The yeast retrotransposon Ty5 uses the anticodon stem-loop of the initiator methionine tRNA as a primer for reverse transcription

Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH o f the acceptor stem, which is complementary to sequences on the retroelemen t mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of t he initiator methionine tRNA (IMT) are complementary to the PBS. We took ad vantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarit y with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restor ed base pairing between the two mutant RNAs restored transposition signific antly. An analysis of 12 imt mutants with base changes outside of the regio n of complementarity failed to identify other tRNA residues important for t ransposition. In addition, assays carried out with heterologous IMTs from S chizosaccharomyces pombe and Arabidopsis thaliana indicated that residues o utside of the anticodon stem-loop have at most a fivefold effect on transpo sition. Our genetic system should make it possible to further define the co mponents required for priming and to understand the mechanism by which Ty5' s novel primer is generated.