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Edited by Jeffrey W. Roberts, Cornell University, Ithaca, NY, and approved December 22, 2016 (received for review October 18, 2016)
Significance
RNA polymerases (RNAPs) from all organisms share a common form of regulation: Regulator proteins bind in a conserved “secondary channel” pore on RNAP and alter RNAP activity. In bacteria, multiple such regulators are present in the same cell, but how these bind without mutual interference is unclear. We directly observed binding of single molecules of secondary channel protein GreB to RNAP transcription complexes. Unexpectedly, GreB was not selectively recruited to RNAPs requiring its transcript proofreading function. Instead, GreB transiently bound to multiple types of complexes, eventually finding via random search RNAPs that require its activity. The observations suggest a paradigm by which a regulator can act while minimizing obstruction of a binding site that must be shared with other proteins.
Abstract
The secondary channel (SC) of multisubunit RNA polymerases (RNAPs) allows access to the active site and is a nexus for the regulation of transcription. Multiple regulatory proteins bind in the SC and reprogram the catalytic activity of RNAP, but the dynamics of these factors’ interactions with RNAP and how they function without cross-interference are unclear. In Escherichia coli, GreB is an SC protein that promotes proofreading by transcript cleavage in elongation complexes backtracked by nucleotide misincorporation. Using multiwavelength single-molecule fluorescence microscopy, we observed the dynamics of GreB interactions with elongation complexes. GreB binds to actively elongating complexes at nearly diffusion-limited rates but remains bound for only 0.3–0.5 s, longer than the duration of the nucleotide addition cycle but far shorter than the time needed to synthesize a complete mRNA. Bound GreB inhibits transcript elongation only partially. To test whether GreB preferentially binds backtracked complexes, we reconstituted complexes stabilized in backtracked and nonbacktracked configurations. By verifying the functional state of each molecular complex studied, we could exclude models in which GreB is selectively recruited to backtracked complexes or is ejected from RNAP by catalytic turnover. Instead, GreB binds rapidly and randomly to elongation complexes, patrolling for those requiring nucleolytic rescue, and its short residence time minimizes RNAP inhibition. The results suggest a general mechanism by which SC factors may cooperate to regulate RNAP while minimizing mutual interference.
Footnotes
↵1L.E.T., L.J.F., and M.L.O. contributed equally to this work.
- ↵2To whom correspondence should be addressed. Email: gelles{at}brandeis.edu.
Author contributions: L.E.T., L.J.F., M.L.O., H.R., S.K., S.K.S., R.A.M., R.L., and J.G. designed research; L.E.T., L.J.F., M.L.O., H.R., S.K., S.K.S., and R.A.M. performed research; L.E.T., L.J.F., and J.G. contributed new reagents/analytic tools; L.E.T., L.J.F., and M.L.O. analyzed data; and L.E.T., L.J.F., M.L.O., and J.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1616525114/-/DCSupplemental.
Dynamics of GreB interactions with RNA polymerase
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