Novel transcript truncating function of Rap1p revealed by synthetic codon-optimized Ty1 retrotransposon

Robert M. Yarrington, Sarah M. Richardson, Cheng Ran Lisa Huang, Jef D. Boeke

    Research output: Contribution to journalArticle

    Abstract

    Extensive mutagenesis via massive recoding of retrotransposon Ty1 produced a synthetic codon-optimized retrotransposon (CO-Ty1). CO-Ty1 is defective for retrotransposition, suggesting a sequence capable of down-regulating retrotransposition. We mapped this sequence to a critical ~20-bp region within CO-Ty1 reverse transcriptase (RT) and confirmed that it reduced Ty1 transposition, protein, and RNA levels. Repression was not Ty1 specific; when introduced immediately downstream of the green fluorescent protein (GFP) stop codon, GFP expression was similarly reduced. Rap1p mediated this down-regulation, as shown by mutagenesis and chromatin immunoprecipitation. A regular threefold drop is observed in different contexts, suggesting utility for synthetic circuits. A large reduction of RNAP II occupancy on the CO-Ty1 construct was observed 39 to the identified Rap1p site and a novel 39 truncated RNA species was observed. We propose a novel mechanism of transcriptional regulation by Rap1p whereby it serves as a transcriptional roadblock when bound to transcription unit sequences.

    Original languageEnglish (US)
    Pages (from-to)523-535
    Number of pages13
    JournalGenetics
    Volume190
    Issue number2
    DOIs
    StatePublished - Feb 1 2012

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    ASJC Scopus subject areas

    • Genetics

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