Studying the replication mechanism of the yeast retrotransposon Ty5 by molecular and computational approaches

Date
2001-01-01
Authors
Gao, Xiang
Major Professor
Advisor
Daniel F. Voytas
Leslie G. Miller
Committee Member
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Zoology and Genetics
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Zoology and Genetics
Abstract

The yeast retrotransposon Ty5 is a Ty1/copia element. It is in the Hemivirus genus of the Pseudoviridae family. The ability to genetically manipulate retrotransposons and the yeast host cell was taken advantage of to explore replication mechanisms unique to Ty5 and common to most retrotransposons. Because of the abundance and diversity of retroelement sequences, along with the fact that many retroelement enzymes have evolved unique functional specificities, computational approaches were also developed to study functional divergence in replication. By screening a randomly mutagenized Ty5 library, two mutations (Y68C, D252N) causing higher transposition frequencies were identified. Both mutations increased Ty5 cDNA levels, but did not have dramatic effects on the steps after cDNA synthesis (i.e. integration and recombination), or protein synthesis, processing, or solubility. The D252N mutation increased the hydrogen bonding potential of the CCHC zinc finger of nucleocapsid protein (NCp), making the Ty5 NCp zinc finger more like Ty1/copia consensus zinc fingers in terms of hydrogen bonding potential. Other mutations that increased the hydrogen bonding potential (D252R, D252K) provided the same fold increase in Ty5 transposition. These results suggest that NCp and its CCHC domain play an important role in Ty5 reverse transcription, and natural occurring mutations in the Ty5 zinc finger repress this function. Hydrogen bonding is suggested to be a universal requirement for the function of retroviral type zinc fingers and cellular zinc fingers. A half-tRNA priming mechanism for Ty5 reverse transcription was also demonstrated. Mutations in the anticodon of tRNAi Met (IMT) and the putative PBS of Ty5 decreased transposition, but transposition was restored when complementarity between the IMT and PBS was restored. A tree-based method and supplemental Split Tester software were developed to study the functional divergence of reverse transcriptase (RT) with respect to half tRNA and full-tRNA priming mechanisms. The domains identified by this computational approach were previously experimentally demonstrated to bind with the tRNA primer/template in HIV RT. Using this software, another domain related to integrase functional specificity, namely whether or not integrase carries out 3'-end processing during integration, was also consistently identified in different integrase datasets. A model describing this functional divergence is proposed.

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