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
of the acceptor stem, which is complementary to sequences
on the retroelement mRNA (the primer binding site, PBS).
However, for some retrotransposons, including the yeast
Ty5 elements, sequences in the anticodon stem-loop of the
initiator methionine tRNA (IMT) are complementary to the
PBS. We took advantage 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 complementarity
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 restored base
pairing between the two mutant RNAs restored transposition
significantly. An analysis of 12 imt mutants with
base changes outside of the region of complementarity failed
to identify other tRNA residues important for transposition.
In addition, assays carried out with heterologous IMTs
from Schizosaccharomyces pombe and Arabidopsis
thaliana indicated that residues outside of the anticodon
stem-loop have at most a fivefold effect on transposition.
Our genetic system should make it possible to further define
the components required for priming and to understand the
mechanism by which Ty5's novel primer is generated.