Structural basis for intrinsic transcription termination.

Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.

Structural basis of RNA polymerase recycling by the Swi2/Snf2 family of ATPase RapA in Escherichia coli.

After transcription termination, cellular RNA polymerases (RNAPs) are occasionally trapped on DNA, impounded in an undefined post-termination complex (PTC), limiting the free RNAP pool and subsequently leading to inefficient transcription. In Escherichia coli, a Swi2/Snf2 family of ATPase called RapA is known to be involved in countering such inefficiency through RNAP recycling; however, the precise mechanism of this recycling is unclear. To better understand its mechanism, here we determined the structures of two sets of E. coli RapA-RNAP complexes, along with the RNAP core enzyme and the elongation complex, using cryo-EM. These structures revealed the large conformational changes of RNAP and RapA upon their association that has been implicated in the hindrance of PTC formation. Our results along with DNA-binding assays reveal that although RapA binds RNAP away from the DNA-binding main channel, its binding can allosterically close the RNAP clamp, thereby preventing its nonspecific DNA binding and PTC formation. Taken together, we propose that RapA acts as a guardian of RNAP by which RapA prevents nonspecific DNA binding of RNAP without affecting the binding of promoter DNA recognition σ factor, thereby enhancing RNAP recycling.

Target site selection and remodelling by type V CRISPR-transposon systems.

Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements1. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion2-7. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ7, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools.

Integration host factor bends and bridges DNA in a multiplicity of binding modes with varying specificity.

Nucleoid-associated proteins (NAPs) are crucial in organizing prokaryotic DNA and regulating genes. Vital to these activities are complex nucleoprotein structures, however, how these form remains unclear. Integration host factor (IHF) is an Escherichia coli NAP that creates very sharp bends in DNA at sequences relevant to several functions including transcription and recombination, and is also responsible for general DNA compaction when bound non-specifically. We show that IHF-DNA structural multimodality is more elaborate than previously thought, and provide insights into how this drives mechanical switching towards strongly bent DNA. Using single-molecule atomic force microscopy and atomic molecular dynamics simulations we find three binding modes in roughly equal proportions: 'associated' (73° of DNA bend), 'half-wrapped' (107°) and 'fully-wrapped' (147°), only the latter occurring with sequence specificity. We show IHF bridges two DNA double helices through non-specific recognition that gives IHF a stoichiometry greater than one and enables DNA mesh assembly. We observe that IHF-DNA structural multiplicity is driven through non-specific electrostatic interactions that we anticipate to be a general NAP feature for physical organization of chromosomes.

TEM observation of compacted DNA of Synechococcus elongatus PCC 7942 using DRAQ5 labeling with DAB photooxidation and osmium black.

To visualize the fine structure of compacted DNA of Synechococcus elongatus PCC 7942, which appears at a specific time in the regular light/dark cycle prior to cell division, ChromEM with some modifications was applied. After staining DNA with DRAQ5, the cells were fixed and irradiated by red laser in the presence of 3,3'-diaminobenzidine and subsequently fixed with OsO4. A system with He-Ne laser (633 nm) was set up for efficient irradiation of the bacterial cells in aqueous solution. The compacted DNA was visualized by transmission electron microscopy, in ultrathin sections as electron dense staining by osmium black.

Structural basis of bacterial σ28 -mediated transcription reveals roles of the RNA polymerase zinc-binding domain.

In bacteria, σ28 is the flagella-specific sigma factor that targets RNA polymerase (RNAP) to control the expression of flagella-related genes involving bacterial motility and chemotaxis. However, the structural mechanism of σ28 -dependent promoter recognition remains uncharacterized. Here, we report cryo-EM structures of E. coli σ28 -dependent transcribing complexes on a complete flagella-specific promoter. These structures reveal how σ28 -RNAP recognizes promoter DNA through strong interactions with the -10 element, but weak contacts with the -35 element, to initiate transcription. In addition, we observed a distinct architecture in which the ß' zinc-binding domain (ZBD) of RNAP stretches out from its canonical position to interact with the upstream non-template strand. Further in vitro and in vivo assays demonstrate that this interaction has the overall effect of facilitating closed-to-open isomerization of the RNAP-promoter complex by compensating for the weak interaction between σ4 and -35 element. This suggests that ZBD relocation may be a general mechanism employed by σ70 family factors to enhance transcription from promoters with weak σ4/-35 element interactions.

