Ty1, a long terminal repeat retrotransposon of Saccharomyces, is structurally and functionally related to retroviruses. However, a differentiating aspect between these retroelements is the diversity of the replication strategies used by long terminal repeat retrotransposons. To understand the structural organization of cis-acting elements present on Ty1 genomic RNA from the GAG region that control reverse transcription, we applied chemoenzymatic probing to RNA/tRNA complexes assembled in vitro and to the RNA in virus-like particles. By comparing different RNA states, our analyses provide a comprehensive structure of the primer-binding site, a novel pseudoknot adjacent to the primer-binding sites, three regions containing palindromic sequences that may be involved in RNA dimerization or packaging and candidate protein interaction sites. In addition, we determined the impact of a novel form of transposon control based on Ty1 antisense transcripts that associate with virus-like particles. Our results support the idea that antisense RNAs inhibit retrotransposition by targeting Ty1 protein function rather than annealing with the RNA genome.

Transposable elements impact genome function by altering gene expression and causing chromosome rearrangements. As a result, organisms have evolved mechanisms, such as RNA-interference, to minimize the level of transposition. However, organisms without the conserved RNAi pathways, like Saccharomyces cerevisiae, must use other mechanisms to prevent transposon movement. Here, we provide evidence that antisense (AS) RNAs from the retrovirus-like element Ty1 inhibit retrotransposition posttranslationally in Saccharomyces. Multiple Ty1AS transcripts overlap Ty1 sequences necessary for copy number control (CNC) and inhibit transposition in trans. Altering Ty1 copy number or deleting sequences in the CNC region that are required for reverse transcription affect Ty1AS RNA level and Ty1 movement. Ty1AS RNAs are enriched in viruslike particles, and are associated with a dramatic decrease in the level of integrase, less reverse transcriptase, and an inability to synthesize Ty1 cDNA. Thus, Ty1AS RNAs are part of an intrinsic mechanism that limits retrotransposition by reducing the level of proteins required for replication and integration.retrotransposon ͉ virus-like particles ͉ RNA interference ͉ Saccharomyces R etrotransposons replicate through an RNA intermediate and can comprise over half the genome in many eukaryotes. To maintain genome stability, forms of RNAi have evolved to inhibit transposition (1). However, Saccharomyces cerevisiae lacks the genes required for RNAi, yet still maintains control over Ty1 retrotransposition. Ty1 elements and retroviruses have similar structures and strategies for gene expression, and encode functionally equivalent Gag and Pol proteins protease (PR), integrase (IN), and reverse transcriptase (RT) (2). Ty1 mRNA has unique subterminal 5Ј (U5) and 3Ј (U3) sequence motifs and a terminally repetitious (R) region. These sequences are required for reverse transcription and integration and form the U3-R-U5 segments of the LTR that bracket the element in the genome. Adjacent to U5 is the primer-binding site (pbs), where tRNA-Met i anneals with Ty1 mRNA to initiate reverse transcription within cytoplasmic virus-like particles (VLPs). Although not completely defined, sequences within GAG also enhance Ty1 mRNA dimer formation and packaging into VLPs (3, 4). The primary translation products are Gag-p49 and Gag-Pol-p199 that are cleaved by PR to form mature Gag-p45, PR-p20, IN-p71, and RT-p63. An association between IN and RT is required for reverse transcription in vivo and RT activity in vitro (5-7). Retrotransposition is completed by IN-mediated integration of Ty1 cDNA into the genome.Despite high levels of Ty1 mRNA and many functional elements in the genome, transposition occurs at a low rate (8-10). Cells also contain a low level of mature Ty1 proteins (11), and few VLPs are present (12). When an active Ty1 element is fused to the GAL1 promoter and carried on a multicopy pGTy1 plasmid (13), galactose induction overcomes the barriers to transposition. However, the mechanism underlying this obse...