EXTENDED INTERACTIONS BETWEEN THE PRIMER TRNA(I)(MET) AND GENOMIC RNAOF THE YEAST TY1 RETROTRANSPOSON

Reverse transcription of the yeast Ty1 retrotransposon is primed by tR NA(i)(Met) base paired to the primer binding site near the 5'-end of T y1 genomic RNA. To understand the molecular basis of the tRNA(i)(Met)- Ty1 RNA interaction the secondary structure of the binary complex was analysed. Enzymatic probes were used to test the conformation of tRNA( i)(Met) and of Ty1 RNA in the free farm and in the complex. A secondar y structure model of the tRNA(i)(Met)-(Ty1) RNA complex consistent wit h the probing data was constructed with the help of a computer program . The model shows that besides interactions between the primer binding site and the last 10 nt at the 3'-end of tRNA(i)(Met), three short re gions of Tyl RNA named boxes 0, 1 and 2.1 interact with the T and D st ems and loops of tRNA(i)(Met) Mutations were made in the boxes or in t he complementary sequences of tRNA(i)(Met) to study the contribution o f these sequences to formation of the complex. We find that interactio n with at least one of the two boxes 0 or 1 is absolutely required for efficient annealing of the two RNAs. Sequence comparison showing that the primary sequence of the boxes is strictly conserved in Tyl and Ty 2 elements and previously published in vivo results underline the func tional importance of the primary sequence of the boxes and suggest tha t extended interactions between genomic Tyl RNA and the primer tRNA(i) (Met) play a role in the reverse transcription pathway.