Watching the Bacteriophage N4 RNA Polymerase Transcription by Time-dependent Soak-trigger-freeze X-ray Crystallography
Watching the Bacteriophage N4 RNA Polymerase Transcription by Time-dependent Soak-trigger-freeze X-ray Crystallography
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DOI：
10.1074/jbc.m112.387712
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发表时间：
2013-02-01
影响因子：
4.8
通讯作者：
Murakami, Katsuhiko S.
中科院分区：
文献类型：
--
作者：
Basu, Ritwika S.;Murakami, Katsuhiko S.
结构生物学的挑战在于理解酶促反应过程中原子级的大分子运动。x射线晶体学可以显示高分辨率的结构；然而，一个明显的限制是它只能显示静态视图。在这里，我们使用时间依赖的浸泡-触发-冻结X射线晶体学，即将核苷酸和二价金属浸泡在噬菌体RNA聚合酶（RNAP）启动子DNA复合体晶体中触发核苷酸转移反应，并在不同的时间点冻结晶体，以捕获转录途径中的实时中间体。在每个晶体结构中，在RNAP活性位点显示出不同强度和形状的核苷酸和金属对应的电子密度图，从而可以从时间角度观察核苷酸和金属结合以及磷酸二酯键的形成。我们的研究提供了底物组装和酶活性位点金属辅因子结合的时间顺序，完成了我们对单亚基rnap的双金属离子机制和保真机制的理解。核苷酸结合金属（Me-B）先于催化金属（Me-A）在活性位点配位。Me-A配位只是暂时的，在磷酸二酯键形成之前建立并在形成后立即解离。我们利用晶体中缓慢的酶促反应捕获了这些难以捉摸的中间体。这些结果表明，简单的随时间变化的浸泡-触发-冻结X射线晶体学为监测酶促反应提供了一种直接的手段。
The challenge for structural biology is to understand atomic-level macromolecular motions during enzymatic reaction. X-ray crystallography can reveal high resolution structures; however, one perceived limitation is that it reveals only static views. Here we use time-dependent soak-trigger-freeze X- ray crystallography, namely, soaking nucleotide and divalent metal into the bacteriophage RNA polymerase (RNAP)-promoter DNA complex crystals to trigger the nucleotidyl transfer reaction and freezing crystals at different time points, to capture real-time intermediates in the pathway of transcription. In each crystal structure, different intensities and shapes of electron density maps corresponding to the nucleotide and metal were revealed at the RNAP active site which allow watching the nucleotide and metal bindings and the phosphodiester bond formation in a time perspective. Our study provides the temporal order of substrate assembly and metal co-factor binding at the active site of enzyme which completes our understanding of the two-metal-ion mechanism and fidelity mechanism in single-subunit RNAPs. The nucleotide-binding metal (Me-B) is coordinated at the active site prior to the catalytic metal (Me-A). Me-A coordination is only temporal, established just before and dissociated immediately after phosphodiester bond formation. We captured these elusive intermediates exploiting the slow enzymatic reaction in crystallo. These results demonstrate that the simple time-dependent soak-trigger-freeze X- ray crystallography offers a direct means for monitoring enzymatic reactions.