Biology subjects

Gelles, J.

Publications and source records attributed to Gelles, J..

17 recordsLinked to original sources

bioRxiv2026

Bacterial RNA polymerase exemplifies a general physical mechanism for accelerating protein-DNA association

Bacterial RNA polymerases (RNAPs) have two flexibly tethered subunit C-terminal domains (-CTDs) that bind DNA. Interaction between -CTDs and some promoter DNA motifs is known to accelerate transcription initiation, but the physical mechanism by which it does so is unclear. We used single-molecule multiwavelength fluorescence microscopy to test how the diffusion-limited binding kinetics of core RNAP to non-promoter DNA differ from those of mutant RNAPs that lack one or both -CTDs. We find that even though -CTDs and their tethers are small compared to the complete RNAP molecule, the presence of two -CTDs accelerates DNA binding by [~]10-fold and [~]55-fold respectively relative to RNAP constructs in which one or both -CTDs are deleted. In contrast, the presence of -CTDs did not have a detectable effect on RNAP-DNA complex lifetimes in the absence of RNA synthesis. We explain how -CTDs achieve the dramatic acceleration of RNAP binding to DNA using a quantitative three-state kinetic model that includes a transient binding intermediate where only the -CTD(s) are bound to DNA, tethering the rest of the RNAP in the vicinity of DNA. The model and assumed parameters are validated using Brownian dynamics simulations of the DNA association reactions for two-, one-, or zero-CTD RNAP constructs. The combination of single-molecule experiments, mathematical theory, and simulations suggests that adding a flexible DNA-binding tether is a general physical mechanism which can accelerate the diffusion-limited binding of a large protein like RNAP to DNA and quantitatively defines the conditions under which this acceleration can occur. Significance StatementLarge enzymes that must associate with DNA to perform their biological functions are expected to bind DNA only slowly because of slow enzyme diffusion. However, some DNA-binding enzymes have one or more additional small DNA-binding domains that can diffuse rapidly but are attached to the enzyme through an unstructured flexible tether. Combining single-molecule experiments, theory, and computation, we present evidence for a general mechanism by which such tethered domains can accelerate enzyme binding to DNA by orders of magnitude, while not significantly changing the duration of the DNA-bound state. We demonstrate this effect for the flexibly-tethered -subunit C-terminal domain of bacterial RNA polymerase. The mechanism may allow the polymerase to rapidly bind DNA while minimizing non-functional sequestration on the genome.

biophysics↗

bioRxiv2025

Dynamics of TFIIH and Spt5 during the transition from transcription initiation to elongation

The transition of RNA polymerase II from initiation to elongation requires the timely exchange of many factors. Some initiation factors and elongation factors contact the same RNA polymerase II surfaces in an apparently mutually exclusive manner. For example, space occupied by the initiation factor TFIIH and elongation factor Spt4/5 (also known as DSIF) are predicted to overlap. To determine whether Spt4/5 actively displaces TFIIH, or binds only after TFIIH dissociates, labeled yeast nuclear extracts were combined with single-molecule fluorescence microscopy to observe their dynamics in real time. TFIIH categorically dissociates before Spt4/5 arrives, with a clear interval in between with neither factor present. Therefore, Spt4/5 binding is not required to dissociate TFIIH from RNA polymerase II.

molecular biology↗

bioRxiv2025

An Orc6 tether mediates ORC binding site switching during replication origin licensing

