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Structural requirements for enzymatic formation of threonylcarbamoyladenosine (t6A) in tRNA: An in vivo study with Xenopus laevis oocytes

Published online by Cambridge University Press:  01 January 1998

ANNIE MORIN
Affiliation:
Laboratoire d'Enzymologie et Biochimie Structurales, C.N.R.S., 1, av. de la Terrasse, F-91198 Gif-sur-Yvette, France
SYLVIE AUXILIEN
Affiliation:
Laboratoire d'Enzymologie et Biochimie Structurales, C.N.R.S., 1, av. de la Terrasse, F-91198 Gif-sur-Yvette, France Present address: Ecole Normale Supérieure de Lyon, Unité de Virologie Humaine, F-69364 Lyon, France.
BRUNO SENGER
Affiliation:
Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, F-67084, France Present address: Biochemie Zentrum Heidelberg (BZH), im Neuenheimer Feld 328, D-69120 Heidelberg, Germany.
RAVINDRA TEWARI
Affiliation:
Physical Chemistry Division, National Chemical Laboratory (C.S.I.R.), Pune 411008, India
HENRI GROSJEAN
Affiliation:
Laboratoire d'Enzymologie et Biochimie Structurales, C.N.R.S., 1, av. de la Terrasse, F-91198 Gif-sur-Yvette, France
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Abstract

We have investigated the specificity of the eukaryotic enzymatic machinery that transforms adenosine at position 37 (3′ adjacent to anticodon) of several tRNAs into threonylcarbamoyladenosine (t6A37). To this end, 28 variants of yeast initiator tRNAMet and yeast tRNAVal, devoid of modified nucleotide, were produced by in vitro transcription with T7 polymerase of the corresponding synthetic tRNA genes and microinjected into the cytoplasm of Xenopus laevis oocytes. Threonylcarbamoyl incorporation was analyzed in tRNA transcripts mutated in the anticodon loop by substitution, deletion, or insertion of nucleotides, or in the overall 3D structure of the tRNA by altering critical tertiary interactions. Specifically, we tested the effects of altering ribonucleotides in the anticodon loop, changes of the loop size, perturbations of the overall tRNA 3D structure due to mutations disruptive of the tertiary base pairs, and truncated tRNAs. The results indicate that, in addition to the targeted A37, only U36 was absolutely required. However, A38 in the anticodon loop considerably facilitates the quantitative conversion of A37 into t6A37 catalyzed by the enzymes present in X. laevis. The anticodon positions 34 and 35 were absolutely “neutral” and can accept any of the four canonical nucleotides A, U, C, or G. The anticodon loop size may vary from six to eight nucleotides, and the anticodon stem may have one mismatch pair of the type A∗C or G∗U at location 30–40 without affecting the efficiency of t6A37 formation and still t6A37 is efficiently formed. Although threonylcarbamoylation of A37 occurred with tRNA having limited perturbations of 3D structure, the overall L-shaped architecture of the tRNA substrate was required for efficient enzymatic conversion of A37 to t6A37. These results favor the idea that unique enzymatic machinery located in the oocyte cytoplasm catalyzes the formation of t6A37 in all U36A37-containing tRNAs (anticodon NNU).

Microinjection of the yeast tRNAMeti into the cytoplasm of X. laevis oocytes also revealed the enzymatic activities for several other nucleotide modifications, respectively m1G9, m2G10, m22G26, m7G46, D47, m5C48/49, and m1A58.

Type
Research Article
Information
RNA , Volume 4 , Issue 1 , January 1998 , pp. 24 - 37
Copyright
© 1998 RNA Society

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