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

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Abstract
Reverse transcription of the Saccharomyces cerevisiaeTy1 retrotransposon is primed by tRNAiMet 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 tRNAiMet. A structural probing study of the interactions between the Ty1 RNA template and the tRNAiMet primer showed that besides interactions between the PBS and the 3′ end of tRNAiMet, three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNAiMet. To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNAiMetwere 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 tRNAiMet contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNAiMet 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 tRNAiMet.