During origin licensing, the origin recognition complex (ORC) loads two Mcm2-7 helicases onto DNA in a head-to-head conformation, establishing the foundation for subsequent bidirectional replication. Single-molecule experiments support a helicase-loading model in which one ORC loads both Mcm2-7 helicases at origins. For this to occur, ORC must release from its initial Mcm2-7 and DNA binding sites, flip over the helicase, and bind the opposite end of the Mcm2-7 complex and adjacent DNA to form the MO complex. Importantly, this binding-site transition occurs without ORC releasing into solution. Using a single-molecule FRET assay, we show that the N-terminal half of Orc6 tethers ORC to the N-terminal tier of Mcm2-7 (Mcm2-7N) during ORCs binding-site transition. This interaction involves both the folded Orc6 N-terminal domain (Orc6N) and the adjacent unstructured linker and forms before ORC releases from its initial Mcm2-7 interaction. The absence of this interaction increases the rate of ORC release into solution, consistent with a tethering function. CDK phosphorylation of ORC inhibits the tethering interaction, providing a mechanism for the known CDK inhibition of MO complex formation. Interestingly, we identify mutations in the Orc6 linker region that support MO complex formation but prevent double-hexamer formation by inhibiting stable second Mcm2-7 recruitment. Our study provides a molecular explanation for a one-ORC mechanism of helicase loading and demonstrates that Orc6 is involved in multiple stages of origin licensing. Significance StatementBidirectional DNA replication is critical for accurate and complete duplication of the genome. Eukaryotic organisms coordinate this through loading of two oppositely-oriented Mcm2-7 replicative helicases at origins of replication. Using single-molecule biochemical studies, we identified and characterized a tethering interaction during helicase loading that enables the helicase loader ORC (origin recognition complex) to flip between two Mcm2-7 and DNA binding sites to load the second helicases in the opposite orientation. This interaction is cell-cycle regulated as part of the mechanisms ensuring replication from a given origin initiates only once. Our findings have important implications for the multiple mechanisms of helicase loading and illustrate how single-molecule studies can complement structural studies to provide a full view of complex molecular assembly events.

biochemistry↗

bioRxiv2024

Mechanisms of synergistic Mediator recruitment in RNA polymerase II transcription activation revealed by single-molecule fluorescence

Transcription activators trigger transcript production by RNA Polymerase II (RNApII) via the Mediator coactivator complex. Here the dynamics of activator, Mediator, and RNApII binding at promoter DNA were analyzed using multi-wavelength single-molecule microscopy of fluorescently labeled proteins in budding yeast nuclear extract. Binding of Mediator and RNApII to the template required activator and an upstream activator sequence (UAS), but not a core promoter. While Mediator and RNApII sometimes bind as a pre-formed complex, more commonly Mediator binds first and subsequently recruits RNApII to form a preinitiation complex precursor (pre-PIC) tethered to activators on the UAS. Interestingly, Mediator occupancy has a highly non-linear response to activator concentration, and fluorescence intensity measurements show Mediator preferentially associates with templates having at least two activators bound. Statistical mechanical modeling suggests this "synergy" is not due to cooperative binding between activators, but instead occurs when multiple DNA-bound activator molecules simultaneously interact with a single Mediator.

biochemistry↗

bioRxiv2024

RapA opens the RNA polymerase clamp to disrupt post-termination complexes and prevent cytotoxic R-loop formation

Following transcript release during intrinsic termination, Escherichia coli RNA polymerase (RNAP) often remains associated with DNA in a post-termination complex (PTC). RNAPs in PTCs are removed from the DNA by the Swi2/Snf2 ATPase RapA. Here, we determined PTC structures on negatively-supercoiled DNA as well as of RapA engaged to dislodge the PTC. We found that core RNAP in the PTC can unwind DNA and initiate RNA synthesis but is prone to producing R-loops. We show that RapA helps control cytotoxic R-loop formation in vivo, likely by disrupting PTCs. Nucleotide binding to RapA triggers a conformational change that opens the RNAP clamp, allowing DNA in the RNAP cleft to reanneal and dissociate. We suggest that analagous ATPases acting on PTCs to suppress transcriptional noise and R-loop formation may be widespread. These results hold significance for the bacterial transcription cycle and highlight a role for RapA in maintaining genome stability.

biophysics↗

bioRxiv2023

Single-molecule analysis of transcription activation: Dynamics of SAGA co-activator recruitment

