Extended Interactions Between the Primer tRNAiMet and Genomic RNA of the Yeast Ty1 Retrotransposon

Abstract

Reverse transcription of the yeast Ty1 retrotransposon is primed by tRNA _i^Met base paired to the primer binding site near the 5′-end of Ty1 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 form and in the complex. A secondary structure model of the tRNA _i^Met -Ty1 RNA complex consistent with 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 regions of Ty1 RNA named boxes 0, 1 and 2.1 interact with the T and D stems and loops of tRNA _i^Met . Mutations were made in the boxes or in the complementary sequences of tRNA _i^Met to study the contribution of these sequences to formation of the complex. We find that interaction 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 Ty1 and Ty2 elements and previously published in vivo results underline the functional importance of the primary sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primer tRNA _i^Met play a role in the reverse transcription pathway.