The Escherichia coli transcription initiation factor σ70 was long believed to dissociate from RNA polymerase after promoter escape, following synthesis of short ~9-11 nt RNAs. However, recent evidence indicates σ70 is retained in elongation complexes (ECs), where it enhances pausing-implying that stochastic or factor-mediated displacement is required for processive elongation. Using fluorescence correlation spectroscopy, we quantified σ70 retention in the presence of elongation factors NusG and NusA, demonstrating that NusG-but not NusA-actively promotes σ70 dissociation. Single-molecule TIRF further revealed that NusG-mediated σ70 dissociation becomes favourable as ECs progress, with lower energy barriers for displacement in later elongation stages. These findings suggest a broader mechanistic paradigm in which elongation factors actively displace initiation factors to regulate transcription-a process that may be conserved across all domains of life.

DNA sequence regulates complex reactions and protein-DNA interactions, yet sequence effects remain poorly understood due to the lack of direct, high-throughput approaches to study sequence-dependence at the single-molecule level. Here, we introduce Single-molecule Phenotyping and In-Situ Sequencing (SPIN-Seq), a DNA-based, protein-free method that links functional and structural properties of a single DNA molecule with the sequence of that same molecule. After performing functional assays on immobilized DNA molecules, SPIN-Seq uses sequencing-by-transient-hybridization on the same surface and instrument to read sequences in each immobilized DNA molecule, directly linking phenotype and genotype. Applying SPIN-Seq, we dissected the interaction of a transcription factor with its target sequence, and revealed the sequence-dependence of RNA polymerase pausing and reaction-path branching during initial transcription. Our method provides powerful, systematic ways to understand complex molecular mechanisms and their sequence dependence. The authors develop SPIN-Seq, a method that directly links the sequence of individual DNA molecules to their functional behavior, revealing how DNA sequence impacts protein-DNA interactions and the kinetics of biological reactions on nucleic acids.

ABSTRACT Bacterial sigma factors (σ) contain a highly conserved structural module, the ‘‘σ-finger’’, which forms a loop that protrudes towards the RNA polymerase (RNAP) active-centre in the open complex and has been implicated in pre-organisation of template DNA, abortive initiation of short RNAs, initiation pausing, and promoter escape. Here, we introduce a novel single-molecule FRET (smFRET) assay to monitor σ-finger motions during transcription initiation and promoter escape. We find that the σ-finger is displaced from its position inside the active site cleft before promoter escape, and after synthesis of RNAs with lengths that are highly dependent on the sequence of the promoter used. Real-time smFRET measurements reveal the presence of significant heterogeneity in the timing of finger displacement and show that different σ-finger conformations in single open transcription complexes are associated with substantially different kinetics in transcription initiation and promoter escape, potentially impacting gene regulation in bacteria.

Single-molecule picometer-resolution nanopore tweezers (SPRNT) enables monitoring of translocation of a nucleic-acid motor protein on a nucleic-acid track with sequence registration, sub-nucleotide spatial resolution, sub-millisecond temporal resolution, and the ability to apply assisting forces. Previous work has demonstrated the ability of SPRNT to monitor the translocation of Escherichia coli RNA polymerase (RNAP) relative to the DNA template strand in transcription elongation and has directly detected a half-translocated state in sequence-dependent transcriptional pausing (Nova, I., Craig, J., Mazumder, A., Laszlo, A., Derrington, I., Noakes, M., Brinkerhoff, H., Yang, S., Vahedian-Movahed, H., Li, L., Zhang, Y., Bowman, J., Mount, J., Huang, J., Ebright., R., and Gundlach, J., in preparation).

Transcription initiation, the first step in gene expression, has been studied extensively in dilute buffer, a condition which fails to consider the crowded environment in live cells. Recent reports indicate the kinetics of promoter escape is altered in crowded conditions for a consensus bacterial promoter. Here, we use a real‐time fluorescence enhancement assay to study the kinetics of unwound bubble formation and promoter escape for three separate promoters. We find that the effect of crowding on transcription initiation is complex, with lower rates of unwound bubble formation, higher rates of promoter escape, and large variations depending on promoter identity. Based on our results, we suggest that altered conditions of crowding inside a live cell can trigger global changes.