Transcription activators are said to stimulate gene expression by "recruiting" coactivators to promoters, yet this term fits several different kinetic models. To directly analyze dynamics of activator-coactivator interactions, single-molecule microscopy was used to image promoter DNA, a transcription activator, and the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex within nuclear extract. SAGA readily, but transiently, binds nucleosome-free DNA without activator, while chromatin template association occurs nearly exclusively when activator is present. On both templates, activator increases SAGA association rates by up to an order of magnitude, and dramatically extends its dwell times. These effects reflect direct interactions with the transactivation domain, as VP16 or Rap1 activation domains produce different SAGA dynamics. Despite multiple bromodomains, acetyl-CoA or histone H3/H4 tail acetylation only modestly improves SAGA binding. Unexpectedly, histone acetylation more strongly affects activator residence. Our studies thus reveal two modes of SAGA interaction with the genome: a short-lived activator-independent interaction with nucleosome-free DNA, and a state tethered to promoter-bound transcription activators that can last up to several minutes.

molecular biology↗

bioRxiv2023

Changing protein-DNA interactions promote ORC binding site exchange during replication origin licensing.

During origin licensing, the eukaryotic replicative helicase Mcm2-7 forms head-to-head double hexamers to prime origins for bidirectional replication. Recent single-molecule and structural studies revealed that one molecule of the helicase loader ORC can sequentially load two Mcm2-7 hexamers to ensure proper head-to-head helicase alignment. To perform this task, ORC must release from its initial high-affinity DNA binding site and "flip" to bind a weaker, inverted DNA site. However, the mechanism of this binding-site switch remains unclear. In this study, we used single-molecule Forster resonance energy transfer (sm-FRET) to study the changing interactions between DNA and ORC or Mcm2-7. We found that the loss of DNA bending that occurs during DNA deposition into the Mcm2-7 central channel increases the rate of ORC dissociation from DNA. Further studies revealed temporally-controlled DNA sliding of helicase-loading intermediates, and that the first sliding complex includes ORC, Mcm2-7, and Cdt1. We demonstrate that sequential events of DNA unbending, Cdc6 release, and sliding lead to a stepwise decrease in ORC stability on DNA, facilitating ORC dissociation from its strong binding site during site switching. In addition, the controlled sliding we observed provides insight into how ORC accesses secondary DNA binding sites at different locations relative to the initial binding site. Our study highlights the importance of dynamic protein-DNA interactions in the loading of two oppositely-oriented Mcm2-7 helicases to ensure bidirectional DNA replication. Significance StatementBidirectional DNA replication, in which two replication forks travel in opposite directions from each origin of replication, is required for complete genome duplication. To prepare for this event, two copies of the Mcm2-7 replicative helicase are loaded at each origin in opposite orientations. Using single-molecule assays, we studied the sequence of changing protein-DNA interactions involved in this process. These stepwise changes gradually reduce the DNA-binding strength of ORC, the primary DNA binding protein involved in this event. This reduced affinity promotes ORC dissociation and rebinding in the opposite orientation on the DNA, facilitating the sequential assembly of two Mcm2-7 molecules in opposite orientations. Our findings identify a coordinated series of events that drive proper DNA replication initiation.

molecular biology↗

bioRxiv2023

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

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. SIGNIFICANCERNA synthesis is an essential conduit of genetic information in all organisms. After transcribing an RNA, the bacterial RNA polymerase (RNAP) must be reused to make subsequent RNAs, but the steps that enable RNAP reuse are unclear. We directly observed the dynamics of individual molecules of fluorescently labeled RNAP and the enzyme RapA as they colocalized with DNA during and after RNA synthesis. Our studies show that RapA uses ATP hydrolysis to remove RNAP from DNA after the RNA is released from RNAP and reveal essential features of the mechanism by which this removal occurs. These studies fill in key missing pieces in our current understanding of the events that occur after RNA is released and that enable RNAP reuse.

biophysics↗

bioRxiv2023

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

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 post-termination RNAP may lead to coupled initiation at nearby operons, but it is unclear whether this can occur over the distance- and time-scales 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 inter-operon coupling can occur and the time required. These quantities depend on previously uncharacterized molecular association and dissociation rate constants between DNA, RNAP and the transcription initiation factor{sigma} 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{sigma} 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 E. 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. SIGNIFICANCE STATEMENTAfter transcribing an operon, a bacterial RNA polymerase can stay bound to DNA, slide along it, and reini-tiate transcription of the same or a different operon. Quantitative single-molecule biophysics experiments combined with mathematical theory demonstrate that this reinitiation process can be quick and efficient over gene spacings typical of a bacterial genome. Reinitiation may provide a mechanism to orchestrate the transcriptional activities of groups of nearby operons.

