CarD stabilizes mycobacterial open complexes via a two-tiered kinetic mechanism.
CarD stabilizes mycobacterial open complexes via a two-tiered kinetic mechanism.
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DOI：
10.1093/nar/gkv078
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发表时间：
2015-03-31
影响因子：
14.9
通讯作者：
Galburt EA
中科院分区：
文献类型：
--
作者：
Rammohan J;Ruiz Manzano A;Garner AL;Stallings CL;Galburt EA
CardD是分枝杆菌中一种重要的全局性转录调节因子。虽然其生物学作用尚不清楚，但CardD通过直接与RNA聚合酶（RNAP）全酶启动子复合物相互作用来发挥作用。在这里，我们使用开放复合物的荧光报告分子，在真实的时间内定量RPo的形成，并显示结核分枝杆菌CardD对RNAP结合复合物在M.结核rrnAP 3核糖体RNA启动子。这些数据表明，牛分枝杆菌RNAP表现出一个不稳定的RPo是由CardD稳定，并建议CardD使用一个两层的，浓度依赖性的机制，通过与开放和封闭的复合物与不同的亲和力。具体而言，开放复合物形成的动力学可以通过一个模型来解释，其中，在饱和浓度的CardD，气泡崩溃的速度减慢，开放的速度加快。CardD突变体的动力学和开放复合物的稳定性进一步阐明了先前显示影响体内CardD依赖性基因调控的关键残基W85、K90和R25所发挥的作用。与M.与牛RNAP相比，大肠杆菌RNAP有效地在rrnAP 3上形成RPo，这表明聚合酶本身之间的重要差异，并突出了转录机制如何在细菌属之间变化。
CarD is an essential and global transcriptional regulator in mycobacteria. While its biological role is unclear, CarD functions by interacting directly with RNA polymerase (RNAP) holoenzyme promoter complexes. Here, using a fluorescent reporter of open complex, we quantitate RPo formation in real time and show that Mycobacterium tuberculosis CarD has a dramatic effect on the energetics of RNAP bound complexes on the M. tuberculosis rrnAP3 ribosomal RNA promoter. The data reveal that Mycobacterium bovis RNAP exhibits an unstable RPo that is stabilized by CarD and suggest that CarD uses a two-tiered, concentration-dependent mechanism by associating with open and closed complexes with different affinities. Specifically, the kinetics of open-complex formation can be explained by a model where, at saturating concentrations of CarD, the rate of bubble collapse is slowed and the rate of opening is accelerated. The kinetics and open-complex stabilities of CarD mutants further clarify the roles played by the key residues W85, K90 and R25 previously shown to affect CarD-dependent gene regulation in vivo. In contrast to M. bovis RNAP, Escherichia coli RNAP efficiently forms RPo on rrnAP3, suggesting an important difference between the polymerases themselves and highlighting how transcriptional machinery can vary across bacterial genera.