Global Conformational Changes Research Articles

Shear-Induced Extensional Response Behaviors of Tethered von Willebrand Factor

We perform single-molecule flow experiments using confocal microscopy and a microfluidic device for shear rates up to 20,000 s−1 and present results for the shear-induced unraveling and elongation of tethered von Willebrand factor (VWF) multimers. Further, we employ companion Brownian dynamics simulations to help explain details of our experimental observations using a parameterized coarse-grained model of VWF. We show that global conformational changes of tethered VWF can be accurately captured using a relatively simple mechanical model. Good agreement is found between experimental results and computational predictions for the threshold shear rate of extension, existence of nonhomogenous fluorescence distributions along unraveled multimer contours, and large variations in extensional response behaviors. Brownian dynamics simulations reveal the strong influence of varying chain length, tethering point location, and number of tethering locations on the underlying unraveling response. Through a complex molecule like VWF that naturally adopts a wide distribution of molecular size and has multiple binding sites within each molecule, this work demonstrates the power of tandem experiment and simulation for understanding flow-induced changes in biomechanical state and global conformation of macromolecules.

Effect of pH and Molecular Length on the Structure and Dynamics of Short Poly(acrylic acid) in Dilute Solution: Detailed Molecular Dynamics Study.

Long MD simulations are carried out using a detailed all-atom force field to investigate the effect of pH or, equivalently, degree of ionization α- (= 0, 50, 100%) and degree of polymerization N (= 20, 23, 46, 70, and 110) on the structure and dynamics of poly(acrylic acid) (PAA) at infinite dilution. To ensure the validity and add to the reliability of our research conclusions, a systematic validation of several molecular mechanics force fields is performed. It is observed that the generalized AMBER force field in combination with the RESP charge fitting method best describes both the structural and dynamical behavior of PAA in comparison to experimentally obtained data. It is found that ⟨ Rg2⟩0.5changes with N as ⟨ Rg2⟩0.5 ∼ Nν, with ν = 0.27 at α- = 0% degree of ionization (acidic conditions), ν = 0.94 at α- = 50% degree of ionization (neutral conditions), and ν = 0.87 at α- = 100% degree of ionization (basic conditions), which is in perfect agreement with theory. The global shape of the PAA chain in the solution is quantified in terms of the three eigenvalues of the average radius-of-gyration tensor, the relative shape anisotropy κ2, and the asphericity parameter b. It is revealed that at α- = 0%, the chain adopts a spherelike conformation, while at α- = 50 and 100%, its conformation is flattened and flexible. In addition, it is revealed that as the degree of ionization increases, the persistence length Lp increases, which suggests that PAA chains become stiffer with increasing pH. The global and local conformational changes of the PAA chain with the degree of ionization are found to be highly related to the solvation of the polymer. Finally, it is revealed that the diffusion coefficient D of the center of mass of PAA also exhibits a power law scaling with N, D ∼ Nν, with ν = 0.25 at α- = 0% degree of ionization, ν = 0.46 at α- = 50% degree of ionization (neutral conditions), and ν = 0.44 at α- = 100% degree of ionization (basic conditions), in excellent agreement with recent experimental data and theoretical predictions.

Use of NMR to probe changes in the flexibility of acid‐stress chaperone HdeB from its inactive to active state

