Cooperative stabilization of Mycobacterium tuberculosis rrnAP3 promoter open complexes by RbpA and CarD.
Cooperative stabilization of Mycobacterium tuberculosis rrnAP3 promoter open complexes by RbpA and CarD.
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DOI：
10.1093/nar/gkw577
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发表时间：
2016-09-06
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;Prusa J;Stallings CL;Galburt EA
分枝杆菌必不可少的转录调控因子RbpA和CarD通过结合起始复合物和增加转录产物的通量来调节转录。这些因子中的每一个都直接与启动子DNA模板和RNA聚合酶（RNAP）全酶相互作用。我们最近用停流荧光法报道了卡介导的开放复合物稳定结核分枝杆菌rrnAP3核糖体启动子的能量学。在这里，我们将这种方法应用于RbpA，并表明RbpA通过与CarD不同的机制稳定rnap -启动子开放复合物（RPo）。此外，这两个因素的浓度依赖性停流实验表明，RbpA和CarD在稳定RPo的能力方面存在正联动（协同）关系。RbpA和CarD之间的正连锁表明，这两个因子可以同时作用于相同的转录起始复合物。最后，当这两种因素同时存在时，开放复合物形成的动力学明显快于单独存在任何一种因素时的动力学，并且接近大肠杆菌RNAP在相同启动子上的动力学。这项工作为这两个基本转录因子的分子机制及其在分枝杆菌生物学和病理学中发挥的关键作用提供了定量框架。
The essential mycobacterial transcriptional regulators RbpA and CarD act to modulate transcription by associating to the initiation complex and increasing the flux of transcript production. Each of these factors interacts directly with the promoter DNA template and with RNA polymerase (RNAP) holoenzyme. We recently reported on the energetics of CarD-mediated open complex stabilization on the Mycobacterium tuberculosis rrnAP3 ribosomal promoter using a stopped-flow fluorescence assay. Here, we apply this approach to RbpA and show that RbpA stabilizes RNAP-promoter open complexes (RPo) via a distinct mechanism from that of CarD. Furthermore, concentration-dependent stopped-flow experiments with both factors reveal positive linkage (cooperativity) between RbpA and CarD with regard to their ability to stabilize RPo. The observation of positive linkage between RbpA and CarD demonstrates that the two factors can act on the same transcription initiation complex simultaneously. Lastly, with both factors present, the kinetics of open complex formation is significantly faster than in the presence of either factor alone and approaches that of E. coli RNAP on the same promoter. This work provides a quantitative framework for the molecular mechanisms of these two essential transcription factors and the critical roles they play in the biology and pathology of mycobacteria.
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影响因子：
14.9
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Davis E;Chen J;Leon K;Darst SA;Campbell EA
通讯作者：
Campbell EA
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Stallings CL
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