Crosslink Mapping at Amino Acid-Base Resolution Reveals the Path of Scrunched DNA in Initial Transcribing Complexes.
Crosslink Mapping at Amino Acid-Base Resolution Reveals the Path of Scrunched DNA in Initial Transcribing Complexes.
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DOI：
10.1016/j.molcel.2015.06.037
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发表时间：
2015-09-03
期刊：
影响因子：
16
通讯作者：
Gourse RL
中科院分区：
文献类型：
--
作者：
Winkelman JT;Winkelman BT;Boyce J;Maloney MF;Chen AY;Ross W;Gourse RL
RNA聚合酶与DNA紧密结合，以高特异性识别启动子，但随后在转录的初始阶段释放这些接触。我们报告了一种位点特异性交联的方法来映射细菌转录中间体的DNA路径在氨基酸和核苷酸分辨率。在通过显示开放复合物（RPO）中的DNA路径与高分辨率X射线结构中的DNA路径相同来验证该方法之后，我们定义了在“紧缩”复合物（RPITC）中添加底物之后的路径。在RPITC中的转录泡内形成的DNA凸起在两条链上的位置不同。我们的数据表明，在含有5-mer RNA的复合物中，非模板链凸起被挤出到溶剂中，而模板链凸起保留在模板链隧道内，对β flap-β′ clamp-σ3.2相互作用施加应力。我们认为，这种压力有助于σ3.2从RNA出口通道的位移，促进启动子逃逸。
RNA polymerase binds tightly to DNA to recognize promoters with high specificity but then releases these contacts during the initial stage of transcription. We report a site-specific crosslinking approach to map the DNA path in bacterial transcription intermediates at amino acid and nucleotide resolution. After validating the approach by showing that the DNA path in open complexes (RPO) is the same as in high resolution X-ray structures, we define the path following substrate addition in ‘scrunched’ complexes (RPITC). The DNA bulges that form within the transcription bubble in RPITC are positioned differently on the two strands. Our data suggest that the non-template strand bulge is extruded into solvent in complexes containing a 5-mer RNA, whereas the template strand bulge remains within the template strand tunnel, exerting stress on the β flap-β′ clamp-σ3.2 interaction. We propose that this stress contributes to σ3.2 displacement from the RNA exit channel, facilitating promoter escape.