Mechanism of sequence-specific pausing of bacterial RNA polymerase
Mechanism of sequence-specific pausing of bacterial RNA polymerase
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DOI：
10.1073/pnas.0900407106
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发表时间：
2009-06-02
影响因子：
11.1
通讯作者：
Kashlev, Mikhail
中科院分区：
文献类型：
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作者：
Kireeva, Maria L.;Kashlev, Mikhail
多亚基RNA聚合酶（RNAP）的序列特异性暂停是转录延伸过程中的限速步骤。平均每100个DNA碱基就会出现一次停顿。已经提出了几种模型来解释停顿，包括三元延伸复合物的回溯，酶沿着DNA的移位延迟，或活性位点的构象变化阻止磷酸二酯键的形成。在这里，我们进行了生化表征先前报道的大肠杆菌RNAP的暂停，并发现它们与回溯或易位延迟无关。相反，暂停的复合物在活性中心含有转录物的3'末端，并且能够结合下一个同源NTP。然而，与完全活性的配合物相比，暂停的配合物中键的形成要慢得多。通过替换编码下一个进入的NTP的碱基和编码新生RNA的3'端的碱基，暂停显著减少，表明RNA的3'端和活性位点中的进入的NTP的（错误）对齐对于暂停至关重要。这些暂停位点在E. coli和Thermus thermophilus RNAPs，但不被Saccharomyces cerevisiae RNAP II识别，表明原核RNAPs可能对活性位点的新生转录本和底物NTP的比对变化更敏感。
Sequence-specific pausing of multisubunit RNA polymerases (RNAPs) represents a rate-limiting step during transcription elongation. Pausing occurs on average every 100 bases of DNA. Several models have been proposed to explain pausing, including backtracking of the ternary elongation complex, delay of translocation of the enzyme along DNA, or a conformational change in the active site preventing formation of the phosphodiester bond. Here, we performed biochemical characterization of previously-reported pauses of Escherichia coli RNAP and found that they are not associated with backtracking or a translocation delay. Instead, the paused complex contains the 3' end of the transcript in the active center and is capable of binding the next cognate NTP. However, bond formation occurs much slower in the paused complex compared with its fully-active counterpart. The pausing is dramatically decreased by a substitution of the base encoding the next incoming NTP and the base encoding the 3' end of the nascent RNA, suggesting that (mis)-alignment of the 3' end of the RNA and the incoming NTP in the active site is crucial for pausing. These pause sites are conserved between E. coli and Thermus thermophilus RNAPs, but are not recognized by Saccharomyces cerevisiae RNAP II, indicating that prokaryotic RNAPs might be more sensitive to the changes in the alignment of the nascent transcript and the substrate NTP in the active site.