Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis.
Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis.
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DOI：
10.1016/j.jmb.2015.09.005
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发表时间：
2015-11-06
影响因子：
5.6
通讯作者：
Buck M
中科院分区：
文献类型：
--
作者：
Zhang N;Schäfer J;Sharma A;Rayner L;Zhang X;Tuma R;Stockley P;Buck M
在细菌RNA聚合酶（RNAP）中，桥式螺旋和开关区形成了一个复杂的网络，具有催化活性中心和主通道。这些相互作用对于催化、水解和钳位结构域运动是重要的。通过瞄准大肠杆菌RNAP中的保守残基，我们能够证明这些区域的功能在σ70依赖和σ54依赖的转录激活过程中是不同的，因此可能是两种转录模式之间关键机制差异的基础。我们进一步证明转录因子DksA直接调节σ54依赖的激活，有正向和负向的作用。这一发现与DksA对σ70依赖性启动子的影响一致。DksA似乎对RNAP与预熔启动子DNA的结合没有显著影响，但对σ54调控启动子初始RNA合成阶段的活性有广泛影响。引人注目的是，去除σ54 I区足以逆转DksA在两个测试启动子中的作用（从刺激到抑制，反之亦然）。我们生成的RNAP突变体也显示出强烈的回溯倾向。这些突变体即使在缺乏非互补核苷酸的情况下，也能将转录水解裂解的速率提高到与水热菌RNAP相当的水平。这些新的表型暗示了桥式螺旋和开关区域作为抗回溯棘轮和RNA水解调节剂的重要功能。桥螺旋和开关区域在RNAP中形成一个复杂的网络。σ70和σ54转录系统不同地使用这种相互作用网络。转录因子DksA和σ54 I区也参与了该网络。该网络的破坏增强了回溯和内在RNA水解。
In bacterial RNA polymerase (RNAP), the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNAP, we are able to show that functions of these regions are differentially required during σ70-dependent and the contrasting σ54-dependent transcription activations and thus potentially underlie the key mechanistic differences between the two transcription paradigms. We further demonstrate that the transcription factor DksA directly regulates σ54-dependent activation both positively and negatively. This finding is consistent with the observed impacts of DksA on σ70-dependent promoters. DksA does not seem to significantly affect RNAP binding to a pre-melted promoter DNA but affects extensively activity at the stage of initial RNA synthesis on σ54-regulated promoters. Strikingly, removal of the σ54 Region I is sufficient to invert the action of DksA (from stimulation to inhibition or vice versa) at two test promoters. The RNAP mutants we generated also show a strong propensity to backtrack. These mutants increase the rate of transcript-hydrolysis cleavage to a level comparable to that seen in the Thermus aquaticus RNAP even in the absence of a non-complementary nucleotide. These novel phenotypes imply an important function of the bridge helix and switch regions as an anti-backtracking ratchet and an RNA hydrolysis regulator. The bridge helix and switch regions form an intricate network in RNAP. The σ70 and σ54 transcription systems differentially use this interaction network. Transcription factor DksA and σ54 Region I also contribute to this network. Disruption of this network enhances backtracking and intrinsic RNA hydrolysis.