Host Cell Control of Yeast Ty3 Element Reverse Transcription

University Of Missouri-Kansas City, Columbia MO

Investigators

Abstract

Retroelements are mobile DNA elements found integrated in the genomes of all eukaryotes and many prokaryotes. The hallmark of retroelements is the copying of an RNA intermediate into DNA by the enzyme reverse transcriptase (RT). Reverse transcription by retroelement RT's has also been a major force in the shaping of eukaryotic genomes. The cDNA's produced from retroelement and cellular mRNA's by reverse transcription insert into host cell chromosomes and constitute a significant fraction of many genomes, creating structural and functional consequences. Two related subgroups of retroelements, widespread in eukaryotes, are retroviruses and the long terminal repeat (LTR) retrotransposons. Because of their limited coding capacity, retroviruses and the LTR retrotransposons depend on their hosts to perform most of the functions necessary for their propagation. Two areas are under investigation that concern host cell control of reverse transcription in the Ty3 LTR retrotransposon of Saccharomyces cerevisiae. First, it has been previously shown that during the G1 phase of the cell cycle, the Ty3 transposition is blocked at reverse transcription. Although Ty3 viruslike particles are produced at equivalent levels in dividing cells and cells arrested in G1, the RT enzyme is inactive in arrested cells. The enzyme becomes active when arrested cells are returned to a dividing state. These data strongly suggest that host factors regulate the activity of Ty3 reverse transcriptase in the cell cycle, which represents a novel cellular control of retroelements. Second, in a genome-wide screen, two mutants that are deficient for Ty3 reverse transcription have been identified. These mutants have been named trt1 and trt2, for Ty3 reverse transcription. RT enzyme isolated from these mutants has reduced activity, suggesting that the proteins encoded by TRT1 and TRT2 directly or indirectly activate the Ty3 RT enzyme. Two goals will be pursued. Goal 1 will be to determine how host genes modulate cell cycle control of Ty3 RT activity. The objectives will be to determine whether Ty3 RT is positively or negatively regulated in the cell cycle, to determine if Ty3 RT is physically modified in response to cell cycle regulation, and to identify host genes that play roles in cell cycle regulation of Ty3 RT activity. In vitro Ty3 RT enzyme assays will be used to assay the mode of regulation, while physical probes will be used to assess Ty3 RT's from dividing and arrested cells for differences. A genetic screen will be used to identify host genes involved in the cell cycle regulation of Ty3 RT activity. Goal 2 will be to identify host genes that activate Ty3 reverse transcription and establish their relationship to each other and to Ty3 RT. The objectives will be to clone the wild-type TRT1 and TRT2 genes, to study the effects of the TRT1 and TRT2 gene products in detail, and to saturate the screen for mutants that exhibit decreased Ty3 reverse transcription. Genetic screens will be used both to isolate the TRT1 and TRT2 genes as well as to isolate additional mutants deficient for Ty3 reverse transcription. Molecular genetic manipulations will then be used to analyze the TRT1 and TRT2 genes. Understanding how reverse transcription is controlled is key to understanding the forces and counterforces that have shaped the genomes of eukaryotes. Although many different aspects of retroelement biology contribute to their mobility, control of reverse transcription is an underexplored area. Moreover, focusing on control of one aspect of the Ty3 life cycle will suggest the extent to which other aspects of the life cycle may be controlled. Understanding what controls Ty3 reverse transcription may lead to ways of controlling the mobility of other retroelements, including retroviruses, with potential therapeutic consequences. It is even reasonable to imagine that mechanisms that regulate Ty3 reverse transcription could serve as models for the control of eukaryotic DNA replication. Finally, the reverse transcriptase enzyme has been harnessed as a tool for use by molecular biologists. Understanding how this enzyme is controlled within the cell can lead to its improved utility.