RNA polymerase sliding on DNA can couple the transcription of nearby bacterial operons

Proc. Natl. Acad. Sci. U.S.A.

Friedman

et al. 2023

Self Cite

Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in the cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryotic Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so are unclear. We used multiwavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the Escherichia coli transcription cycle. In our experiments, RapA at <5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post termination complex (PTC)—consisting of core RNA polymerase (RNAP)-bound sequence nonspecifically to double-stranded DNA—and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription reinitiation in proteobacterial genomes.

mentioning

confidence: 99%

Recycling of bacterial RNA polymerase by the Swi2/Snf2 ATPase RapA

Inlow

Proc. Natl. Acad. Sci. U.S.A.

Friedman

et al. 2023

Self Cite

“…Jin and co-workers proposed that RapA allows recovery of RNAP from a “posttranscription or posttermination” refractory state, but the composition of this refractory state and its position in the transcription cycle remained unclear (17, 22, 23). Here we provide direct evidence that RapA binds to the post-termination RNAP-DNA complex (PTC), an intermediate in the transcription cycle that follows intrinsic termination (10, 12, 13, 15). Within seconds of its binding to the PTC, RapA effectively removes core RNAP from DNA.…”

Section: Discussionmentioning

confidence: 97%

“…In bacterial transcription, there is evidence for re-initiation after intrinsic termination from experiments both in vitro and in vivo (10, 12, 13, 15). These studies, among others (43–46), are consistent with the proposal that there is a balance between re-initiation (local) and conventional initiation (global) in cells.…”

Section: Discussionmentioning

confidence: 99%

“…While in the PTC state, RNAP may hop or slide along DNA in a random walk and flip 180 degrees on DNA, allowing re-initiation of either sense or anti-sense transcription from a suitably situated promoter upon σ factor binding (10, 12, 13). However, these re-initiation pathways are limited to 1-D diffusion to proximal promoters on the same template, and efficiency of re-initiation through this pathway depends on terminator-promoter distance and σ factor availability (15). The mechanism by which RNAP dissociates from DNA during intrinsic termination and is efficiently recycled to the cytoplasmic pool of RNAP remains unknown.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Recycling of Bacterial RNA Polymerase by the Swi2/Snf2 ATPase RapA

Inlow¹,

Friedman³

et al. 2023

Preprint

Self Cite

Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryote Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so is unclear. We used multi-wavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the E. coli transcription cycle. In our experiments, RapA at < 5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post-termination complex (PTC) -- consisting of core RNA polymerase (RNAP) bound to dsDNA -- and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription re-initiation in proteobacterial genomes.

“…Source data for the single-molecule experiments and code for the search time computation data have been deposited in Zenodo ( https://doi.org/10.5281/zenodo.7976266 ) ( 47 ).…”

Section: Data Materials and Software Availabilitymentioning

confidence: 99%

RNA polymerase sliding on DNA can couple the transcription of nearby bacterial operons

Proc. Natl. Acad. Sci. U.S.A.

Inlow

Friedman

et al. 2023

Self Cite

DNA transcription initiates after an RNA polymerase (RNAP) molecule binds to the promoter of a gene. In bacteria, the canonical picture is that RNAP comes from the cytoplasmic pool of freely diffusing RNAP molecules. Recent experiments suggest the possible existence of a separate pool of polymerases, competent for initiation, which freely slide on the DNA after having terminated one round of transcription. Promoter-dependent transcription reinitiation from this pool of posttermination RNAP may lead to coupled initiation at nearby operons, but it is unclear whether this can occur over the distance and timescales needed for it to function widely on a bacterial genome in vivo. Here, we mathematically model the hypothesized reinitiation mechanism as a diffusion-to-capture process and compute the distances over which significant interoperon coupling can occur and the time required. These quantities depend on molecular association and dissociation rate constants between DNA, RNAP, and the transcription initiation factor σ 70 ; we measure these rate constants using single-molecule experiments in vitro. Our combined theory/experimental results demonstrate that efficient coupling can occur at physiologically relevant σ 70 concentrations and on timescales appropriate for transcript synthesis. Coupling is efficient over terminator–promoter distances up to ∼1,000 bp, which includes the majority of terminator–promoter nearest neighbor pairs in the Escherichia coli genome. The results suggest a generalized mechanism that couples the transcription of nearby operons and breaks the paradigm that each binding of RNAP to DNA can produce at most one messenger RNA.