Substitution of uridine in vivo by the intrinsic photoactivable probe 4-thiouridine in Escherichia coli RNA

Its use for E. coli ribosome structural analysis

Authors

  • Alain FAVRE,

    Corresponding author
    1. Groupe de Photobiologie Moléculaire, Institut Jacques Monod, Centre National de la Recherche Scientifique, Université Paris VII
      Correspondence to A. Favre, Groupe de Photobiologie Moléculaire, Institut Jacques Monod, CNRS, Université Paris VII, 2, Place Jussieu, F-75251 Paris Cedex 05, France
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  • Roberto BEZERRA,

    1. Groupe de Photobiologie Moléculaire, Institut Jacques Monod, Centre National de la Recherche Scientifique, Université Paris VII
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  • Eliane HAJNSDORF,

    1. Groupe de Photobiologie Moléculaire, Institut Jacques Monod, Centre National de la Recherche Scientifique, Université Paris VII
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  • Yolande LEMAIGRE DUBREUIL,

    1. Groupe de Photobiologie Moléculaire, Institut Jacques Monod, Centre National de la Recherche Scientifique, Université Paris VII
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  • Alain EXPERT-BEZANÇON

    1. Groupe de Photobiologie Moléculaire, Institut Jacques Monod, Centre National de la Recherche Scientifique, Université Paris VII
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Correspondence to A. Favre, Groupe de Photobiologie Moléculaire, Institut Jacques Monod, CNRS, Université Paris VII, 2, Place Jussieu, F-75251 Paris Cedex 05, France

Abstract

In vivo incorporation of the uridine-photoactivable analogue, 4-thiouridine, into the ribosomal RNA of an Escherichia coli pyrD strain has been demonstrated. It is highly dependent on the exogeneous uridine and 4-thiouridine concentrations as well as on temperature. We have defined conditions allowing the substitution of 13 ± 2% of the uridine residues in bulk RNA by 4-thiouridine. On a high-Mg2+ sucrose gradient, 33 ± 3% of ribonucleic particles sediment as 70S ribosomes, the remaining being in the form of non-associated 50S and 30S particles containing immature rRNA. The thiolated 70S ribosomes tolerate a 4–5% substitution level (40 thiouridine molecules/particle).

Surprisingly, 3–4% of ribosomal proteins, about two protein molecules/particle, were spontaneously covalently bound to 4-thiouridine-substituted rRNA. Specific 366-nm photoactivation increased this proportion to 10–12%, i.e. up to six or seven ribosomal protein molecules/particle. The photochemical cross-linking proceeds with aparent first-order kinetics with a quantum yield close to 5 × 10–3. Although extensive photodynamic breakage of rRNA occurs under aerobic conditions, both the kinetics and yield of ribosomal protein cross-linking were independent of oxygenation conditions. The thiolated (4.5%) 70S ribosomes allowed the poly(U)-directed poly(Phe)synthesis at 48% the control rate. Photoactivation decreased this activity to 28% and 10% when performed under nitrogen and in aerated conditions, respectively.

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