[9] Photolabile derivatives of oligonucleotides as probes of ribosomal structure

Cooperman, Barry S.; Alexander, Rebecca W.; Bukhtiyarov, Yuri; Vladimirov, S.N.; Druzina, Zhanna; Wang, Ruo; Zuño, Nora

doi:10.1016/s0076-6879(00)18048-6

Cited by 4 publications

(7 citation statements)

References 22 publications

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“…Except as specified below, materials were obtained and methods were performed as described elsewhere (3,4).…”

Section: Methodsmentioning

confidence: 99%

“…Preparation of PHONT-labeled 50S RNA annealed with 32 P-labeled oligo cDNA promer was carried out as described (3), except the first heating step was carried out at 65ЊC rather than 70ЊC, and better results were obtained with reverse transcriptase from Sekkagaku in place of that from Promega. Extracted RNase H fragments (see below) were analyzed similarly, except that the rRNA was labeled with p*PHONT.…”

Section: Methodsmentioning

confidence: 99%

“…Since this seminal article, photoinduced crosslinking and the related approach of photoaffinity labeling have been used extensively to explore binding sites of a large number of biological macromolecules (2), particularly those deemed too large for direct structure elucidation. In recent years we have used photoinduced crosslinking to examine ribosome structure (3)(4)(5)(6). In our studies radioactive, photolabile derivatives of oligonucleotides (PHONTs), 2 having sequences complementary to rRNA sequences, are bound to their targeted sequences in intact ribosomes or ribosomal subunits, and, on photolysis, incorporate into ribosomal components that can subsequently be identified.…”

Section: Introductionmentioning

confidence: 99%

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Large-Scale Motions within Ribosomal 50S Subunits as Demonstrated Using Photolabile Oligonucleotides

Seo

Cooperman

2002

Bioorganic Chemistry

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“…Except as specified below, materials were obtained and methods were performed as described elsewhere (3,4).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Large-Scale Motions within Ribosomal 50S Subunits as Demonstrated Using Photolabile Oligonucleotides

Seo

Cooperman

2002

Bioorganic Chemistry

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“…RNA extraction from 70S ribosomes, and PAGE analysis of RNase H fragments were carried out essentially as described (Cooperman et al 2000;Seo and Cooperman 2002). Interestingly, the time required to photolyze the s 4 U of Ph-ASL 33 (t 1/2 ∼3-4 min), as determined by the decrease in A 330 , was significantly longer than to photolyze the s 4 U of Ph-ASL 37 (t 1/2 < 1 min).…”

Section: Photolysismentioning

confidence: 99%

“…The sites of 16S rRNA modifications were analyzed by primer extension assay (Cooperman et al 2000) using the oligodeoxynucleotide complementary to 16S nucleotides 984-969 as the primer. …”

Section: Kethoxal Modificationmentioning

confidence: 99%

Photolabile anticodon stem–loop analogs of tRNA^Phe as probes of ribosomal structure and structural fluctuation at the decoding center

Druzina

Cooperman²

2004

RNA

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With the recent availability of high-resolution structures of bacterial ribosomes, studies of ribosome-catalyzed protein biosynthesis are now focusing on the nature of conformational changes that occur as the ribosome exerts its complex catalytic function. Photocrosslinking can be relevant for this purpose by providing clues to ribosomal structural fluctuations and dynamics. Here we describe crosslinking experiments on 70S ribosomes using two photolabile anticodon stem-loop derivatives of Escherichia coli tRNA Phe carrying a 4-thiouridine in either position 33 or 37 and denoted Ph-ASLs. One or both of these Ph-ASLs bind to the tRNA A-, P-, and E-sites on the ribosome, with both binding to and photocrosslinking from the E-site showing strong dependence on the presence of a tRNA in the P-site. Both Ph-ASLs crosslink to the extreme 3-end of 16S rRNA from both the P-and E-sites, providing direct confirmatory evidence in solution for the folding back of the 3-end toward the decoding region. This suggests that the 3-end of 16S rRNA may act as a switch in controlling mRNA access to the decoding center, a phenomenon of potential relevance for the translation of leaderless mRNA. E-site bound Ph-ASLs also form photocrosslinks to nucleotides 1395-1398, 1399-1400, and 1491-1494 at the top of helix 44 of 16S rRNA, indicating movement of the decoding center from a position between the A-and P-sites seen in the crystal structure to one neighboring the E-site.

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Probing RNA Structure by Photoaffinity Crosslinking with 4‐Thiouridine and 6‐Thioguanosine

Harris¹,

Christian²

2005

Handbook of RNA Biochemistry

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[9] Photolabile derivatives of oligonucleotides as probes of ribosomal structure

Cited by 4 publications

References 22 publications

Large-Scale Motions within Ribosomal 50S Subunits as Demonstrated Using Photolabile Oligonucleotides

Large-Scale Motions within Ribosomal 50S Subunits as Demonstrated Using Photolabile Oligonucleotides

Photolabile anticodon stem–loop analogs of tRNA^Phe as probes of ribosomal structure and structural fluctuation at the decoding center

Probing RNA Structure by Photoaffinity Crosslinking with 4‐Thiouridine and 6‐Thioguanosine

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[9] Photolabile derivatives of oligonucleotides as probes of ribosomal structure

Cited by 4 publications

References 22 publications

Large-Scale Motions within Ribosomal 50S Subunits as Demonstrated Using Photolabile Oligonucleotides

Large-Scale Motions within Ribosomal 50S Subunits as Demonstrated Using Photolabile Oligonucleotides

Photolabile anticodon stem–loop analogs of tRNAPhe as probes of ribosomal structure and structural fluctuation at the decoding center

Probing RNA Structure by Photoaffinity Crosslinking with 4‐Thiouridine and 6‐Thioguanosine

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Photolabile anticodon stem–loop analogs of tRNA^Phe as probes of ribosomal structure and structural fluctuation at the decoding center