A conformational switch in response to Chi converts RecBCD from phage destruction to DNA repair.

The RecBCD complex plays key roles in phage DNA degradation, CRISPR array acquisition (adaptation) and host DNA repair. The switch between these roles is regulated by a DNA sequence called Chi. We report cryo-EM structures of the Escherichia coli RecBCD complex bound to several different DNA forks containing a Chi sequence, including one in which Chi is recognized and others in which it is not. The Chi-recognized structure shows conformational changes in regions of the protein that contact Chi and reveals a tortuous path taken by the DNA. Sequence specificity arises from interactions with both the RecC subunit and the sequence itself. These structures provide molecular details for how Chi is recognized and insights into the changes that occur in response to Chi binding that switch RecBCD from bacteriophage destruction and CRISPR spacer acquisition to constructive host DNA repair.

Mechanism of DNA End Sensing and Processing by the Mre11-Rad50 Complex.

DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils.

Projection structures reveal the position of the DNA within DNA-Dps Co-crystals.

One of the universal mechanisms for the response of Escherichia coli to stress is the increase of the synthesis of specific histone-like proteins that bind the DNA, Dps. As a result, two-and three-dimensional crystalline arrays may be observed in the cytoplasm of starving cells. Here, we determined the conditions to obtain very thin two-dimensional DNA-Dps co-crystals in vitro, and studied their projection structures, using electron microscopy. Analysis of the projection maps of the free Dps crystals revealed two lattice types: hexagonal and rectangular. We used the fluorescently labeled DNA to prove that the DNA is present within the co-crystals with Dps in vitro, and visualized its position using transmission electron microscopy. Molecular modeling confirmed the DNA position within the crystal. We have also suggested a structural model for the DNA-Dps co-crystal dissolving in the presence of Mg2+ ions.

Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding.

A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex1-3. To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12-14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed4-6. Here we present cryo-electron microscopy structures of bacterial RNAP-promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2-universal structural features of RNAP-in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life.

Role of a Stem-Loop Structure in Helicobacter pylori cagA Transcript Stability.

Helicobacter pylori CagA is a secreted effector protein that contributes to gastric carcinogenesis. Previous studies showed that there is variation among H. pylori strains in the steady-state levels of CagA and that a strain-specific motif downstream of the cagA transcriptional start site (the +59 motif) is associated with both high levels of CagA and premalignant gastric histology. The cagA 5' untranslated region contains a predicted stem-loop-forming structure adjacent to the +59 motif. In the current study, we investigated the effect of the +59 motif and the adjacent stem-loop on cagA transcript levels and cagA mRNA stability. Using site-directed mutagenesis, we found that mutations predicted to disrupt the stem-loop structure resulted in decreased steady-state levels of both the cagA transcript and the CagA protein. Additionally, these mutations resulted in a decreased cagA mRNA half-life. Mutagenesis of the +59 motif without altering the stem-loop structure resulted in reduced steady-state cagA transcript and CagA protein levels but did not affect cagA transcript stability. cagA transcript stability was not affected by increased sodium chloride concentrations, an environmental factor known to augment cagA transcript levels and CagA protein levels. These results indicate that both a predicted stem-loop structure and a strain-specific +59 motif in the cagA 5' untranslated region influence the levels of cagA expression.

Bovine macrophage-derived extracellular traps act as early effectors against the abortive parasite Neospora caninum.

