A specific terminal structure is required for Ty1 transposition.

Abstract

Yeast retrotransposon Ty1 directs the synthesis of virus-like particles (VLPs) consisting of Ty1-encoded proteins, RNA, and reverse transcripts. Ty1 reverse transcripts, tagged with a selectable marker and found within VLPs, are capable of transposing into naked target DNA in vitro. Cassettes consisting of a Ty long terminal repeat (LTR), or delta, marked with supF, and flanked by appropriate restriction sites were constructed. These artificial substrates, whose termini resemble those of linear, full-length Ty1 reverse transcripts, can be coincubated with VLPs (containing unmarked reverse transcripts), resulting in the very efficient integration of the artificial substrate. The results suggest that Ty DNA is limiting for transposition in vivo, suggesting that inefficient reverse transcription regulates Ty1 transposition. Analysis of the transposition of these model substrates, which resemble in vivo Ty1 transposition intermediates or differ from them in subtle ways, shows that Ty transposition proceeds by the linkage of the 3' hydroxyl residue of the reverse transcript to target DNA.