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Proc Natl Acad Sci U S A. 2016 Dec 27;113(52):14994-14999. doi: 10.1073/pnas.1613673114. Epub 2016 Dec 12.

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

Author information

1
Department of Microbiology, The Ohio State University, Columbus, OH 43210.
2
The Center for RNA Biology, The Ohio State University, Columbus, OH 43210.
3
Department of Biochemistry, University of Turku, Turku FIN-20014, Finland.
4
Department of Microbiology, The Ohio State University, Columbus, OH 43210; artsimovitch.1@osu.edu.

Abstract

Upon RNA polymerase (RNAP) binding to a promoter, the σ 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 β' clamp domain plays the most prominent role. In this work, we investigate the role of the β 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.

KEYWORDS:

RNA polymerase; beta pincer; discriminator; promoter; transcription

PMID:
27956639
PMCID:
PMC5206560
DOI:
10.1073/pnas.1613673114
[Indexed for MEDLINE]
Free PMC Article

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