Host factors that affect the transposition of Ty3 in Saccharomyces cerevisiae: Defining the role of TFIIIC95 in Ty3 integration.

摘要

Ty3 is a position-specific, transposable element of the budding yeast Saccharomyces cerevisiae. Although Ty3 lacks an obligate extracellular phase in its lifecycle, genomic arrangement and intracellular processes of Ty3 are similar to those of retroviruses.; In order to identify the cellular factors that affect the transposition of Ty3, a disruption mutagenesis approach was utilized to conduct a genetic screen. A total of 27,000 transformants were screened for transposition phenotype, and candidate mutants from the primary screen were retested to confirm the phenotype. After two rounds of screening, the genomic DNA disrupted in 62 mutants were cloned, sequenced and identified by the blast search of the Saccharomyces Genome Database. The known genes identified include cell cycle regulators, transcription and chromatin factors, protein kinases, nuclear pore proteins, RNA-binding proteins and factors involved in stability and degradation of proteins. Mutation in most of these genes resulted in reduced Ty3 transposition compared to the wild-type strain. Further characterization of a mutant that contained an insertion in the LHP1 gene, which encodes a RNA-binding protein, revealed that Ty3 transposition into a target plasmid was reduced by eight-fold.; Another mutant isolated had a mutation in the TFC1 gene, which produced a truncated 95 kD subunit of TFIIIC (TFIIIC95) and reduced the apparent retrotransposition of Ty3 into a target plasmid by 11-fold. Although TFIIIC95 is conserved and essential, no defect in growth nor transcription of tRNAs is detected in the mutant strain. Steps of the Ty3 lifecycle, such as protein expression, proteolytic processing, virus-like particle formation, and reverse transcription are not affected by the mutation. However, severe orientation bias in Ty3 integration into a divergent tDNA target was observed in the mutant strain. Investigation of the molecular basis for orientation bias showed that the carboxyl-terminus of TFIIIC95 lacking in the mutant is critical for interaction with Ty3 IN. These results argue for a direct role of TFIIIC in Ty3 integration in vivo, and suggest that the ends of this retrovirus-like element are not equivalent in target interaction. (Abstract shortened by UMI.)