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首页> 外文期刊>Molecular and Cellular Biology >G Clustering Is Important for the Initiation of Transcription-Induced R-Loops In Vitro, whereas High G Density without Clustering Is Sufficient Thereafter
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G Clustering Is Important for the Initiation of Transcription-Induced R-Loops In Vitro, whereas High G Density without Clustering Is Sufficient Thereafter

机译：G聚类对于转录诱导的R环的体外启动很重要，而此后没有聚类的高G密度就足够了

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R-loops form cotranscriptionally in vitro and in vivo at transcribed duplex DNA regions when the nascent RNA is G-rich, particularly with G clusters. This is the case for phage polymerases, as used here (T7 RNA polymerase), as well as RNA polymerases in bacteria, Saccharomyces cerevisiae, avians, mice, and humans. The nontemplate strand is left in a single-stranded configuration within the R-loop region. These structures are known to form at mammalian immunoglobulin class switch regions, thus exposing regions of single-stranded DNA for the action of AID, a single-strand-specific cytidine deaminase. R-loops form by thread-back of the RNA onto the template DNA strand, and here we report that G clusters are extremely important for the initiation phase of R-loop formation. Even very short regions with one GGGG sequence can initiate R-loops much more efficiently than random sequences. The high efficiencies observed with G clusters cannot be achieved by having a very high G density alone. Annealing of the transcript, which is otherwise disadvantaged relative to the nontemplate DNA strand because of unfavorable proximity while exiting the RNA polymerase, can offer greater stability if it occurs at the G clusters, thereby initiating an R-loop. R-loop elongation beyond the initiation zone occurs in a manner that is not as reliant on G clusters as it is on a high G density. These results lead to a model in which G clusters are important to nucleate the thread-back of RNA for R-loop initiation and, once initiated, the elongation of R-loops is primarily determined by the density of G on the nontemplate DNA strand. Without both a favorable R-loop initiation zone and elongation zone, R-loop formation is inefficient.
机译：当新生的RNA富含G，尤其是带有G簇时，R-环在转录的双链DNA区域在体外和体内共转录。此处使用的噬菌体聚合酶（T7 RNA聚合酶），以及细菌，酿酒酵母，禽类，小鼠和人类中的RNA聚合酶就是这种情况。非模板链以单链构型保留在R环区域内。已知这些结构在哺乳动物免疫球蛋白类转换区形成，从而使单链DNA区域暴露于AID（一种单链特异性胞苷脱氨酶）的作用。 R环通过将RNA穿线回到模板DNA链上而形成，在这里我们报道G簇对于R环形成的起始阶段极为重要。即使是只有一个GGGG序列的非常短的区域，也可以比随机序列更有效地启动R环。仅通过具有非常高的G密度无法实现用G簇观察到的高效率。转录本的退火相对于非模板DNA链而言是不利的，因为退出RNA聚合酶时不利的邻近性，如果它在G簇上发生，则可以提供更高的稳定性，从而启动R环。 R环延伸超出起始区域的方式不像依赖于高G密度那样依赖于G簇。这些结果导致了一个模型，其中G簇对于R环起始的RNA形成核的成核非常重要，一旦启动，R环的延伸主要取决于非模板DNA链上G的密度。如果没有良好的R环起始区和延伸区，R环的形成效率很低。

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