2-aminoethyl) -1,6 -hexamethylenediaminediplatinum(II)] I dichloride (Pt2AmCIa) has been used in investigation of rRNA-protein cross-links in free 70 S ribosomes and in the initiation complex 70 S ribosome.…”

Section: Introductionmentioning

confidence: 99%

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Differences in 23 S rRNA‐protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex

et al. 1989

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rRNA-protein cross-links in free E. coil 35S-labeled 70 S ribosomes and in the initiation complex 3sS-labeled 70 S ribosome-AUGUr • fMet-tRNAff et were studied with the aid of a new type of binuclear Pt(II) compound -dichlorotetraammine(1,6-hexamethylenediaminediplatinum) dichloride. The use of this reagent allowed us to reveal differences in the rRNA-protein neighbourhood in free 70 S ribosomes and in the initiation complex. Proteins L3, L6, L23 and L25 were shown to cross-link to 23 S rRNA only in the initiation complex, whereas proteins L1, L13, L14, L16, L17, L18, L22, L28 and S1 did so in both free ribosomes and the complex. 16 S rRNA was found to be cross-linked preferentially to a single protein, S 1, in both states of the ribosomes.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Differences in 23 S rRNA‐protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex

et al. 1989

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“…Then the complex and free 70 S ribosomes were treated with 1 mM PtzAmCL for 50 min at 20°C. Separation of platinated ribosomes into subunits was performed after dissociation of the initiation complex induced by addition of EDTA as described in [8]. rRNA-protein cross-links were isolated according to [8].…”

Section: Methodsmentioning

confidence: 99%

“…The activity of ribosomes in poly(U)-directed (Phe)z synthesis was determined according to [5]. Initiation complex was obtained as in [8] with the following alterations:…”

Section: Methodsmentioning

confidence: 99%

rRNA‐protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex Probing by bifunctional Pt(II)‐containing reagent

Chistyakov¹,

Abdukayumov²,

Веньяминова³

et al. 1988

FEBS Letters

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The cleavable homobifunctional reagent dichloro[N,N,K,W-tetrakis(2-aminoethyl)-1,6-hexamethylenediamminediplatinum(lI)] dichloride was used for studying rRNA-protein cross-links in free 35S-labelled 70 S ribosomes and within initiation complex ribosome AUGU, fMet-tRNAr". It was shown that the sets of proteins cross-linked to 16 S and 23 S rRNA in free 70 S ribosomes and in 70 S initiation complex do not differ significantly. The authors are the first to demonstrate most of the 23 S rRNA-protein cross-links and some 16 S rRNA-protein cross-links, in particular those with L7/L12 protein.

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Transit of tRNA through the Escherichia coli ribosome: cross‐linking of the 3′ end of tRNA to ribosomal proteins at the P and E sites

et al. 2002

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Photoreactive derivatives of yeast tRNA Phe containing 2-azidoadenosine at their 3P P termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)-programmed ribosomes, deacylated tRNA Phe , Phe-tRNA Phe and N-acetyl-Phe-tRNA Phe probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNA Phe bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNA Phe probe also labeled protein L1. Crosslinking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit. ß

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Photoaffinity modification of E-site of Escherichia coli ribosomes

Cited by 4 publications

References 13 publications

Differences in 23 S rRNA‐protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex

Differences in 23 S rRNA‐protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex

rRNA‐protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex Probing by bifunctional Pt(II)‐containing reagent

Transit of tRNA through the Escherichia coli ribosome: cross‐linking of the 3′ end of tRNA to ribosomal proteins at the P and E sites

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