Mishra Saurabh, Sen Ranjan
Laboratory of Transcription, Center for DNA Fingerprinting and Diagnostics, Tuljaguda Complex, 4-1-714 Mozamjahi Road, Nampally, Hyderabad 500 001, India Graduate Studies, Manipal University, India.
Laboratory of Transcription, Center for DNA Fingerprinting and Diagnostics, Tuljaguda Complex, 4-1-714 Mozamjahi Road, Nampally, Hyderabad 500 001, India
Nucleic Acids Res. 2015 Jul 13;43(12):5744-58. doi: 10.1093/nar/gkv479. Epub 2015 May 18.
Interaction of the lambdoid phage N protein with the bacterial transcription elongation factor NusA is the key component in the process of transcription antitermination. A convex surface of E. coli NusA-NTD, located opposite to its RNA polymerase-binding domain (the β-flap domain), directly interacts with N in the antitermination complex. We hypothesized that this N-NusA interaction induces allosteric effects on the NusA-RNAP interaction leading to transformation of NusA into a facilitator of the antitermination process. Here we showed that mutations in β-flap domain specifically defective for N antitermination exhibited altered NusA-nascent RNA interaction and have widened RNA exit channel indicating an intricate role of flap domain in the antitermination. The presence of N reoriented the RNAP binding surface of NusA-NTD, which changed its interaction pattern with the flap domain. These changes caused significant spatial rearrangement of the β-flap as well as the β' dock domains to form a more constricted RNA exit channel in the N-modified elongation complex (EC), which might play key role in converting NusA into a facilitator of the N antitermination. We propose that in addition to affecting the RNA exit channel and the active center of the EC, β-flap domain rearrangement is also a mechanistic component in the N antitermination process.
λ噬菌体N蛋白与细菌转录延伸因子NusA的相互作用是转录抗终止过程中的关键组成部分。大肠杆菌NusA-NTD的一个凸面，位于其RNA聚合酶结合结构域（β-侧翼结构域）的对面，在抗终止复合物中直接与N相互作用。我们推测这种N-NusA相互作用会对NusA-RNAP相互作用产生变构效应，导致NusA转变为抗终止过程的促进因子。在这里我们表明，在N抗终止方面存在特异性缺陷的β-侧翼结构域中的突变表现出NusA与新生RNA相互作用的改变，并且RNA出口通道变宽，这表明侧翼结构域在抗终止中具有复杂的作用。N的存在使NusA-NTD的RNAP结合表面重新定向，这改变了其与侧翼结构域的相互作用模式。这些变化导致β-侧翼以及β'对接结构域发生显著的空间重排，从而在N修饰的延伸复合物（EC）中形成更狭窄的RNA出口通道，这可能在将NusA转变为N抗终止的促进因子中起关键作用。我们提出，除了影响RNA出口通道和EC的活性中心外，β-侧翼结构域的重排也是N抗终止过程中的一个机制组成部分。
