ROLE OF RNA PRIMERS IN INITIATION OF MINUS-STRAND AND PLUS-STRAND DNA-SYNTHESIS OF THE YEAST RETROTRANSPOSON TY1

The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae is a lon g terminal repeat mobile genetic element that transposes through an RN A intermediate. Initiation of minus-strand and plus strand DNA synthes is are two critical steps during reverse transcription of the retrotra nsposon genome. Initiation of minus-strand DNA synthesis of the Ty1 el ement is primed by the cytoplasmic initiator methionine tRNA base pair ed to the primer binding site near the 5' end of the genomic RNA. A st ructural probing study of the primer tRNA-Ty1 RNA binary complex revea ls that besides interactions between the primer binding site and the l ast 10 nucleotides at the 3' end of the primer tRNA, three short regio ns of Ty1 RNA named box 0, box 1 and box 2.1 interact with the T and D stems and loops of the primer tRNA. Some in vivo results underline th e functional importance of the nucleotide sequence of the boxes and su ggest that extended interactions between genomic Ty1 RNA and the prime r tRNA play a role in the reverse transcription pathway. Plus-strand D NA synthesis is initiated from an RNase H resistant oligoribonucleotid e spanning a purine-rich sequence, the polypurine tract (PPT). Two sit es of initiation located at the 5' boundary of the 3' long terminal re peat (PPT 1) acid near the middle of the TyB (pol) gene in the integra se coding sequence (PPT1) have been identified in the genome of Ty1. T he two PPTs have an identical sequence, TGGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abo lish initiation of plus strand DNA synthesis. Ty1 elements bearing a m utated PPT2 sequence are not defective for transposition whereas mutat ions in PPT1 abolish transposition.