期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：52
页码：14994-14999
DOI：10.1073/pnas.1613673114
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe nontemplate DNA strand in the transcription bubble interacts with RNA polymerase and accessory factors to control initiation, elongation, transcription-coupled repair, and translation. During initiation, {sigma} subunit interactions with the nontemplate DNA regulate promoter complex formation and lifetime, abortive synthesis, and start site selection. Here, we show that the {beta} subunit gate loop contacts with an adjacent segment of the nontemplate discriminator region play a similar role during initiation. The deletion of the gate loop alters the structure and properties of promoter complexes and has pleiotropic effects on RNA chain elongation and termination. We propose that, acting in concert with accessory factors, the gate loop mediates the clamp closure and guides the nontemplate strand in initiation and elongation complexes. Upon RNA polymerase (RNAP) binding to a promoter, the {sigma} factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the {beta}' clamp domain plays the most prominent role. In this work, we investigate the role of the {beta} gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.