Macrophages are multipurpose phagocytes and are considered to be irreplaceable during the early host innate immune response against microbial and parasitic pathogens. However, no report has investigated the novel anti-parasitic mechanism of macrophage-derived extracellular traps (ETs) against the abortive apicomplexan parasite Neospora caninum (N. caninum) in cattle. Scanning electron microscopy (SEM) was used to visualize and characterize N. caninum tachyzoite-induced macrophage-triggered ETs in exposed bovine macrophages. Fluorescence confocal microscopy was used to confirm the classical backbone structure of DNA embedded with histone 3 (H3) and myeloperoxidase (MPO) in N. caninum tachyzoite-induced macrophage-derived ETs. Furthermore, the lactate dehydrogenase (LDH) levels in the supernatants of parasite-exposed macrophages were detected by a LDH Cytotoxicity Assay® kit. The results clearly demonstrated that N. caninum tachyzoites triggered bovine macrophage-derived ET-like structures. Inhibiting assays revealed that N. caninum tachyzoite-induced macrophage-mediated ET formation may be an ERK 1/2- and p38 MAPK-dependent cell death process. In conclusion, the present study is the first report on the formation of ETs in bovine macrophages against N. caninum tachyzoites and adds new data on the possible role of macrophages in vivo infection by capturing invasive stages and exposing them to other leukocytes.

High-resolution 3D models of Caulobacter crescentus chromosome reveal genome structural variability and organization.

High-resolution three-dimensional models of Caulobacter crescentus nucleoid structures were generated via a multi-scale modeling protocol. Models were built as a plectonemically supercoiled circular DNA and by incorporating chromosome conformation capture based data to generate an ensemble of base pair resolution models consistent with the experimental data. Significant structural variability was found with different degrees of bending and twisting but with overall similar topologies and shapes that are consistent with C. crescentus cell dimensions. The models allowed a direct mapping of the genomic sequence onto the three-dimensional nucleoid structures. Distinct spatial distributions were found for several genomic elements such as AT-rich sequence elements where nucleoid associated proteins (NAPs) are likely to bind, promoter sites, and some genes with common cellular functions. These findings shed light on the correlation between the spatial organization of the genome and biological functions.

Dynamics of the Interaction of RecG Protein with Stalled Replication Forks.

As a guardian of the bacterial genome, the RecG DNA helicase repairs DNA replication and rescues stalled replication. We applied atomic force microscopy (AFM) to directly visualize dynamics of RecG upon the interaction with replication fork substrates in the presence and absence of SSB using high-speed AFM. We directly visualized that RecG moves back and forth over dozens of base pairs in the presence of SSB. There is no RecG translocation in the absence of SSB. Computational modeling was performed to build models of Escherichia coli RecG in a free state and in complex with the fork. The simulations revealed the formation of complexes of RecG with the fork and identified conformational transitions that may be responsible for RecG remodeling that can facilitate RecG translocation along the DNA duplex. Such complexes do not form with the DNA duplex, which is in line with experimental data. Overall, our results provide mechanistic insights into the modes of interaction of RecG with the replication fork, suggesting a novel role of RecG in the repair of stalled DNA replication forks.

Visualizing bacterial DNA replication and repair with molecular resolution.

Although DNA replication and repair in bacteria have been extensively studied for many decades, in recent years the development of single-molecule microscopy has provided a new perspective on these fundamental processes. Because single-molecule imaging super-resolves the nanometer-scale dynamics of molecules, and because single-molecule imaging is sensitive to heterogeneities within a sample, this nanoscopic microscopy technique measures the motions, localizations, and interactions of proteins in real time without averaging ensemble observations, both in vitro and in vivo. In this Review, we provide an overview of several recent single-molecule fluorescence microscopy studies on DNA replication and repair. These experiments have shown that, in both Escherichia coli and Bacillus subtilis the DNA replication proteins are highly dynamic. In particular, even highly processive replicative DNA polymerases exchange to and from the replication fork on the scale of a few seconds. Furthermore, single-molecule investigations of the DNA mismatch repair (MMR) pathway have measured the complex interactions between MMR proteins, replication proteins, and DNA. Single-molecule imaging will continue to improve our understanding of fundamental processes in bacteria including DNA replication and repair.

Structural basis of bacterial transcription activation.

In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3',5'-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo-electron microscopy structure of an intact Escherichia coli class I TAC containing a CAP dimer, a σ70-RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide. The structure shows how CAP wraps the upstream DNA and how the interactions recruit RNAP. Our study provides a structural basis for understanding how activators activate transcription through the class I recruitment mechanism.

