Nucleotide residues of tRNA, directly interacting with proteins within the complex of the 30 S subunit of <i>E. coli</i> ribosome with poly(U) and NAcPhe‐tRNA<sup>Phe</sup>

Abdurashidova, G.G.; Цветкова, Е. А.; Budowsky, E.I.

doi:10.1016/0014-5793(89)80149-8

Cited by 18 publications

(12 citation statements)

References 14 publications

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“…However, some other data also suggest that this protein can approach the tRNA binding domain . It can be cross-linked to S13 and S9 [29] and to A21 of tRNA bound to the small subunit [12].…”

Section: Resultsmentioning

confidence: 99%

“…Protein S19 has been cross-linked to P and Asite bound tRNA via photoreactive probes attached to acp3U47 and s4U8, respectively [2,11]. Proteins S5, S7 and S9 form covalent bonds to A21, U45 and U60, respectively, upon UV irradiation of a AcPhe-tRNAPhe complex with the 30S ribosomal subunit [12]. Several other proteins have been found cross-linked to tRNA but the cross-linking sites in tRNA have not been defined unequivocally (see [1] for review).…”

Section: Introductionmentioning

confidence: 99%

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Neighbourhood of the central fold of the tRNA molecule bound to theE.coliribosome—affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop

Podkowiński

Górnicki

1991

Nucl Acids Res

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The neighbourhood of the dihydrouridine loop of tRNA molecule bound to E. coli ribosome has been studied by affinity labeling, using modified tRNAs carrying photoreactive azidonitrophenyl probes attached to the 3-(3-amino-3-carboxypropyl)-uridine located at position 20:1 of Lupin methionine elongator tRNA. The maximum distance between the pyrimidine ring and the azido group estimated for the two probes employed in this study is 10-11 A and 18-19 A, respectively. Cross-linking of the uncharged, modified tRNAs has been studied with poly(A, U, G) as a message, under conditions directing uncharged tRNAs preferentially to the ribosomal P-site. Modified tRNAs bind covalently to both ribosomal subunits with high yields upon irradiation of the respective non-covalent complexes. Proteins S7, L33 and L1 have been consistently found cross-linked to tRNAs modified with both probes, and S5 and L5 to tRNA modified with the longer probe. Surprisingly, an S5-tRNA cross-linking product is reproducibly found in a protein fraction prepared from the purified 50S subunit. Cross-linking to rRNAs is significant only for the longer probe and is stimulated 2-4 fold in the presence of poly(A,U,G). The cross-linking sites are located between nucleotides 1302 and 1398 in 16S rRNA and between nucleotides 2281 and 2358 in 23S rRNA.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Neighbourhood of the central fold of the tRNA molecule bound to theE.coliribosome—affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop

Podkowiński

Górnicki

1991

Nucl Acids Res

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“…It is conceivable that, despite the somewhat distant position of their mass centres from the cleft, these proteins could extend towards the 1390-1400 region sufficiently to explain the simultaneous cross-linking that was observed. In this context it is also of interest that protein S5 has been cross-linked to the D-loop of tRNA (Abdurashidova et al, 1989). The simultaneous cross-linking to positions 532 and the 1390-1400 region of the 16S RNA is, however, very hard to account for.…”

Section: Discussionmentioning

confidence: 99%

The path of mRNA through the Escherichia coli ribosome; site-directed cross-linking of mRNA analogues carrying a photo-reactive label at various points 3′ to the decoding site.

et al. 1991

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mRNA analogues approximately 40 bases long were prepared by T7 transcription from synthetic DNA templates. Each message contained the sequence ACC‐GCG (coding for threonine and alanine, respectively), together with a single thio‐U residue located at a variable position on the 3′‐side of these coding triplets. The thio‐U residue was either substituted with 4‐azidophenacyl bromide to introduce a photo‐reactive group, or was left unsubstituted for direct UV cross‐linking. After binding to Escherichia coli 70S ribosomes in the presence of tRNA‐Thr or tRNA‐Ala, the thio‐U residue or azidophenyl group was photo‐activated and the products of cross‐linking (which was exclusively to the 30S subunit) were analysed. Immunological analysis of the cross‐linked proteins showed that S5 and S3, together with S1, were the targets of cross‐linking at positions close to the decoding site, with the cross‐linking to S3 and S1 persisting at positions further away. Analysis of the 16S RNA showed cross‐links to the region of bases 1390–1400 in all cases, but in one instance (with the reactive nucleotide 11 bases from the decoding site) simultaneous cross‐linking was observed to the latter region and to position 532; these two RNA regions are far apart in current three‐dimensional models of the 30S subunit.

