title: Novel transcript truncating function of Rap1p revealed by synthetic codon-optimized Ty1 retrotransposon.
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 3' to the identified Rap1p site and a novel 3' 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.
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Yarrington
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Richardson
authors.forename: Cheng Ran
authors.initials: CR
authors.lastname: Lisa Huang
authors.forename: Jef D
authors.initials: JD
authors.lastname: Boeke
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003062:Codon; D015966:Gene Expression Regulation, Fungal; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D059372:Nucleotide Motifs; D054730:Protein Interaction Domains and Motifs; D012333:RNA, Messenger / Q000378:metabolism; D018626:Retroelements / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D003062:Codon; C440829:RAP1 protein, S cerevisiae; D012333:RNA, Messenger; D018626:Retroelements; D029701:Saccharomyces cerevisiae Proteins; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D014157:Transcription Factors