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Sequences Outside Recognition Sets Are Not Neutral for tRNA Aminoacylation

EVIDENCE FOR NONPERMISSIVE COMBINATIONS OF NUCLEOTIDES IN THE ACCEPTOR STEM OF YEAST tRNAPhe*
  • Magali Frugier
    Footnotes
    Affiliations
    Unité Propre de Recherche n° 9002 du CNRS, “Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance”, Institut de Biologie Moléculaire et Cellulaire, 15, rue René Descartes, 67084 Strasbourg Cedex, France
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  • Mark Helm
    Footnotes
    Affiliations
    Unité Propre de Recherche n° 9002 du CNRS, “Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance”, Institut de Biologie Moléculaire et Cellulaire, 15, rue René Descartes, 67084 Strasbourg Cedex, France
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  • Brice Felden
    Footnotes
    Affiliations
    Unité Propre de Recherche n° 9002 du CNRS, “Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance”, Institut de Biologie Moléculaire et Cellulaire, 15, rue René Descartes, 67084 Strasbourg Cedex, France
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  • Richard Giegé
    Correspondence
    To whom correspondence should be addressed: IBMC du CNRS, 15, rue René Descartes, 67084 Strasbourg Cedex, France. Tel.: 33 (0)3 88 41 70 59; Fax: 33 (0)3 88 60 22 18
    Affiliations
    Unité Propre de Recherche n° 9002 du CNRS, “Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance”, Institut de Biologie Moléculaire et Cellulaire, 15, rue René Descartes, 67084 Strasbourg Cedex, France
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  • Catherine Florentz
    Affiliations
    Unité Propre de Recherche n° 9002 du CNRS, “Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance”, Institut de Biologie Moléculaire et Cellulaire, 15, rue René Descartes, 67084 Strasbourg Cedex, France
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  • Author Footnotes
    * This work was supported in part by funds from the CNRS, Ministère de la Recherche et de l'Enseignement Supérieur (MRES), and Université Louis Pasteur, Strasbourg.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Supported by a fellowship from Association pour la Recherche contre le Cancer.
    § Supported by a fellowship from the Fonds der Chemischen Industrie and a TMR fellowship from the European Community.
    ¶ Supported by a fellowship from MRE and present address: Howard Hughes Medical Institute of Human Genetics, Bldg. 553, Salt Lake City, UT 84112.
Open AccessPublished:May 08, 1998DOI:https://doi.org/10.1074/jbc.273.19.11605
      Phenylalanine identity of yeast tRNAPhe is governed by five nucleotides including residues A73, G20, and the three anticodon nucleotides (Sampson et al., 1989, Science 243, 1363–1366). Analysis of in vitro transcripts derived from yeast tRNAPhe and Escherichia colitRNAAla bearing these recognition elements shows that phenylalanyl-tRNA synthetase is sensitive to additional nucleotides within the acceptor stem. Insertion of G2-C71 has dramatic negative effects in both tRNA frameworks. These effects become compensated by a second-site mutation, the insertion of the wobble G3-U70 pair, which by itself has no effect on phenylalanylation. From a mechanistic point of view, the G2-C71/G3-U70 combination is not a “classical” recognition element since its antideterminant effect is compensated for by a second-site mutation.
      This enlarges our understanding of tRNA identity that appears not only to be the outcome of a combination of positive and negative signals forming the so-called recognition/identity set but that is also based on the presence of nonrandom combinations of sequences elsewhere in tRNA. These sequences, we name “permissive elements,” are retained by evolution so that they do not hinder aminoacylation. Likely, no nucleotide within a tRNA is of random nature but has been selected so that a tRNA can fulfill all its functions efficiently.
      The specificity of transfer RNA aminoacylation is a crucial step in protein synthesis. Investigations during the last years have shown that the aminoacylation identity of a tRNA is linked to the presence of specific sets of signals allowing both discrimination by cognate aminoacyl-tRNA synthetases (aaRSs),
      The abbreviations used are: aaRS, aminoacyl-tRNA synthetase; AlaRS, AspRS, PheRS, SerRS, ValRS, alanyl-, aspartyl-, phenylalanyl-, seryl-, and valyl-tRNA synthetases; DTE, diifthioerythritol.
      1The abbreviations used are: aaRS, aminoacyl-tRNA synthetase; AlaRS, AspRS, PheRS, SerRS, ValRS, alanyl-, aspartyl-, phenylalanyl-, seryl-, and valyl-tRNA synthetases; DTE, diifthioerythritol.
      the positive elements, and rejection by noncognate synthetases, the negative elements or antideterminants (
      • Giegé R.
      • Puglisi J.D.
      • Florentz C.
      ,
      • Saks M.E.
      • Sampson J.R.
      • Abelson J.N.
      ,
      • McClain W.H.
      ). The completeness of a set of positive elements has generally been tested by co-transplantation of the corresponding nucleotides into one or several noncognate host tRNAs that acquire the new aminoacylation properties. In several instances, this approach allowed detection of special requirements for the optimal expression of a given aminoacylation identity set within a host tRNA. Thus, minor elements and conformational features were shown to contribute to aminoacylation identities (e.g. Refs.
      • McClain W.
      • Foss K.
      • Jenkins R.A.
      • Schneider J.
      ,
      • Francklyn C.
      • Musier-Forsyth K.
      • Schimmel P.
      ,
