| The identification of the critical components of the Ty1 RNA and element-encoded proteins has allowed and will continue to allow detailed in vivo genetic studies and biochemical characterization in vitro. Genetic and biochemical studies of the replication of the Ty1 retrotransposon in Saccharomyces cerevisiae are described here. We describe a modular mini-Ty1 element encoding the minimal sequence capable of being retrotransposod by the Ty1 proteins, supplied in trans by a helper construct. Using a genetic screening strategy, we recovered transposition-deficient modular mini-Ty1-HIS3 elements; many of these possessed mutations in sequences required in cis for Ty1 replication and integration. Moreover, two distinct clusters of mutations mapped to the GAGGAGA sequence at the extreme 5′ end of the Ty1 transcript and the complementary downstream UCUCCUC sequence, 264 nucleotides into the RNA. Disruption of the reverse complementarity of these two sequences decreased Ty1 cDNA accumulation and transposition, despite normal levels of Ty1 RNA and proteins. Restoration of complementarity rescued cDNA production and transposition to wild-type levels. We propose that the interaction between the 5′ GAGGAGA and UCUCCUC sequences allows Ty1 RNA to form a dimeric structure, similar to but structurally distinct from the retroviral “kissing-loop” dimerization motif.; We show that mutations in PMR1, a yeast gene encoding a calcium/manganese exporter, dramatically decrease Ty1 retrotransposition. Ty1 cDNA is reduced in pmr1 mutant cells, despite normal levels of Ty RNA and proteins. The transposition defect results from Mn2+ accumulation, which in turn inhibits reverse transcription. Intracellular accumulation of Mn2+ in pmr1 cells directly affects Ty 1 reverse transcriptase (RT) activity. Trace amounts of Mn2+ potently inhibit Ty1 RT and HIV-1 RT in vitro when the preferred cation, Mg2+, is present in saturating amounts. Both Mn2+ and Mg2+ alone activate Ty1 RT cooperatively with Hill coefficients of 2, providing strong kinetic evidence for a dual divalent cation requirement at the RT active site. These findings emphasize the importance of metal ion clusters in catalysis and suggest a novel class of RT inhibitors, based on alteration of intracellular metal ion concentrations. |