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“…Identification of tRNA residues cross-linked with proteins Position in tRNA of nucleoside residues cross-linked with protein was determined as described (14) in the material, isolated by adsorption to immune complexes. After limited random hydrolysis of phosphodiester bonds the oligonucleotides containing the cross-linked protein may be removed from the mixture by phenol deproteinization.…”

Section: Ribosomalmentioning

confidence: 99%

“…than one tRNA molecule occupying different tRNA-binding sites in the ribosome. For identification of the protein cross-linked with tRNA and determination of tRNA residue involved in the cross-link [32P]-label was introduced (after deacylation (14)) into the 3'-end of tRNA using [32P]pCp and T4 RNA ligase (15).…”

mentioning

confidence: 99%

Direct tRNA-protein interactions in ribosomal complexes

Abdurashidova¹,

Цветкова²,

Budowsky³

1991

Nucl Acids Res

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Nucleotide residues in E. coli tRNA(Phe) interacting directly with proteins in pre- and posttranslocated ribosomal complexes have been identified by UV-induced cross-linking. In the tRNA(Phe) molecule located in the Ab-site (pretranslocated complex) residues A9, G18, A26 and U59 are cross-linked with proteins S10, L27, S7 and L2, respectively. In tRNA(Phe) located in the Pt-site (posttranslocated complex) residues C17, G44, C56 and U60 are cross-linked with proteins L2, L5, L27 and S9, respectively. The same cross-links (except for G44-L5) have been found for tRNA in the Pb-site of the pretranslocated ribosomal complex. None of the tRNA(Phe) residues cross-linked with proteins in the complexes examined by us are involved in the stabilization of the secondary structure, but residues A9, G18, A26, G44 and C56 participate in stabilization of tRNA tertiary structure. Since translocation of tRNA(Phe) from Ab- to P-site is accompanied by changes of tRNA contacts with proteins L2 and L27, we postulate that this translocation is coupled with tRNA turn around the axis joining the anticodon loop with the CCA-end of the molecule. This is in agreement with the idea about the presence of a kink in mRNA between codons located in the ribosomal A- and P-sites. In all E. coli tRNAs with known primary structure positions 18 and 56, interacting with L27 protein, when tRNA is located either in A- or P-site, are invariant, whereas positions 17 and 60, interacting with proteins only when tRNA is in the P-site, are strongly conserved. In positions 9, 26 and 59 purines are the preferred residues. In most E. coli tRNAs deviations from the consensus in these three positions is strongly correlated.

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Nucleotide residues of tRNA, directly interacting with proteins within the complex of the 30 S subunit of E. coli ribosome with poly(U) and NAcPhe‐tRNA^Phe

Cited by 18 publications

References 14 publications

Neighbourhood of the central fold of the tRNA molecule bound to theE.coliribosome—affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop

Neighbourhood of the central fold of the tRNA molecule bound to theE.coliribosome—affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop

The path of mRNA through the Escherichia coli ribosome; site-directed cross-linking of mRNA analogues carrying a photo-reactive label at various points 3′ to the decoding site.

Direct tRNA-protein interactions in ribosomal complexes

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Nucleotide residues of tRNA, directly interacting with proteins within the complex of the 30 S subunit of E. coli ribosome with poly(U) and NAcPhe‐tRNAPhe

Cited by 18 publications

References 14 publications

Neighbourhood of the central fold of the tRNA molecule bound to theE.coliribosome—affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop

Neighbourhood of the central fold of the tRNA molecule bound to theE.coliribosome—affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop

The path of mRNA through the Escherichia coli ribosome; site-directed cross-linking of mRNA analogues carrying a photo-reactive label at various points 3′ to the decoding site.

Direct tRNA-protein interactions in ribosomal complexes

Contact Info

Product

Resources

About

Nucleotide residues of tRNA, directly interacting with proteins within the complex of the 30 S subunit of E. coli ribosome with poly(U) and NAcPhe‐tRNA^Phe