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Mol Cell Biol, February 1998, p. 799-806, Vol. 18, No. 2
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Interactions between Ty1 Retrotransposon RNA and the T and D Regions of the tRNA_i^Met Primer Are Required for Initiation of Reverse Transcription In Vivo

S. Friant,¹ T. Heyman,² A. S. Byström,³ M. Wilhelm,¹ and F. X. Wilhelm^1,*

Unité Propre de Recherche 9002 du Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, 67084 Strasbourg,¹ and Unité Mixte de Recherche 216 du Centre National de la Recherche Scientifique, Institut Curie-Biologie, Centre Universitaire, 91405 Orsay,² France, and Department of Microbiology, Umea University, S-901 87 Umea, Sweden³

Received 6 June 1997/Returned for modification 25 July 1997/Accepted 18 November 1997

Reverse transcription of the Saccharomyces cerevisiae Ty1 retrotransposon is primed by tRNA_i^Met base paired to the primer binding site (PBS) near the 5' end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNA_i^Met. A structural probing study of the interactions between the Ty1 RNA template and the tRNA_i^Met primer showed that besides interactions between the PBS and the 3' end of tRNA_i^Met, three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNA_i^Met. To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNA_i^Met were tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNA_i^Met contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNA_i^Met and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNA_i^Met.

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^* Corresponding author. Mailing address: IBMC, 15 rue René Descartes, 67084 Strasbourg, France. Phone: 33 (0) 3 88 41 70 06. Fax: 33 (0) 3 88 60 22 18. E-mail: wilhelm{at}ibmc.u-strasbg.fr.

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