Probing the mechanical stability of bridged DNA-H-NS protein complexes by single-molecule AFM pulling.

Atomic force microscopy (AFM) has proven to be a powerful tool for the study of DNA-protein interactions due to its ability to image single molecules at the nanoscale. However, the use of AFM in force spectroscopy to study DNA-protein interactions has been limited. Here we developed a high throughput, AFM based, pulling assay to measure the strength and kinetics of protein bridging of DNA molecules. As a model system, we investigated the interactions between DNA and the Histone-like Nucleoid-Structuring protein (H-NS). We confirmed that H-NS both changes DNA rigidity and forms bridges between DNA molecules. This straightforward methodology provides a high-throughput approach with single-molecule resolution which is widely applicable to study cross-substrate interactions such as DNA-bridging proteins.

Compaction and condensation of DNA mediated by the C-terminal domain of Hfq.

Hfq is a bacterial protein that is involved in several aspects of nucleic acids metabolism. It has been described as one of the nucleoid associated proteins shaping the bacterial chromosome, although it is better known to influence translation and turnover of cellular RNAs. Here, we explore the role of Escherichia coli Hfq's C-terminal domain in the compaction of double stranded DNA. Various experimental methodologies, including fluorescence microscopy imaging of single DNA molecules confined inside nanofluidic channels, atomic force microscopy, isothermal titration microcalorimetry and electrophoretic mobility assays have been used to follow the assembly of the C-terminal and N-terminal regions of Hfq on DNA. Results highlight the role of Hfq's C-terminal arms in DNA binding, change in mechanical properties of the double helix and compaction of DNA into a condensed form. The propensity for bridging and compaction of DNA by the C-terminal domain might be related to aggregation of bound protein and may have implications for protein binding related gene regulation.

Cryo-EM Structure of a Relaxase Reveals the Molecular Basis of DNA Unwinding during Bacterial Conjugation.

Relaxases play essential roles in conjugation, the main process by which bacteria exchange genetic material, notably antibiotic resistance genes. They are bifunctional enzymes containing a trans-esterase activity, which is responsible for nicking the DNA strand to be transferred and for covalent attachment to the resulting 5'-phosphate end, and a helicase activity, which is responsible for unwinding the DNA while it is being transported to a recipient cell. Here we show that these two activities are carried out by two conformers that can both load simultaneously on the origin of transfer DNA. We solve the structure of one of these conformers by cryo electron microscopy to near-atomic resolution, elucidating the molecular basis of helicase function by relaxases and revealing insights into the mechanistic events taking place in the cell prior to substrate transport during conjugation.

Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model using ex situ atomic force microscopy.

The influence of heparin and heparan sulphate (HepS) on the appearance and analysis of open promoter complex (RPo) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ70RNAP) on linear DNA using ex situ imaging by atomic force microscopy (AFM) has been investigated. Introducing heparin or HepS into the reaction mix significantly reduces non-specific interactions of the σ70RNAP and RNAP after RPo formation allowing for better interpretation of complexes shown within AFM images, particularly on DNA templates containing more than one promoter. Previous expectation was that negatively charged polysaccharides, often used as competitive inhibitors of σRNAP binding and RPo formation, would also inhibit binding of the DNA template to the mica support surface and thereby lower the imaging yield of active RNAP-DNA complexes. We found that the reverse of this was true, and that the yield of RPo formation detected by AFM, for a simple tandem gene model containing two λPR promoters, increased. Moreover and unexpectedly, HepS was more efficient than heparin, with both of them having a dispersive effect on the sample, minimising unwanted RNAP-RNAP interactions as well as non-specific interactions between the RNAP and DNA template. The success of this method relied on the observation that E. coli RNAP has the highest affinity for the mica surface of all the molecular components. For our system, the affinity of the three constituent biopolymers to muscovite mica was RNAP>Heparin or HepS>DNA. While we observed that heparin and HepS can inhibit DNA binding to the mica, the presence of E. coli RNAP overcomes this effect allowing a greater yield of RPos for AFM analysis. This method can be extended to other DNA binding proteins and enzymes, which have an affinity to mica higher than DNA, to improve sample preparation for AFM studies.