Expression and processing of proteins encoded by the Saccharomyces retrotransposon Ty5.

Retroelements (retrotransposons and retroviruses) have two genes in common: gag, which specifies structural proteins that form a virus or virus-like particle, and pol, which specifies catalytic proteins required for replication. For many retroelements, gag and pol are present on separate reading frames. Their expression is highly regulated, and the ratio of Gag to Pol is critical for retroelement replication. The Saccharomyces retrotransposon Ty5 contains a single open reading frame, and we characterized Gag and Pol expression by generating transpositionally active Ty5 elements with epitope tags at the N terminus or C terminus or within the integrase coding region. Immunoblot analysis identified two Gag species (Gag-p27 and Gag-p37), reverse transcriptase (Pol-p59), and integrase (Pol-p80), all of which are largely insoluble in the absence of urea or ionic detergent. These proteins result from proteolytic processing of a polyprotein, because elements with mutations in the presumed active site of Ty5 protease express a single tagged protein (Gag-Pol-p182). Protease mutants are also transpositionally inactive. In a time course experiment, we monitored protein expression, proteolytic processing, and transposition of a Ty5 element with identical epitope tags at its N and C termini. Both transposition and the abundance of Gag-p27 increased over time. In contrast, the levels of Gag-p37 and reverse transcriptase peaked after approximately 14 h of induction and then gradually decreased. This may be due to differences in stability of Gag-p27 relative to Gag-p37 and reverse transcriptase. The ratio of Ty5 Gag to Pol averaged 5:1 throughout the time course experiment, suggesting that differential protein stability regulates the amounts of these proteins.

The pheromone response pathway activates transcription of Ty5 retrotransposons located within silent chromatin of Saccharomyces cerevisiae.

The Saccharomyces retrotransposon Ty5 integrates preferentially into transcriptionally inactive regions (silent chromatin) at the HM loci and telomeres. We found that silent chromatin represses basal Ty5 transcription, indicating that these elements are encompassed by silent chromatin in their native genomic context. Because transcription is a requirement for transposition, integration into silent chromatin would appear to prevent subsequent rounds of replication. Using plasmid-borne Ty5-lacZ constructs, we found that Ty5 expression is haploid specific and is repressed 10-fold in diploid strains. Ty5 transcription is also regulated by the pheromone response pathway and is induced approximately 20-fold upon pheromone treatment. Deletion analysis of the Ty5 LTR promoter revealed that a 33 bp region with three perfect matches to the pheromone response element is responsible for both mating pheromone and cell-type regulation. Transcriptional repression of Ty5 by silent chromatin can be reversed by pheromone treatment, which leads to transcription and transposition. Ty5 replication, therefore, is normally repressed by silent chromatin and appears to be induced during mating. This is the first example of transcriptional activation of a gene that naturally resides within silent chromatin.