HdeB is an intrinsically disordered periplasmic chaperone essential to the acid resistance of numerous pathogenic bacteria. It, along with its homolog, HdeA, binds to vulnerable periplasmic proteins at low pH and prevents them from irreversibly aggregating in the acidic stomach. The activity of these two proteins is responsible for the survivability of pathogenic bacteria and resulting proliferation of dysentery. At physiological pH, HdeB is an inactive folded homodimer and remains a structurally unchanged, fully folded dimer in its active state (at pH 4–5), suggesting that the mechanism of chaperone activation is a result of changes in its dynamics (internal motions). The long‐term goal of this study is to gain insight into HdeB's unique chaperoning mechanism by characterizing its pH dependent changes in structure and dynamics. In order to achieve atomic level specificity and detect subtle conformational changes, we are utilizing Nuclear Magnetic Resonance (NMR) spectroscopy to compare HdeB's flexibility and structure in its inactive and active states at pH 6.0 and 4.5, respectively. Isotopically labeled HdeB was recombinantly expressed and purified. Subsequently, protein backbone and side chain dynamics were examined at pH 6 and pH 4.5 utilizing R2 and R1 relaxation experiments, which identify fast (ps–ns) time scale motions within the structure; this indicates local motions, such as methyl rotations. Additionally, intermediate (μs‐ms) time scale motions, which reflect global conformational changes and flexibility, were identified using CPMG relaxation dispersion experiments. Notable changes in dynamics were observed with activation. At pH 6.0, most residues with μs‐ms motions are clustered at the dimer binding interface (BC loop and helix B). With the transition to pH 4.5, μs‐ms motions expand within the dimer binding interface (W27, T33, G37, T40, K53) and extend beyond it (L16δ, I55δ). Furthermore, at pH 4.5, Helix B decreases in solvent protection, suggesting decreased burial and a loosening of the dimer binding interface. Since this helix is extremely hydrophobic, loosening exposes residues that are well suited to interacting with the exposed hydrophobic regions of an unfolding client protein, likely enabling interactions crucial to substrate binding. Interestingly, the largest changes with activation were observed near multiple titratable residues, making us suspect a protonation change. Continuing research will focus on experimentally determining acid dissociation constants to confirm this.Support or Funding InformationWe gratefully acknowledge the support of the NIH for research funds (SC3‐GM116745) and BUILD PODER for research support to L.A., as well as the NSF for funding the purchase of our NMR spectrometer (CHE‐1040134).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biophysical characterization of heme binding to the intrinsically disordered region of Bach1.

Transcriptional repressor Bach1 plays an important role in antioxidant response. Bach1 function is regulated by heme binding to the four cysteine-proline (CP) motifs in Bach1, which leads to inhibition of its activity. Three of these CP motifs are located N-terminal to the bZip (basic leucine zipper) domain that is responsible for DNA binding. Based on sequence analysis, the region surrounding these CP motifs was expected to be intrinsically disordered. Bach1 is one of few known intrinsically disordered proteins that accept multiple heme molecules for functional regulation, but the molecular mechanisms of heme binding and functional regulation remain unclear. Uncovering these mechanisms is important for understanding Bach1-mediated antioxidant response. Biophysical characterization revealed that 5-coordinated heme binding was unique to the CP motifs within the heme-binding region of Bach1, whereas 6-coordinated binding occurred nonspecifically. Comparison of the wild-type protein and a CP motif mutant indicated that the level of 6-coordinated heme binding was reduced in the absence of 5-coordinated heme binding. Analytical ultracentrifugation showed that the CP motif mutant protein had a more elongated conformation than the wild-type protein, suggesting that cysteines within the CP motifs contribute to intramolecular interactions in Bach1. Thus, heme binding at the CP motifs induces a global conformational change in the Bach1 heme-binding region, and this conformational change, in turn, regulates the biological activity of Bach1.

Two-Step Structural Changes in Orange Carotenoid Protein Photoactivation Revealed by Time-Resolved Fourier Transform Infrared Spectroscopy

The orange carotenoid protein (OCP), which is essential in cyanobacterial photoprotection, is the first photoactive protein containing a carotenoid as an active chromophore. Static and time-resolved Fourier transform infrared (FTIR) difference spectroscopy under continuous illumination at different temperatures was applied to investigate its photoactivation mechanism. Here, we demonstrate that in the OCP, the photo-induced conformational change involves at least two different steps, both in the second timescale at 277 K. Each step involves partial reorganization of α-helix domains. At early illumination times, the disappearance of a nonsolvent-exposed α-helix (negative 1651 cm-1 band) is observed. At longer times, a 1644 cm-1 negative band starts to bleach, showing the disappearance of a solvent-exposed α-helix, either the N-terminal extension and/or the C-terminal tail. A kinetic analysis clearly shows that these two events are asynchronous. Minor modifications in the overall FTIR difference spectra confirm that the global protein conformational change consists of-at least-two asynchronous contributions. Comparison of spectra recorded in H2O and D2O suggests that internal water molecules may contribute to the photoactivation mechanism.

