INTERACTIONS BETWEEN TY1 RETROTRANSPOSON RNA AND THE T-REGIONS AND D-REGIONS OF THE TRNA(I)(MET) PRIMER ARE REQUIRED FOR INITIATION OF REVERSE TRANSCRIPTION IN-VIVO

Reverse transcription of the Saccharomyces cerevisiae Ty1 retrotranspo son 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 comp lementary to the last 10 nucleotides of the acceptor stem of tRNA(i)(M et). A structural probing study of the interactions between the Ty1 RN A template and the tRNA(i)(Met) primer showed that besides interaction s 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 import ant for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNA(i)(Met) were tested for the ability to su pport 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 pair ing between the primer,and the template restore transposition, Analysi s of the reverse transcription intermediates generated inside Ty1 viru s-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity bet ween Ty1 RNA and primer tRNA(i)(Met).