Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism
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Curated by eLife
Evaluation Summary:
This work aims to provide insight into the molecular mechanism by which RNA polymerase separates the two strands of DNA, generating a single-stranded template for RNA synthesis. Using single-molecule analysis, the authors examined two conformational transitions taking place during RNA transcription initiation: DNA unwinding and RNAP clamp movements. Pending addition of some important controls, the paper will help to distinguish between two competing hypotheses within the literature. The work will be of relevance to a wide range of researchers interested in the molecular basis of gene expression and gene regulation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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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 analyse 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.
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Evaluation Summary:
This work aims to provide insight into the molecular mechanism by which RNA polymerase separates the two strands of DNA, generating a single-stranded template for RNA synthesis. Using single-molecule analysis, the authors examined two conformational transitions taking place during RNA transcription initiation: DNA unwinding and RNAP clamp movements. Pending addition of some important controls, the paper will help to distinguish between two competing hypotheses within the literature. The work will be of relevance to a wide range of researchers interested in the molecular basis of gene expression and gene regulation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to …
Evaluation Summary:
This work aims to provide insight into the molecular mechanism by which RNA polymerase separates the two strands of DNA, generating a single-stranded template for RNA synthesis. Using single-molecule analysis, the authors examined two conformational transitions taking place during RNA transcription initiation: DNA unwinding and RNAP clamp movements. Pending addition of some important controls, the paper will help to distinguish between two competing hypotheses within the literature. The work will be of relevance to a wide range of researchers interested in the molecular basis of gene expression and gene regulation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This article describes single-molecule measurements of two conformational changes that take place during transcription initiation: DNA unwinding and RNAP clamp movements. The work is technically sound and the incredibly clean single-molecule traces we have grown accustomed to from this group are beautiful.
The claim is that the data exclude a model where the RNAP clamp needs to acquire more open conformations in order for DNA unwinding to take place. The measurements appear to support this claim, but fall short in completely excluding the model due to some missing experiments.
The two main data sets presented show that within the time required for DNA unwinding, no RNAP conformational changes consistent with the open-most clamp structure are observed. However, as this is performed on a single, unnatural …
Reviewer #1 (Public Review):
This article describes single-molecule measurements of two conformational changes that take place during transcription initiation: DNA unwinding and RNAP clamp movements. The work is technically sound and the incredibly clean single-molecule traces we have grown accustomed to from this group are beautiful.
The claim is that the data exclude a model where the RNAP clamp needs to acquire more open conformations in order for DNA unwinding to take place. The measurements appear to support this claim, but fall short in completely excluding the model due to some missing experiments.
The two main data sets presented show that within the time required for DNA unwinding, no RNAP conformational changes consistent with the open-most clamp structure are observed. However, as this is performed on a single, unnatural promoter sequence without presenting negative controls for specificity, no broad conclusions can be made. This severely limits the impact that this work would have on the field of bacterial transcription.
There is also a danger in referring to "open" and "closed" states in general as it appears that a range of clamp openings are possible. Therefore, what someone labels as "open" from structural work may or may not be what is considered "open" in these relative fluorescent experiments.
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Reviewer #2 (Public Review):
The structure of cellular multi-subunit RNA polymerases is highly conserved, with a notable feature being their "crab claw" shape. Since the structure of RNA polymerase was first determined there has been a great deal of interest in how the opening and closing of that claw might affect the steps in transcription initiation, elongation and termination. Transcription initiation poses particular problems, as RNA polymerase must unwind the double-stranded DNA to create a region of single-stranded DNA that can be used as a template for RNA synthesis. Different models have proposed that RNA polymerase opens up to bind double-stranded DNA that is then unwound, or that it is unwound first and then a single strand is loaded into the narrower opening in a "closed claw". There is experimental evidence in favour of each …
Reviewer #2 (Public Review):
The structure of cellular multi-subunit RNA polymerases is highly conserved, with a notable feature being their "crab claw" shape. Since the structure of RNA polymerase was first determined there has been a great deal of interest in how the opening and closing of that claw might affect the steps in transcription initiation, elongation and termination. Transcription initiation poses particular problems, as RNA polymerase must unwind the double-stranded DNA to create a region of single-stranded DNA that can be used as a template for RNA synthesis. Different models have proposed that RNA polymerase opens up to bind double-stranded DNA that is then unwound, or that it is unwound first and then a single strand is loaded into the narrower opening in a "closed claw". There is experimental evidence in favour of each of these models. In this paper single molecule techniques are used to monitor both the unwinding of the double stranded DNA and the opening of RNA polymerase. The results suggest that RNA polymerase does not need to adopt an open conformation in order to unwind and load DNA, and hence that unwinding is likely to occur outside the main cleft of the enzyme. The work also detects a post-loading step in which the RNAP closes more tightly on the DNA, which is proposed to act to "lock" the polymerase onto the DNA. The results presented in the paper are an important addition to our understanding of this key step in gene expression.
Strengths
The work has been done with RNA polymerase from Escherichia coli, which is perhaps one of the best-studied RNA polymerases, and uses the most prominent sigma factor binding to a canonical promoter. The system has therefore been chosen to be off wide interest and applicability, and also sets the groundwork for similar studies in other systems. The development of ideas is logical and multiple lines of evidence are used to test the validity of the conclusions. For example, on finding a difference in kinetics between the unwinding of the upstream and downstream ends of the bubble the assumption that this represents directional unwinding is tested by increasing the strength of the intervening base pairing, and the expected changes in rate are seen. Similarly, the requirement (or otherwise) for clamp opening is demonstrated both by single molecule FRET experiments and by testing the effect of an inhibitor of clamp opening. The experiments are presented clearly in both the text and the figures, and the results are convincing. Overall the correlation between the rates of strand opening and clamp locking is compelling, and the conclusion that the clamp need not open to form the open complex, and closes more tightly after bubble opening seems to me to be convincing.
Weaknesses
I have noted above that the experimental system was carefully and appropriately chosen. And the authors note that the tools developed here can subsequently be applied to other systems, and list some of those that they feel may be of interest. This acknowledges one potential weakness of the study, which is that it studies essentially a single promoter, and what is true of this system may not be true of all. Given that the main conclusion is that there is not an absolute requirement for clamp opening in order for an open complex to form I feel a single system is sufficient to make this claim. Further limitations of the experimental systems also leave some potential room for argument: the time resolution of the experiments means that very rapid opening would not be detected, and as DNA opening and RNA polymerase opening were measured in separate experiments the conclusions rely in part on correlation between results gained in slightly different conditions. But I do not think that these caveats undermine the authors' conclusions. Finally, the smFRET experiments monitor the relative position of two residues on either side of the clamp. These report on clamp opening (as stated in the paper) but do not reflect movement of other parts of RNA polymerase that might facilitate entry of double-stranded DNA into the cleft. Movement of the beta lobe is of particular interest in this context, given prior structural and modelling work: Unarta et al PNAS 2021 118 e2024324118, Chen et al Mol Cell 2020 78 275 and references therein. If, as suggested in these other papers, movement of the beta lobe might allow double-stranded DNA to be accommodated in the RNA polymerase cleft without substantial clamp opening the logical link between the data presented here and an "unwind then load" model might be weakened.
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