Transcription initiation at a consensus bacterial promoter proceeds via a 'bind-unwind-load-and-lock' mechanism
Abstract
Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load-and-lock' model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.
Data availability
All information for replication is included in the submission and data corresponding to each figure are provided as source data files. MATLAB software packages TwoTone and ebFRET are available on Github (https://github.com/annawang692/TwoTone2018 and http://ebfret.github.io/).
Article and author information
Author details
Funding
Wellcome Trust (110164/Z/15/Z)
- Achillefs Kapanidis
NIH Office of the Director (GM041376)
- Richard H Ebright
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Mazumder et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,050
- views
-
- 268
- downloads
-
- 14
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Structural Biology and Molecular Biophysics
Ciliary rootlets are striated bundles of filaments that connect the base of cilia to internal cellular structures. Rootlets are critical for the sensory and motile functions of cilia. However, the mechanisms underlying these functions remain unknown, in part due to a lack of structural information of rootlet organization. In this study, we obtain 3D reconstructions of membrane-associated and purified rootlets from mouse retina using cryo-electron tomography. We show that flexible protrusions on the rootlet surface, which emanate from the cross-striations, connect to intracellular membranes. In purified rootlets, the striations were classified into amorphous (A)-bands, associated with accumulations on the rootlet surface, and discrete (D)-bands corresponding to punctate lines of density that run through the rootlet. These striations connect a flexible network of longitudinal filaments. Subtomogram averaging suggests the filaments consist of two intertwined coiled coils. The rootlet’s filamentous architecture, with frequent membrane-connecting cross-striations, lends itself well for anchoring large membranes in the cell.
-
- Structural Biology and Molecular Biophysics
Although the αC-β4 loop is a stable feature of all protein kinases, the importance of this motif as a conserved element of secondary structure, as well as its links to the hydrophobic architecture of the kinase core, has been underappreciated. We first review the motif and then describe how it is linked to the hydrophobic spine architecture of the kinase core, which we first discovered using a computational tool, local spatial Pattern (LSP) alignment. Based on NMR predictions that a mutation in this motif abolishes the synergistic high-affinity binding of ATP and a pseudo substrate inhibitor, we used LSP to interrogate the F100A mutant. This comparison highlights the importance of the αC-β4 loop and key residues at the interface between the N- and C-lobes. In addition, we delved more deeply into the structure of the apo C-subunit, which lacks ATP. While apo C-subunit showed no significant changes in backbone dynamics of the αC-β4 loop, we found significant differences in the side chain dynamics of K105. The LSP analysis suggests disruption of communication between the N- and C-lobes in the F100A mutant, which would be consistent with the structural changes predicted by the NMR spectroscopy.