Yeast TY1 retrotransposon: The minus-strand primer binding site and a cis-acting domain of the TY1 RNA are both important for packaging of primer tRNA inside virus-like particles

Abstract

Reverse transcription of the yeast retrotransposon Ty1 is primed by the cytoplasmic initiator methionine tRNA (tRNAjiMet). The primer tRNAiMet is packaged inside virus-like particles (VLPs) and binds to a 10 nucleotides minus-strand primer binding site, the (-)PBS, complementary to its 3′ acceptor stem. We have found that three short sequences of the Ty1 RNA (box 1, box 2.1 and box 2.2) located 3′ to the (-)PBS are complementary to other regions of the primer tRNAjiMet (TψC and DHL) stems and loops). Reconstitution of reverse transcription in vitro with T7 transcribed Ty1 RNA species and tRNAjiMet purified from yeast cells shows that the boxes do not affect the efficiency of reverse transcription. Thus the role of the boxes on packaging of the primer tRNAjiMet into the VLPs was investigated by analysing the level of tRNAiMet packaged into mutant VLPs. Specific nucleotide changes in the (-)PBS or in the boxes that do not change the protein coding sequence but disrupt the complementarity with the primer tRNAiMet diminish or abolish the packaging of the tRNAjiMet within the VLPs. We propose that base pairing between the primer tRNAjiMet and the Ty1 RNA is of major importance for tRNAjMet packaging into the VLPs. Moreover the intactness of the boxes is essential for retrotransposition as shown by the transposition defect of a Ty1 element harboring an intact (-)PBS and mutated boxes. © 1994 Oxford University Press.