RNA polymerases (RNAPs) carry out the first step in the central dogma of molecular biology by transcribing DNA into RNA. Despite their importance, much about how RNAPs work remains unclear, in part because the small (3.4 Angstrom) and fast (~40 ms/nt) steps during transcription were difficult to resolve. Here, we used high-resolution nanopore tweezers to observe the motion of single Escherichia coli RNAP molecules as it transcribes DNA ~1,000 times improved temporal resolution, resolving single-nucleotide and fractional-nucleotide steps of individual RNAPs at saturating nucleoside triphosphate concentrations. We analyzed RNAP during processive transcription elongation and sequence-dependent pausing at the yrbL elemental pause sequence. Each time RNAP encounters the yrbL elemental pause sequence, it rapidly interconverts between five translocational states, residing predominantly in a half-translocated state. The kinetics and force-dependence of this half-translocated state indicate it is a functional intermediate between pre- and post-translocated states. Using structural and kinetics data, we show that, in the half-translocated and post-translocated states, sequence-specific protein–DNA interaction occurs between RNAP and a guanine base at the downstream end of the transcription bubble (core recognition element). Kinetic data show that this interaction stabilizes the half-translocated and post-translocated states relative to the pre-translocated state. We develop a kinetic model for RNAP at the yrbL pause and discuss this in the context of key structural features.

Photobleaching of fluorescent probes limits the observation span of typical single-molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Förster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach should enable capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.

The Cover Feature shows a REFRESHing strategy to circumvent photobleaching: just like lightbulbs which only light up when placed on a fixture, fluorogenic single-stranded DNAs fluoresce only when hybridized to a target on a molecule of interest. These “molecular light-bulbs” are replaced regularly before they blow, so we never have to worry about being in the “dark“ again – or losing a molecule to bleaching. Cover design: Nathaniel Heather. More information can be found in the Research Article by Mirjam Kümmerlin, Abhishek Mazumder, and Achillefs N. Kapanidis.

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

Photobleaching of fluorescent probes limits the observation span of a typical single molecule fluorescence measurement and hinders simultaneous observation of dynamics across timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes throughout the experiment via transient binding of fluorescently labelled single-stranded DNAs to complementary target DNA strands attached to the target molecule. We show our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the photobleaching time under our conditions. We also show our method is adaptable to FRET and study the conformational dynamics of DNA Holliday Junctions for extended periods. By adjusting the temporal resolution and observation span, we envision to capture the conformational dynamics of proteins and nucleic acids over wide range of timescales.

ABSTRACT Protein interactions with nucleic acids are central to all genetic processes and many biotechnological applications. While many sequence-dependent protein-DNA interactions have been studied in detail using single-molecule methods, there is no standard high-throughput way to link the complex single-molecule kinetics of protein-DNA interactions with the DNA sequence of a single molecule. Here we provide the missing link by introducing a single-molecule imaging method (Gap-Seq) that interrogates DNA sequences via transient binding of short fluorescent DNA to a single DNA molecule previously used to characterise a protein-DNA interaction. In Gap-Seq, we identify a base by the degree of binding of 6-9 nt-long DNAs to surface-immobilised DNA substrates featuring a short single-stranded gap. To facilitate detection, we also developed a fluorescence quenching strategy that allows single-molecule detection at up to 500 nM of unbound fluorescent DNA. We link single-base differences on single DNA molecules to the kinetics of protein-DNA interactions by studying the interaction of a transcription activator with its cognate site. Finally, we show that our assay can address mixed sequences by distinguishing between two different sequences immobilised on the same field of view, paving the way for interrogation of sequence libraries for both mechanistic work and biotechnological applications.

The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3-0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes >1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.

