Molecular mechanism of transcription inhibition by peptide antibiotic microcin J25
Molecular mechanism of transcription inhibition by peptide antibiotic microcin J25
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DOI：
10.1016/j.molcel.2004.05.017
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发表时间：
2004-06-18
期刊：
影响因子：
16
通讯作者：
Severinov, K
中科院分区：
文献类型：
--
作者：
Adelman, K;Yuzenkova, J;Severinov, K
21个氨基酸的肽Microcin J25（MccJ 25）抑制细菌RNA聚合酶（RNAP）的转录。MccJ 25抗性突变聚集在RNAP二级通道中，通过该通道，认为进入的NTP底物到达催化中心，并且新生RNA的3'端可能穿入回溯转录复合物中。次级通道也接受转录物切割因子GreA和GreB。在这里，我们证明MccJ 25抑制GreA/GreB依赖的转录物切割，阻碍形成回溯复合物，并可以交联到延伸复合物中新生RNA的X-末端。这些结果将MccJ 25结合位点置于二级通道内。此外，单分子测定显示MccJ 25结合到转录RNAP暂时停止转录延长，但对暂停之间的延长速度没有影响。单分子数据的动力学分析，使我们能够提出一个模型的MccJ 25的转录抑制，设想完全闭塞的二级通道结合抑制剂。
21 amino acid peptide Microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3' end of the nascent RNA is likely to thread in backtracked transcription complexes. The secondary channel also accepts transcript cleavage factors GreA and GreB. Here, we demonstrate that MccJ25 inhibits GreA/GreB-dependent transcript cleavage, impedes formation of backtracked complexes, and can be crosslinked to the X-end of the nascent RNA in elongation complexes. These results place the MccJ25 binding site within the secondary channel. Moreover, single-molecule assays reveal that MccJ25 binding to a transcribing RNAP temporarily stops transcript elongation but has no effect on the elongation velocity between pauses. Kinetic analysis of single-molecule data allows us to put forward a model of transcription inhibition by MccJ25 that envisions the complete occlusion of the secondary channel by bound inhibitor.