biophysics↗

bioRxiv2023

Regulation of replication origin licensing by ORC phosphorylation reveals a two-step mechanism for Mcm2-7 ring closing

Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 are well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. Phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO-complex formation and provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO-complex formation. Significance StatementEach time a eukaryotic cell divides (by mitosis) it must duplicate its chromosomal DNA exactly once to ensure that one full copy is passed to each resulting cell. Both under-replication or over-replication result in genome instability and disease or cell death. A key mechanism to prevent over-replication is the temporal separation of loading of the replicative DNA helicase at origins of replication and activation of these same helicases during the cell division cycle. Here we define the mechanism by which phosphorylation of the primary DNA binding protein involved in these events inhibits helicase loading. Our studies identify multiple steps of inhibition and provide new insights into the mechanism of helicase loading in the uninhibited condition.

molecular biology↗

bioRxiv2021

A Helicase-tethered ORC Flip Enables Bidirectional Helicase Loading

Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2-7 during helicase loading. We demonstrate that a single ORC molecule can recruit both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between the first and second helicase recruitment, we observe a rapid change in interactions between ORC and the first Mcm2-7. In quick succession ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.

molecular biology↗

bioRxiv2021

Bayesian machine learning analysis of single-molecule fluorescence colocalization images

Multi-wavelength single-molecule fluorescence colocalization (CoSMoS) methods allow elucidation of complex biochemical reaction mechanisms. However, analysis of CoSMoS data is intrinsically challenging because of low image signal-to-noise ratios, non-specific surface binding of the fluorescent molecules, and analysis methods that require subjective inputs to achieve accurate results. Here, we use Bayesian probabilistic programming to implement Tapqir, an unsupervised machine learning method that incorporates a holistic, physics-based causal model of CoSMoS data. This method accounts for uncertainties in image analysis due to photon and camera noise, optical non-uniformities, non-specific binding, and spot detection. Rather than merely producing a binary "spot/no spot" classification of unspecified reliability, Tapqir objectively assigns spot classification probabilities that allow accurate downstream analysis of molecular dynamics, thermodynamics, and kinetics. We both quantitatively validate Tapqir performance against simulated CoSMoS image data with known properties and also demonstrate that it implements fully objective, automated analysis of experiment-derived data sets with a wide range of signal, noise, and non-specific binding characteristics.

biophysics↗

bioRxiv2021

Single molecule studies reveal branched pathways for activator-dependent pre-initiation complex assembly

RNA polymerase II (Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly. HIGHLIGHTSSingle molecule microscopy reveals unexpected dynamics of RNA Pol II and GTFs Multiple Pol IIs cluster on UAS/enhancer-bound activators before binding the core promoter Pol II, TFIIF, and TFIIE, but not TFIIH, can pre-assemble at the UAS/enhancer Activators increase the rates of Pol II and GTF association with DNA eTOC BlurbSingle-molecule microscopy experiments by Baek et al. show that RNA polymerase II and basal transcription factors TFIIF and TFIIE preassemble on UAS/enhancer-bound activators, poised for loading into initiation complexes with TFIIH at the core promoter. Transcription activators kinetically enhance factor recruitment, creating a localized cluster of polymerases at the UAS/enhancer.

molecular biology↗

bioRxiv2020

DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7

The committed step of eukaryotic DNA replication occurs when the replicative Mcm2-7 helicase pairs that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45 and GINS are individually recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). The multiple phosphorylation sites on the Mcm2-7 tails promote DDK-dependent modulation of the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity results in increased frequency of CMG formation. Our findings reveal molecular mechanisms sensitizing helicase activation to DDK levels with implications for the control of replication origin efficiency and timing.