Dynamics of the full-length yeast Hsp90 dimer

The Hsp90 chaperone is a complex homodimeric biological assembly that assists in the folding of proteins. It can undergo global conformational changes between an open (Adenosindiphosphat, ADP-bound) and closed (Adenosintriphosphat, ATP-bound) state that are of functional importance. How the conformational transitions are triggered and coupled to chaperone function is not well understood. Molecular dynamics simulations in explicit solvent starting from either the closed conformation or the open conformation in different nucleotide bound states and in the apo (without nucleotide) state were performed. On the time scale of ~300 ns the simulations starting from the closed state stayed close to the starting conformation independent of the nucleotide bound state. In case of the open structure the simulations indicated large global fluctuations including movements towards a more closed state, however, no complete transitions to the closed state were observed. The analysis of conformational fluctuations indicates only modest differences in local fluctuations of the monomers for open vs. closed state and global dimer changes are mediated by small local motions of the C-terminal Hsp90 segments.

How cardiolipin modulates the dynamics of respiratory complex I.

Cardiolipin modulates the activity of membrane-bound respiratory enzymes that catalyze biological energy transduction. The respiratory complex I functions as the primary redox-driven proton pump in mitochondrial and bacterial respiratory chains, and its activity is strongly enhanced by cardiolipin. However, despite recent advances in the structural biology of complex I, cardiolipin-specific interaction mechanisms currently remain unknown. On the basis of millisecond molecular simulations, we suggest that cardiolipin binds to proton-pumping subunits of complex I and induces global conformational changes that modulate the accessibility of the quinone substrate to the enzyme. Our findings provide key information on the coupling between complex I dynamics and activity and suggest how biological membranes modulate the structure and activity of proteins.

Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly.

Allosteric mechanism of the circadian protein Vivid resolved through Markov state model and machine learning analysis.

The fungal circadian clock photoreceptor Vivid (VVD) contains a photosensitive allosteric light, oxygen, voltage (LOV) domain that undergoes a large N-terminal conformational change. The mechanism by which a blue-light driven covalent bond formation leads to a global conformational change remains unclear, which hinders the further development of VVD as an optogenetic tool. We answered this question through a novel computational platform integrating Markov state models, machine learning methods, and newly developed community analysis algorithms. Applying this new integrative approach, we provided a quantitative evaluation of the contribution from the covalent bond to the protein global conformational change, and proposed an atomistic allosteric mechanism leading to the discovery of the unexpected importance of A’α/Aβ and previously overlooked Eα/Fα loops in the conformational change. This approach could be applicable to other allosteric proteins in general to provide interpretable atomistic representations of their otherwise elusive allosteric mechanisms.

Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography

Supercritical fluid chromatography (SFC) has become the fastest growing analytical tool for chiral and achiral small-molecule pharmaceutical separations. The benefits from savings in cost (as a result of lower solvent and energy consumption), and time have made SFC a proven effective tool for solving many analytical problems for small-molecules over the years. There is, however, a gap in the application of SFC for larger biomolecules, proteins and peptides. There has been a notable increase of protein- and peptide-based drug therapies that contain a higher-order structure important to their efficacy. These studies leverage the use of size exclusion chromatography coupled with hydrogen–deuterium exchange (SEC-HDX) methodology and circular dichroism (CD) spectroscopy to probe global conformational structures of model peptides and proteins following purification by preparative SFC. It was demonstrated that bradykinin and insulin can be used in SFC purification, and moreover, insulin was able to recover its original higher-order structure when compared to pre-purification insulin by three orthogonal techniques: 1) calculated percent alpha-helicity based on CD spectra, 2) alpha-helix – temperature hysteresis analysis by CD and 3) SEC-HDX-MS at different temperatures. However, it was shown that the higher order structures of the other three model proteins used in the study (ubiquitin, cytochrome C, and apomyoglobin) were significantly modified during SFC purification and were unable to re-fold to their original conformations. The present workflow was applied successfully to several peptide therapeutic programs at our comp any and in addition can be applied for small proteins.

