CarD and RbpA modify the kinetics of initial transcription and slow promoter escape of the Mycobacterium tuberculosis RNA polymerase
CarD and RbpA modify the kinetics of initial transcription and slow promoter escape of the Mycobacterium tuberculosis RNA polymerase
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DOI：
10.1093/nar/gkz449
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发表时间：
2019-07-26
影响因子：
14.9
通讯作者：
Galburt, Eric A.
中科院分区：
文献类型：
--
作者：
Jensen, Drake;Manzano, Ana Ruiz;Galburt, Eric A.
结核分枝杆菌（Mycobacterium tuberculosis，Mtb）是结核病的病原体，在受到宿主来源的应激时，其产生独特的转录调控机制。分枝杆菌RNA聚合酶（RNAP）的转录起始先前已显示出相对于大肠杆菌（Eco）RNAP表现出不同的开放复合物动力学和稳定性。然而，转录起始速率还取决于开放复合物形成后的动力学，例如初始核苷酸掺入和随后的启动子逃逸。在这里，使用实时荧光分析，我们提出了第一个深入的动力学分析的初始转录和启动子逃逸的结核分枝杆菌RNAP。我们表明，在生态RNAP，结核分枝杆菌显示较慢的初始核苷酸掺入，但更快的整体启动子逃逸动力学的结核分枝杆菌rrnAP3启动子。此外，在必要的转录因子CardD和RbpA的背景下，Mtb启动子逃逸通过对最初转录复合物的差异效应而减慢。最后，基于他们的能力，以增加开放复合物形成的速度和降低启动子逃逸的速度，我们建议，CardD和RbpA能够激活或镇压取决于给定的启动子的基础启动动力学的限速步骤。
The pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, enacts unique transcriptional regulatory mechanisms when subjected to host-derived stresses. Initiation of transcription by the Mycobacterial RNA polymerase (RNAP) has previously been shown to exhibit different open complex kinetics and stabilities relative to Escherichia coli (Eco) RNAP. However, transcription initiation rates also depend on the kinetics following open complex formation such as initial nucleotide incorporation and subsequent promoter escape. Here, using a real-time fluorescence assay, we present the first in-depth kinetic analysis of initial transcription and promoter escape for the Mtb RNAP. We show that in relation to Eco RNAP, Mtb displays slower initial nucleotide incorporation but faster overall promoter escape kinetics on the Mtb rrnAP3 promoter. Furthermore, in the context of the essential transcription factors CarD and RbpA, Mtb promoter escape is slowed via differential effects on initially transcribing complexes. Finally, based on their ability to increase the rate of open complex formation and decrease the rate of promoter escape, we suggest that CarD and RbpA are capable of activation or repression depending on the rate-limiting step of a given promoter's basal initiation kinetics.