MULTIPLE MOLECULAR DETERMINANTS FOR RETROTRANSPOSITION IN A PRIMER TRANSFER-RNA

Retroviruses and long terminal repeat-containing retroelements use hos t-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement R NA. Usually, one or more specific tRNAs, including the primer, are sel ected and packaged within the virion. The reverse transcriptase (RT) i nteracts with the primer tRNA and initiates DNA synthesis. The structu ral and sequence features of primer tRNAs important for these specific interactions are poorly understood. We have developed a genetic assay in which mutants of tRNA(i)(Met), the primer for the Ty1 retrotranspo son of Saccharomyces cerevisiae, can be tested for the ability to serv e as primers in the reverse transcription process. This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis. We find that mutations in the TX C loop and the acceptor stem regions of the tRNA(i)(Met) affect transp osition most severely. Conversely, mutations in the anticodon region h ave only minimal effects on transposition. Further study of the accept or stem and other mutants demonstrates that complementarity to the ele ment primer binding site is a necessary but not sufficient requirement for effective tRNA priming. Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as add itional recognition determinants. Ty3 and Ty1, two very distantly rela ted retrotransposons, require similar molecular determinants in this p rimer tRNA for transposition.