DNA Origami-Protein Interactions and the Role of Steric Hindrance

DNA origami -- a novel class of synthetic DNA nanostructures -- are designed to fold into predetermined shapes that can undergo dynamic changes. Here, we characterize the structure and dynamics of a DNA origami with a triangular-shape, using Langevin-type molecular dynamics simulations, and the coarse-grained OxDNA model. In particular, we focus on two different network geometries of DNA strands and analyse the different fluctuations properties. We link the local and global conformational changes, and the local DNA network properties, to the binding propensity of the DNA cleaving protein HinP1I. Finally, we use the Michaelis-Menten theory to connect the binding propensity predictions with time-dependent gel electrophoresis experiments.

Versatile Analysis of DNA–Biomolecule Interactions in Solution by Hydrodynamic Separation and Single Molecule Detection

DNA can interact with a wide array of molecules with a range of binding affinities, stoichiometry, and size-scales. We present a sensitive, quantitative, and versatile platform for sensing and evaluating these diverse DNA-biomolecule interactions and DNA conformational changes in free solution. Single molecule free solution hydrodynamic separation utilizes differences in hydrodynamic mobility to separate bound DNA-biomolecule complexes from unbound DNA and determine the associated size change that results from binding. Single molecule detection enables highly quantitative analysis of the fraction of DNA in the bound and unbound state to characterize binding behavior including affinity, stoichiometry, and cooperativity. A stacked injection scheme increases throughput to enable practical analysis of DNA-biomolecule interactions using only picoliters of sample per measurement. To demonstrate analysis of DNA-protein interactions on a local scale, we investigate binding of the E. coli single stranded binding protein to two DNA oligos both individually and in direct competition. We show that stoichiometry and cooperativity is a function of DNA length and verify these differences in binding characteristics through direct competition. To demonstrate analysis of DNA-small molecule interactions and global conformational changes, we also assess DNA condensation with the polyamine spermidine. We use hydrodynamic mobility to evaluate the size of spermidine-condensed DNA and single molecule burst analysis to evaluate DNA packing within the condensed globules relative to free-coiled DNA. This platform thus presents a versatile tool capable of quantitative and sensitive evaluation of diverse biomolecular interactions, complex properties, and binding characteristics.

Cryo-EM reveals two distinct serotonin-bound conformations of full-length 5-HT3A receptor.

Serotonin receptor (5-HT3AR)1, a cationic pentameric ligand-gated ion channel (pLGIC), is the clinical target for management of nausea and vomiting associated with radiation and chemotherapies2. Upon binding, serotonin induces a global conformational change encompassing the ligand-binding extracellular domain (ECD), the transmembrane domain (TMD), and the intracellular domain (ICD), the molecular details of which are unclear. Here, we present two serotonin-bound structures of the full-length 5-HT3AR in distinct conformations at 3.32 Å and 3.89 Å resolutions that reveal the mechanism underlying channel activation. When compared to Apo-5-HT3AR, serotonin-bound states underwent a large twisting motion in the ECD and TMD leading to the opening of a 165 Å long permeation pathway. Notably, this motion results in creation of lateral portals for ion permeation at the interface of the TMD and ICD. Combined with molecular dynamics simulations, these structures provide novel insights into conformational coupling across domains and functional modulation.

The 2β Splice Variation Alters the Structure and Function of the Stromal Interaction Molecule Coiled-Coil Domains.

Stromal interaction molecule (STIM)-1 and -2 regulate agonist-induced and basal cytosolic calcium (Ca2+) levels after oligomerization and translocation to endoplasmic reticulum (ER)-plasma membrane (PM) junctions. At these junctions, the STIM cytosolic coiled-coil (CC) domains couple to PM Orai1 proteins and gate these Ca2+ release-activated Ca2+ (CRAC) channels, which facilitate store-operated Ca2+ entry (SOCE). Unlike STIM1 and STIM2, which are SOCE activators, the STIM2β splice variant contains an 8-residue insert located within the conserved CCs which inhibits SOCE. It remains unclear if the 2β insert further depotentiates weak STIM2 coupling to Orai1 or independently causes structural perturbations which prevent SOCE. Here, we use far-UV circular dichroism, light scattering, exposed hydrophobicity analysis, solution small angle X-ray scattering, and a chimeric STIM1/STIM2β functional assessment to provide insights into the molecular mechanism by which the 2β insert precludes SOCE activation. We find that the 2β insert reduces the overall α-helicity and enhances the exposed hydrophobicity of the STIM2 CC domains in the absence of a global conformational change. Remarkably, incorporation of the 2β insert into the STIM1 context not only affects the secondary structure and hydrophobicity as observed for STIM2, but also eliminates the more robust SOCE response mediated by STIM1. Collectively, our data show that the 2β insert directly precludes Orai1 channel activation by inducing structural perturbations in the STIM CC region.