cell biology↗

bioRxiv2020

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

In eukaryotes, RNA polymerase II (RNApII) transcribes messenger RNA from template DNA. Decades of experiments have identified the proteins needed for transcription activation, initiation complex assembly, and productive elongation. However, the dynamics of recruitment of these proteins to transcription complexes, and of the transitions between these steps, are poorly understood. We used multi-wavelength single-molecule fluorescence microscopy to directly image and quantitate these dynamics in a budding yeast nuclear extract that reconstitutes activator-dependent transcription in vitro. A strong activator (Gal4-VP16) greatly stimulated reversible binding of individual RNApII molecules to template DNA, with no detectable involvement of RNApII-containing condensates. Binding of labeled elongation factor Spt4/5 to DNA typically followed RNApII binding, was NTP-dependent, and was correlated with association of mRNA-binding protein Hek2, demonstrating specificity of Spt4/5 binding to elongation complexes. Quantitative kinetic modeling shows that only a fraction of RNApII binding events are productive and implies a rate-limiting step, probably associated with recruitment of general transcription factors, needed to assemble a transcription-competent pre-initiation complex at the promoter. Spt4/5 association with transcription complexes was slowly reversible, with DNA-bound RNApII molecules sometimes binding and releasing Spt4/5 multiple times. The average Spt4/5 residence time was of similar magnitude to the time required to transcribe an average length yeast gene. These dynamics suggest that a single Spt4/5 molecule remains associated during a typical transcription event, yet can dissociate from RNApII to allow disassembly of abnormally long-lived (i.e., stalled) elongation complexes. Significance StatementThe synthesis of a eukaryotic messenger RNA molecule involves the association of RNA polymerase and dozens of accessory proteins on DNA. We used differently colored fluorescent dyes to tag DNA, RNA polymerase II, and the elongation factor Spt4/5 in yeast nuclear extract, and then observed the assembly and dynamics of individual molecules of the proteins with single DNA molecules by microscopy. The observations quantitatively define an overall pathway by which transcription complexes form and evolve in response to an activator protein. They suggest how molecular complex dynamics may be tuned to optimize efficient RNA production.

biochemistry↗

bioRxiv2020

Mechanism of upstream promoter element stimulation of transcription at a ribosomal RNA promoter determined by single-molecule imaging

DNA elements upstream of transcription promoters play a role in regulating transcription initiation in all organisms. In bacteria, upstream A-T rich sequences called UP elements can stimulate transcription through contact with the subunit C-terminal domain (CTD) of core RNA polymerase (RNAP), but the kinetic mechanisms by which they do so remain unclear. We investigated the role of the UP element in stimulating initiation from the strong E. coli 16s rRNA promoter using single-molecule fluorescence microscopy to visualize {sigma}70RNAP holoenzyme binding and the formation nascent RNA by oligonucleotide probe hybridization on individual DNA molecules containing the rrnB P1 promoter. By directly detecting initial binding of {sigma}70RNAP to promoter and monitoring the lifetimes of promoter-polymerase complexes, the experiments reveal the kinetic mechanism of polymerase recruitment to the promoter and the subsequent conformational change that stabilizes binding. The presence of UP stimulated the rate of initial binding of polymerase to promoter by at least six-fold, and this stimulation was fully sufficient to account for the increase in initiation rate by UP. Thus, UP likely functions at this strong promoter simply by acting as a binding target for the rapidly reorienting CTD domain tethered to the core polymerase. In contrast, there were only minor effects of UP on the measured rates of the conformational change or the dissociation rates of the initial {sigma}70RNAP promoter complexes. These studies define a paradigmatic kinetic mechanism for stimulation of transcription initiation by direct CTD-DNA interactions. This mechanism can serve as a building block of more complex regulatory architectures in which CTD promotes transcription through interactions with both DNA and protein activators.

biochemistry↗

bioRxiv2019

Alternative transcription cycle for bacterial RNA polymerase

RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. We used single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remained bound to DNA and sometimes exhibited one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarted transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this \"secondary initiation\" in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.

biophysics↗