Molecular Signatures of retrotransposon Ty3 insertion into the RNA Polymerase III transcription initiation site (Integrase)

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.类Gypsy转座元件Ty3可插入于由RNA聚合酶3（RNA polymerase 3，RNAP3）转录的基因的转录起始位点近端。本研究采用带有随机条形码的Ty3（random-barcode Ty3），对特定位点的Ty3插入事件进行定量计数。令人意外的是，对酵母基因组的饱和转座（saturation transposition）分析显示，即便属于同功受体家族的转运RNA基因（transfer RNA genes，tDNAs），其被Ty3靶向的频率也存在显著差异。Ty3整合酶的异位表达实验表明，其可独立于其他Ty3蛋白靶向整合位点。整合酶、RNAP3与转录因子Brf1（factor Brf1）在各靶点的结合程度差异，并未达到整合频率差异的同等幅度。元数据分析（metadata analysis）显示，组蛋白H3第4位赖氨酸乙酰化修饰（H3K4Ac）与插入频率呈正相关。仅携带75 bp 5'侧翼序列与tDNA靶点的高拷贝质粒（high copy plasmids），即可重现Ty3的靶向频率特征。按插入频率对插入事件进行加权分析后，确定了一段富含A/T且后续紧跟碱基C的序列，作为基因近端的链转移位点。该位点紧邻RNAP3转录起始位点基序（CAA）中的腺嘌呤序列。近期解析的RNAP3起始复合物（RNAP3 initiation complexes）中的DNA结构显示，在起始复合物内，转录起始位点在紧邻基因近端Ty3链转移位点的位置发生剧烈弯折。我们提出Ty3整合过程分为两个步骤：第一步，宿主转录因子Brf1与整合酶结合；第二步，整合酶利用YR位点的柔性，二者共同决定了Ty3靶向频率的广泛差异。