Combination of circular dichroism spectroscopy and size-exclusion chromatography coupled with HDX-MS for studying global conformational structures of peptides in solution

Misfolding of therapeutic peptides and proteins can lead to numerous issues, ranging in severity, including loss of function, aggregation, immunogenicity, and cytotoxicity. A primary component of protein folding is secondary structure, including α-helices and β-sheets. Many native peptides and proteins are predominately α-helical therefore, it is of critical importance to develop robust and reliable analytical tools to investigate protein higher order structure, including the percentage of α-helix under various conditions, to evaluate protein folding and prevent the negative effects of misfolding. However, given the complexity of protein folding and higher order structure, it is unlikely that one technique will provide a comprehensive analysis. To bridge this gap, this study presents the combination of two orthogonal techniques – circular dichroism (CD) and size-exclusion chromatography-hydrogen-deuterium exchange-mass spectrometry (SEC-HDX-MS) to investigate global peptide and protein conformations. Also, the incorporation of trifluoroethanol (TFE), a known stabilizer of α-helical structures, into the analyses, aims to enhance the discrimination power of these two techniques by increasing the alpha helical stability range of study. CD data was used to estimate the percent of α-helix content and its thermal stability while online SEC-HDX-MS screening compared global conformational changes of each peptide based on a difference in the number of deuterons exchanged to protons, ΔHDX. The workflow described in this report can be very beneficial in pharmaceutical development. The model peptides were chosen to demonstrate the workflow with commercially available compounds. The goal of this study was to show a proof-of-concept for direct correlation of these methodologies and to estimate the percentage of α-helix content at a particular ΔHDX, which is indicative of the state of protein folding.

Global conformational changes in IgG-Fc upon mutation of the FcRn-binding site are not associated with altered antibody-dependent effector functions.

Antibody engineering is important for many diagnostic and clinical applications of monoclonal antibodies. We recently reported a series of fragment crystallizable (Fc) mutations targeting the neonatal Fc receptor (FcRn) site on a Lewis Y (Ley) binding IgG1, hu3S193. The hu3S193 variants displayed shortened in vivo half-lives and may have potential for radioimaging or radiotherapy of Ley-positive tumors. Here, we report Fc crystal structures of wild-type hu3S193, seven FcRn-binding site variants, and a variant lacking C1q binding or complement-dependent cytotoxicity (CDC) activity. The Fc conformation of the FcRn-binding sites was similar for wild-type and all mutants of hu3S193 Fc, which suggests that FcRn interactions were directly affected by the amino acid substitutions. The C1q-binding site mutant Fc was nearly identical with the wild-type Fc. Surprisingly, several hu3S193 Fc variants showed large changes in global structure compared with wild-type Fc. All hu3S193 Fc mutants had similar antibody-dependent cellular cytotoxicity, despite some with conformations expected to diminish Fc gamma receptor binding. Several hu3S193 variants displayed altered CDC, but there was no correlation with the different Fc conformations. All versions of hu3S193, except the C1q-binding site mutant, bound C1q, suggesting that the altered CDC of some variants could result from different propensities to form IgG hexamers after engaging Ley on target cells. Overall, our findings support the concept that the antibody Fc is both flexible and mobile in solution. Structure-based design approaches should take into account the conformational plasticity of the Fc when engineering antibodies with optimal effector properties.

