Targets of Ty3 transposition in Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE31820 | GEO | 2011/09/02
SECONDARY ACCESSION(S): PRJNA145135
REPOSITORIES: GEO
Similar Datasets
Project description:The goal of these experiments was to define the targets of Ty3 transposition in Saccharomyces cerevisiae. Ty3 is a retroviruslike element that is found at the transcription initiation site of chromosomal tRNA genes. A Ty3 that can be induced by growth in galactose-containing medium and which was marked by an insertion of HIS3 downstream of the second open reading frame of the element (POL3) was induced to undergo transposition by plating cells onto galactose containing medium and replica-plating onto medium selective for cells that had undergone transposition. These cells were collected, DNA was extracted, and inverse PCR was performed using primers inside the Ty3 element in order to generate a library of insertion sites flanked by Illumina sequence-compatible primers.
2011-09-02 | E-GEOD-31820 | biostudies-arrayexpress
Project description:Targets of Ty3 transposition in Saccharomyces cerevisiae
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Project description:The 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.
2018-04-15 | GSE97893 | GEO
Project description:The 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.
2018-04-15 | GSE97892 | GEO
Project description:The 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.
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