Allosteric control of catalysis by the F loop of RNA polymerase
Allosteric control of catalysis by the F loop of RNA polymerase
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DOI：
10.1073/pnas.0905402106
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发表时间：
2009-11-10
影响因子：
11.1
通讯作者：
Kulbachinskiy, Andrey
中科院分区：
文献类型：
--
作者：
Miropolskaya, Nataliya;Artsimovitch, Irina;Kulbachinskiy, Andrey
细菌RNA聚合酶（RNAP）在催化过程中经历协调的构象变化。特别是，协同折叠的触发环和重排的桥螺旋在RNAP活性中心已牵连在核苷酸添加和RNAP易位。在中等温度下，嗜热栖热菌（Thermus aquaticus）RNAP的催化速率与其最接近的嗜温亲戚耐辐射异常球菌（Deinococcus radiodurans）相比显着降低。在这里，我们表明，这种差异的一部分是由第三个元素，F环，这是相邻的桥螺旋的N末端，并直接接触折叠触发环。T中F环和桥螺旋的相邻片段中的氨基酸残基的取代。aquaticus RNAP为它们的D. radiodurans对应物显著提高了催化速率（在20摄氏度时高达40倍）。F环的缺失显著损害了核苷酸添加和焦磷酸解的速率，但它对内在RNA切割只有中等影响。链球菌溶血素是一种阻断触发环折叠的抗生素，它不能抑制突变酶添加核苷酸。对溶血链球菌素的抗性可能是由于其功能靶标的丧失，即触发环的折叠，其已经被F环缺失所削弱。我们的研究结果表明，F环是必不可少的触发环在核苷酸添加过程中的正确折叠，并管理在不同的细菌RNAP的温度适应性。
Bacterial RNA polymerases (RNAPs) undergo coordinated conformational changes during catalysis. In particular, concerted folding of the trigger loop and rearrangements of the bridge helix at the RNAP active center have been implicated in nucleotide addition and RNAP translocation. At moderate temperatures, the rate of catalysis by RNAP from thermophilic Thermus aquaticus is dramatically reduced compared with its closest mesophilic relative, Deinococcus radiodurans. Here, we show that a part of this difference is conferred by a third element, the F loop, which is adjacent to the N terminus of the bridge helix and directly contacts the folded trigger loop. Substitutions of amino acid residues in the F loop and in an adjacent segment of the bridge helix in T. aquaticus RNAP for their D. radiodurans counterparts significantly increased the rate of catalysis ( up to 40-fold at 20 degrees C). A deletion in the F loop dramatically impaired the rate of nucleotide addition and pyrophosphorolysis, but it had only a moderate effect on intrinsic RNA cleavage. Streptolydigin, an antibiotic that blocks folding of the trigger loop, did not inhibit nucleotide addition by the mutant enzyme. The resistance to streptolydigin likely results from the loss of its functional target, the folding of the trigger loop, which is already impaired by the F-loop deletion. Our results demonstrate that the F loop is essential for proper folding of the trigger loop during nucleotide addition and governs the temperature adaptivity of RNAPs in different bacteria.