Mapping the functional anatomy of Orai1 transmembrane domains for CRAC channel gating

Store-operated Orai1 channels are activated through a unique inside-out mechanism involving binding of the endoplasmic reticulum Ca2+ sensor STIM1 to cytoplasmic sites on Orai1. Although atomic-level details of Orai structure, including the pore and putative ligand binding domains, are resolved, how the gating signal is communicated to the pore and opens the gate is unknown. To address this issue, we used scanning mutagenesis to identify 15 residues in transmembrane domains (TMs) 1-4 whose perturbation activates Orai1 channels independently of STIM1. Cysteine accessibility analysis and molecular-dynamics simulations indicated that constitutive activation of the most robust variant, H134S, arises from a pore conformational change that opens a hydrophobic gate to augment pore hydration, similar to gating evoked by STIM1. Mutational analysis of this locus suggests that H134 acts as steric brake to stabilize the closed state of the channel. In addition, atomic packing analysis revealed distinct functional contacts between the TM1 pore helix and the surrounding TM2/3 helices, including one set mediated by a cluster of interdigitating hydrophobic residues and another by alternative ridges of polar and hydrophobic residues. Perturbing these contacts via mutagenesis destabilizes STIM1-mediated Orai1 channel gating, indicating that these bridges between TM1 and the surrounding TM2/3 ring are critical for conveying the gating signal to the pore. These findings help develop a framework for understanding the global conformational changes and allosteric interactions between topologically distinct domains that are essential for activation of Orai1 channels.

Crystal structure of an assembly intermediate of respiratory Complex II

In approximately 10% of flavoenzymes, the flavin is covalently attached. Despite the importance of this covalent cofactor for a number of biological functions, the mechanism of covalent flavinylation is currently poorly understood. In this work, we determined the crystal structure of a captured assembly intermediate of covalent flavinylation using the Escherichia coli Complex II FrdA subunit crosslinked with its assembly factor, SdhE. Two global conformational changes in FrdA subunit were observed as compared to prior structures of the assembled Complex II protein: rotation of the capping domain, and the destabilization of two large loops proximal to the active site of the FrdA subunit. Together, these changes create a tunnel to the active site. This FrdA‐SdhE structure was used as a basis to develop a mechanism of covalent flavinylation that is supported by spectroscopic and kinetic analyses.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biophysical characterization of hit compounds for mechanism-based enzyme activation.

Across all families of enzymes, only a dozen or so distinct classes of non-natural small molecule activators have been characterized, with only four known modes of activation among them. All of these modes of activation rely on naturally evolved binding sites that trigger global conformational changes. Among the enzymes that are of greatest interest for small molecule activation are the seven sirtuin enzymes, nicotinamide adenine dinucleotide (NAD+)-dependent protein deacylases that play a central role in the regulation of healthspan and lifespan in organisms ranging from yeast to mammals. However, there is currently no understanding of how to design sirtuin-activating compounds beyond allosteric activators of SIRT1-catalyzed reactions that are limited to particular substrates. Here, we introduce a general mode of sirtuin activation that is distinct from the known modes of enzyme activation. Based on the conserved mechanism of sirtuin-catalyzed deacylation reactions, we establish biophysical properties of small molecule modulators that can in principle result in enzyme activation for diverse sirtuins and substrates. Building upon this framework, we propose strategies for the identification, characterization and evolution of hits for mechanism-based enzyme activating compounds.

RNA Polymerase Accommodates a Pause RNA Hairpin by Global Conformational Rearrangements that Prolong Pausing

Sequence-specific pausing by RNA polymerase (RNAP) during transcription plays crucial and diverse roles in gene expression. In bacteria, RNA structures are thought to fold within the RNA exit channel of the RNAP and can increase pause lifetimes significantly. The biophysical mechanism of pausing is uncertain. We used single-particle cryo-EM to determine structures of paused complexes, including a 3.8-Å structure of an RNA hairpin-stabilized, paused RNAP that coordinates RNA folding in the his operon attenuation control region of E.coli. The structures revealed a half-translocated pause state (RNA post-translocated, DNA pre-translocated) that can explain transcriptional pausing and a global conformational change of RNAP that allosterically inhibits trigger loop folding and can explain pause hairpin action. Pause hairpin interactions with the RNAP RNA exit channel suggest how RNAP guides the formation of nascent RNA structures.