Bacterial RNA Polymerases (RNAPs) bind to transcription initiation factors called σ factors to start promoter-specific transcription. Within the σ factor lies a highly conserved structural module, the “σ-finger”, which forms a loop that protrudes towards the RNAP active-center and that interacts with the template strand DNA. The close proximity of the σ-finger to the “heart” of transcription is implicated in the pre-organisation of the template strand DNA, in the synthesis of the first short RNAs, and in the pausing of transcription upon synthesis of a 6-mer RNA. However, the σ-finger also blocks entry of the nascent RNA to the RNAP exit channel and must be displaced during initial transcription as the first step in promoter escape. Despite recent structural studies, σ-finger conformational changes during late transcription initiation and promoter escape are still unknown. Here we report a novel single-molecule FRET ruler that monitors the conformational dynamics of the E.coli σ70-finger from the early stages of transcription initiation to promoter escape. Our results using the σ-finger FRET ruler on transcription complexes formed on a derivative of the lac promoter show that the σ-finger adopts three conformations, which interconvert with rates 0.01-1.5s−1. Intriguingly, we find the σ-finger first displaced in complexes trapped in synthesis of abortive transcripts up to a 10-mer RNA. This observation is likely driven by collision of the σ-finger with the 5’-end of the growing nascent RNA. Based on our results, we propose a new model describing the relation of the conformational changes in the σ-finger to the dynamics of transcription initiation and promoter escape. Archaeal and eukaryotic transcription initiation complexes also contain structural modules that block the RNAP exit channel, so it is therefore likely that this model applies to all kingdoms of life.

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.

RNA polymerase (RNAP), the protein machine at the heart of transcription, initiates RNA synthesis after a sequence of conformational changes that separate the DNA double helix around the transcription start site and form a catalytically active complex with promoter DNA (the “open complex” or “RPo”). Open complex formation has been studied extensively using ensemble biochemistry and structural biology; however, its mechanism still remains a mystery due to presence of many transient intermediates, and structural/functional heterogeneity. Here, we address these limitations by studying bacterial RPo formation in real-time and at the single-molecule level. To achieve this, we have developed novel single-molecule fluorescence assays (based on single-molecule FRET and on Cy3 fluorescence enhancement) to monitor DNA melting and loading in the main RNAP channel, and capture nanoscale conformational changes of the RNAP clamp, a mobile module implicated in DNA loading during RPo formation. In contrast to proposed models for RPo formation, our results show that the clamp remains closed during the entire process; further, we show the DNA opens in two sequential rate-limiting steps roughly linked to the opening of the upstream and downstream halves of the transcription bubble. We also show that the complexes formed on-pathway to the RPo differ kinetically and functionally to complexes formed after isomerization of the RPo to different species; this observation stresses the importance of capturing intermediates during real-time reactions. Our results allow us to reject the prevailing RPo formation models (which postulate an obligatory clamp opening/closing that allows loading of double-stranded DNA to the main RNAP channel), and to propose a model where DNA first melts outside the main channel, and is then loaded as single-stranded DNA. Our methods and biological observations are general should apply to many other processes involving DNA opening.

Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) is a technique that enables observation of single enzyme movement along nucleic acids under an applied force. SPRNT provides measurements of enzyme position along a nucleic acid substrate with sub-Angstrom spatial and millisecond temporal resolution, while simultaneously providing the DNA sequence within the enzyme. We use SPRNT to monitor many E. coli RNA Polymerase (RNAP) core complexes during transcription elongation and pausing with an assisting force. We determine that during elongation at low [NTP], RNAP primarily stalls in a post-translocated state, with brief deviations forward to a hyper-translocated state and backwards to a pre-translocated state. The rates and frequencies of these transitions vary significantly with DNA sequence and the magnitude of assisting force. During transcription pausing at an elemental pause sequence, we observe transitions between five distinct enzyme states (backtracked, pre-, half-, post-, and hyper translocated), including a half-translocated state between pre and post. We develop a model for RNAP pausing and elongation by varying the applied force and monitoring RNAP mutants with SPRNT.

The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an "unfolded"/"open" state that allows an NTP substrate to enter the active center and a "folded"/"closed" state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.

Single-molecule picometer resolution nanopore tweezers (SPRNT) is a single-molecule technique that enables observation of enzyme movement along nucleic acids under an applied force. SPRNT measures enzyme position with sub-Angstrom spatial and millisecond temporal resolution, while simultaneously providing DNA sequence within the enzyme. We use SPRNT to monitor many E. coli RNA Polymerase (RNAP) core complexes during transcription elongation and pausing with an assisting force. We determine that during elongation at low [NTP], RNAP primarily stalls in a post-translocated state, with brief deviations forward to a hyper-translocated state and backwards to a pre-translocated state. The rates and frequencies of these transitions vary with DNA sequence. During transcription pausing at a pre-determined pause sequence, we observe RNAP entering a partially-translocated state, with brief deviations to backtracked, pre-, post- and hyper-translocated states. We develop a model for RNAP pausing by varying the applied force and monitoring RNAP mutants with SPRNT.