ENAapplication/xmlftp.sra.ebi.ac.uk/vol1/fastq/SRR545/004/SRR5453804/SRR5453804_2.fastq.gzftp.sra.ebi.ac.uk/vol1/fastq/SRR545/003/SRR5453803/SRR5453803_2.fastq.gzftp.sra.ebi.ac.uk/vol1/fastq/SRR545/001/SRR5453801/SRR5453801_1.fastq.gzftp.sra.ebi.ac.uk/vol1/fastq/SRR545/006/SRR5453806/SRR5453806_1.fastq.gzftp.sra.ebi.ac.uk/vol1/fastq/SRR545/002/SRR5453802/SRR5453802_1.fastq.gzftp.sra.ebi.ac.uk/vol1/fastq/SRR545/004/SRR5453804/SRR5453804_1.fastq.gzftp.sra.ebi.ac.uk/vol1/fastq/SRR545/005/SRR5453805/SRR5453805_1.fastq.gzftp.sra.ebi.ac.uk/vol1/fastq/SRR545/003/SRR5453803/SRR5453803_1.fastq.gzftp.sra.ebi.ac.uk/vol1/fastq/SRR545/006/SRR5453806/SRR5453806_2.fastq.gzftp.sra.ebi.ac.uk/vol1/fastq/SRR545/005/SRR5453805/SRR5453805_2.fastq.gzftp.sra.ebi.ac.uk/vol1/fastq/SRR545/002/SRR5453802/SRR5453802_2.fastq.gzftp.sra.ebi.ac.uk/vol1/fastq/SRR545/001/SRR5453801/SRR5453801_2.fastq.gzprimaryOK2000000GenomicsSuzanne Sandmeyer, Biological Chemistry, University of California Irvinehttps://www.ebi.ac.uk/ena/browser/view/PRJNA383299Saccharomyces cerevisiaeThe Gypsy-like element Ty3 inserts proximal to the transcription start sites of genes transcribed by RNA polymerase 3 (RNAP3). In this study, a random-barcode Ty3 was used to count Ty3 insertions at specific sites. Surprisingly, saturation transposition of the yeast genome showed that tDNAs even within isoacceptor families are targeted at widely different frequencies. Ectopic expression of Ty3 integrase showed that it localizes to integration targets independent of other Ty3 proteins. Binding of integrase, RNAP3 and factor Brf1 at individual targets did not differ to the same extent as integration. Metadata analysis showed that histone modification H3K4Ac correlated positively with insertion frequency. Targeting frequency could be reconstituted on high copy plasmids containing only 75 bp of 5’ flanking sequence plus the tDNA target. Weighting of insertions according to frequency identified an A/T-rich sequence followed by C as the site of gene-proximal strand transfer. This site lies immediately adjacent to the adenines of the RNAP3 transcription start site motif (CAA). Recent structures of DNA in RNAP3 initiation complexes show that in the initiation complex the transcription start site is sharply bent at the position adjacent to the gene-proximal Ty3 strand transfer. We propose that Ty3 integration occurs in two steps: in the first, host Brf1 engages integrase; in the second, integrase exploits YR flexibility, and that together, these steps determine the wide range of Ty3 targeting frequencies. Overall design: The Ty3 Integrase was cloned into Estradiol-inducible plasmid pKP3915 with a N-terminal 3X FLAG sequence to make plasmid pKP4010. For ChIP-seq, pKP4010 or pKP3915 (empty vector control) were transformed into the S. cerevisiae strain BY4741 and selected for on synthetic media lacking Uracil. Prior to harvesting cells for immuoprecipitation, 10 µM β-estradiol was added to cultures to induce integrase expression for approximately six hours.ENASite, HERV-K(III) Pol protein, Polymerase III, RT, Start Site, RNA, Transcription, HERV-K10 Pol protein, Ribonuclease H, HERV-K_5q33.3 provirus ancestral Pol protein, HERV-K_3q27.2 provirus ancestral Pol protein, Transcription Initiation Sites, INS, Transcription Start Sites, HERV-K(C7) Pol protein, HERV-K113 Pol protein, Transcription Start, Start Sites, Transcription Start Site, HERV-K102 Pol protein, Integrase, Transcription Initiation, Reverse transcriptase, HERV-K107 Pol protein, Polymerase C, HERV-K115 Pol protein, IN, HERV-K_11q22.1 provirus ancestral Pol protein, Initiation Site, HERV-K108 Pol protein, HERV-K(C19) Pol protein, Initiation Sites, HERV-K_7p22.1 provirus ancestral Pol protein, HERV-K_1q22 provirus ancestral Pol protein., DNA Dependent RNA Polymerase III, DNA-Dependent RNA Polymerase III, HERV-K_19q11 provirus ancestral Pol protein, Sites, RNA Polymerase C, 3.1.26.4, HERV-K_8p23.1 provirus ancestral Pol protein, HERV-K(HML-2.HOM) Pol protein, HERV-K_19p13.11 provirus ancestral Pol protein, 2.7.7.49, RNase HSaccharomyces oviformis, Baker's Yeasts, Yeast, S cerevisiae, Candida robusta, Saccharomyces capensis, Saccharomyces diastaticus, Saccharomyces cerevisiae 'var. diastaticus', Baker's, Brewer's, S. cerevisiae., baker's yeast, Mycoderma cerevisiae, Baker, Baker's Yeast, brewer's yeast, Saccharomyces uvarum var. melibiosus, Brewer's Yeast, Saccharomyces italicus, Baker Yeast0.00.00.00.00.00falseSaccharomyces cerevisiaeMolecular Signatures of retrotransposon Ty3 insertion into the RNA Polymerase III transcription initiation site (Integrase)2022-05-122018-04-16PRJNA383299GSE97892312490624932