IRIM, CNRS, Univ Montpellier, 1919 route de Mende, 34293 Montpellier, France.
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Nucleic Acids Res. 2018 Nov 2;46(19):10106-10118. doi: 10.1093/nar/gky714.
The transcriptional activator RbpA associates with Mycobacterium tuberculosis RNA polymerase (MtbRNAP) during transcription initiation, and stimulates formation of the MtbRNAP-promoter open complex (RPo). Here, we explored the influence of promoter motifs on RbpA-mediated activation of MtbRNAP containing the stress-response σB subunit. We show that both the 'extended -10' promoter motif (T-17G-16T-15G-14) and RbpA stabilized RPo and allowed promoter opening at suboptimal temperatures. Furthermore, in the presence of the T-17G-16T-15G-14 motif, RbpA was dispensable for RNA synthesis initiation, while exerting a stabilization effect on RPo. On the other hand, RbpA compensated for the lack of sequence-specific interactions of domains 3 and 4 of σB with the extended -10 and the -35 motifs, respectively. Mutations of the positively charged residues K73, K74 and R79 in RbpA basic linker (BL) had little effect on RPo formation, but affected MtbRNAP capacity for de novo transcription initiation. We propose that RbpA stimulates transcription by strengthening the non-specific interaction of the σ subunit with promoter DNA upstream of the -10 element, and by indirectly optimizing MtbRNAP interaction with initiation substrates. Consequently, RbpA renders MtbRNAP promiscuous in promoter selection, thus compensating for the weak conservation of the -35 motif in mycobacteria.
转录激活因子 RbpA 在转录起始过程中与结核分枝杆菌 RNA 聚合酶（MtbRNAP）结合，并刺激 MtbRNAP-启动子开放复合物（RPo）的形成。在这里，我们探讨了启动子基序对 RbpA 介导的含应激反应σB 亚基的 MtbRNAP 激活的影响。我们表明，“扩展-10”启动子基序（T-17G-16T-15G-14）和 RbpA 均稳定 RPo 并允许在非最佳温度下启动子打开。此外，在存在 T-17G-16T-15G-14 基序的情况下，RbpA 对于 RNA 合成起始是可有可无的，而对 RPo 则发挥稳定作用。另一方面，RbpA 补偿了 σB 结构域 3 和 4 与扩展-10 和-35 基序的序列特异性相互作用的缺乏。RbpA 碱性连接子（BL）中带正电荷的残基 K73、K74 和 R79 的突变对 RPo 的形成影响不大，但影响 MtbRNAP 从头转录起始的能力。我们提出，RbpA 通过增强 σ 亚基与-10 元件上游启动子 DNA 的非特异性相互作用，以及通过间接优化 MtbRNAP 与起始底物的相互作用来刺激转录。因此，RbpA 使 MtbRNAP 在启动子选择中变得杂乱无章，从而补偿了分枝杆菌中-35 基序的弱保守性。