      • Frugier M.
      • Florentz C.
      • Schimmel P.
      • Giegé R.
      ,
      • Hou Y.-M.
      • Westhof E.
      • Giegé R.
      ,
      • Becker H.D.
      • Giegé R.
      • Kern D.
      ).
      Recognition elements required for phenylalanylation of yeast tRNAPhe were defined in the pioneering work of Uhlenbeck and co-workers (
      • Sampson J.R.
      • Uhlenbeck O.C.
      ,
      • Sampson J.R.
      • DiRenzo A.B.
      • Behlen L.S.
      • Uhlenbeck O.C.
      ) as a set of five major elements. These elements correspond to G20, G34, A35, A36, and A73, and their competence to confer phenylalanine (Phe) identity was first demonstrated by transplantation into four host tRNAs that all acquired optimal phenylalanylation capacities (
      • Sampson J.R.
      • DiRenzo A.B.
      • Behlen L.S.
      • Uhlenbeck O.C.
      ). Nucleotides involved in tertiary interactions were shown not to contribute to identity by a direct effect (
      • Sampson J.
      • DiRenzo A.B.
      • Behlen L.S.
      • Uhlenbeck O.C.
      ). Alternatively, expression of Phe identity in the yeast tRNAAsp context has revealed that PheRS is sensitive to fine local structural features, such as the D-loop and variable region structures (
      • Perret V.
      • Florentz C.
      • Puglisi J.D.
      • Giegé R.
      ). Finally, in neither study based on sequence comparisons of natural or engineered Phe accepting species, nucleotides within the acceptor stem helix were found important for specificity.
      In a previous work, we have been able to create a chimeric tRNA, efficiently recognized and aminoacylated at once by three different aminoacyl-tRNA synthetases including yeast PheRS (
      • Frugier M.
      • Florentz C.
      • Schimmel P.
      • Giegé R.
      ), and found that effective phenylalanylation of this tRNA was dependent, among other features, on the sequence of base pair 2–71 within the acceptor stem. Engineering of a tRNA with multiple specificities was based on the synthesis of a chimeric tRNAAsp containing the recognition sets for yeast PheRS (the five residues listed above), forEscherichia coli AlaRS (the G3-U70 base pair,e.g. Refs.
      • Hou Y.-M.
      • Schimmel P.
      and
      • McClain W.H.
      • Foss K.
      ), and for yeast ValRS (A73 and A35; Ref.
      • Florentz C.
      • Dreher T.W.
      • Rudinger J.
      • Giegé R.
      ). Notice that the valine identity residues A73 and A35 are common to the Phe recognition set. Simultaneous optimization of alanylation and phenylalanylation efficiencies could be achieved by insertion of specific structural features (the length of the α- and β-domains within the D-loop shaped to 4 and 2 nucleotides and the length of the variable region extended to 5 nucleotides) and mutation of base pair 2–71 in the amino acid acceptor stem from C-G to G-C (
      • Frugier M.
      • Florentz C.
      • Schimmel P.
      • Giegé R.
      ).
      Whereas the structural changes introduced were directed by already established yeast PheRS requirements (
      • Sampson J.
      • DiRenzo A.B.
      • Behlen L.S.
      • Uhlenbeck O.C.
      ,
      • Perret V.
      • Florentz C.
      • Puglisi J.D.
      • Giegé R.
      ), replacement of base pair C2-G71 by G2-C71 in the chimeric tRNAAsp transcript was guided by our present understanding of E. colitRNAAla identity. Indeed, this base pair is important for optimal alanine identity expression (
      • Francklyn C.
      • Musier-Forsyth K.
      • Schimmel P.
      ,
      • Shi J.P.
      • Francklyn C.
      • Hill K.
      • Schimmel P.
      ,
      • McClain W.
      • Foss K.
      • Jenkins R.A.
      • Schneider J.
      ) but was not expected to be of any influence on phenylalanylation (
      • Sampson J.R.
      • DiRenzo A.B.
      • Behlen L.S.
      • Uhlenbeck O.C.
      ,
      • Sampson J.R.
      • Behlen L.S.
      • DiRenzo A.B.
      • Uhlenbeck O.C.
      ). Enhancement of phenylalanylation activity by insertion of a G2-C71 base pair was effective in three different structural contexts, all containing the G3-U70 base pair required for efficient alanylation (
      • Frugier M.
      • Florentz C.
      • Schimmel P.
      • Giegé R.
      ).
      Here, we basically investigate the role of base pairs 2–71 and 3–70 in Phe identity in yeast tRNAPhe as well as in the frameworks of yeast tRNAAsp and E. colitRNAAla. The kinetic data collected for various T7 transcripts demonstrate that these base pairs are involved in an optimal expression of the yeast tRNAPhe recognition set. Several possible roles played by these nucleotides are discussed. They do clearly not behave as positive recognition elements as commonly defined in the field. The new outcome of our studies is that the sequence of a tRNA, apart from the recognition elements and the consensus nucleotides involved in the establishment of the three-dimensional structure, is not random for aminoacylation. Some combinations are tolerated, others are not. Thus, specificity is linked to the set of positive and negative recognition elements as well as to an adequate sequence combination within the remaining domains of the tRNA.

      Acknowledgments

      We thank M. Baltzinger (CNRS/UPR9005, Strasbourg) for the generous gift of yeast PheRS, F. W. Studier (Brookhaven National Laboratory) for T7 RNA polymerase clone, A. Hoeft (IBMC, Strasbourg) for DNA oligonucleotide synthesis, and O. Uhlenbeck ( University of Colorado, Boulder) for critical reading of a first version of this manuscript and constructive comments.

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