Structural Basis for Transcript Elongation Control by NusG Family Universal Regulators.

NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and coordinating ρ-dependent termination and translation. The NusG paralog RfaH engages the EC only at operon polarity suppressor (ops) sites and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryoelectron microscopy (cryo-EM) to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base-pairing of the upstream duplex DNA to suppress backtracking, explaining stimulation of elongation genome-wide. The RfaH-opsEC structure reveals how RfaH confers operon specificity through specific recognition of an ops hairpin in the single-stranded nontemplate DNA and tighter binding to the EC to exclude NusG. Tight EC binding by RfaH sterically blocks the swiveled RNAP conformation necessary for hairpin-stabilized pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.

Pre-termination Transcription Complex: Structure and Function.

Rho is a general transcription termination factor playing essential roles in RNA polymerase (RNAP) recycling, gene regulation, and genomic stability in most bacteria. Traditional models of transcription termination postulate that hexameric Rho loads onto RNA prior to contacting RNAP and then translocates along the transcript in pursuit of the moving RNAP to pull RNA from it. Here, we report the cryoelectron microscopy (cryo-EM) structures of two termination process intermediates. Prior to interacting with RNA, Rho forms a specific "pre-termination complex" (PTC) with RNAP and elongation factors NusA and NusG, which stabilize the PTC. RNA exiting RNAP interacts with NusA before entering the central channel of Rho from the distal C-terminal side of the ring. We map the principal interactions in the PTC and demonstrate their critical role in termination. Our results support a mechanism in which the formation of a persistent PTC is a prerequisite for termination.

Unlocking translational machinery for antitubercular drug development.

Targeting translational factor proteins (TFPs) presents significant promise for the development of innovative antitubercular drugs. Previous insights from antibiotic binding mechanisms and recently solved 3D crystal structures of Mycobacterium tuberculosis (Mtb) elongation factor thermo unstable-GDP (EF-Tu-GDP), elongation factor thermo stable-EF-Tu (EF-Ts-EF-Tu), and elongation factor G-GDP (EF-G-GDP) have opened up new avenues for the design and development of potent antituberculosis (anti-TB) therapies.

An α helix to ß barrel domain switch transforms the transcription factor RfaH into a translation factor.

NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a ß barrel in NusG and an α hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here, we show that the released RfaH-CTD completely refolds from an all-α to an all-ß conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated. PAPERFLICK:

Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P.

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

Structural basis of mitochondrial receptor binding and constriction by DRP1.

Mitochondrial inheritance, genome maintenance and metabolic adaptation depend on organelle fission by dynamin-related protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogues mitochondrial dynamics proteins of 49 and 51 kDa (MID49 and MID51) and mitochondrial fission factor (MFF); however, the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryo-electron microscopy structure of full-length human DRP1 co-assembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a marked elongation and rotation of the GTPase domain, bundle-signalling element and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes the polymerization of a linear DRP1 filament with MID49 or MID51. After co-assembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery.

Structure of Gcn1 bound to stalled and colliding 80S ribosomes.

The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.

The Structural and Molecular Mechanisms of Mycobacterium tuberculosis Translational Elongation Factor Proteins.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

Structural basis for the dynamics of human methionyl-tRNA synthetase in multi-tRNA synthetase complexes.

In mammals, eight aminoacyl-tRNA synthetases (AARSs) and three AARS-interacting multifunctional proteins (AIMPs) form a multi-tRNA synthetase complex (MSC). MSC components possess extension peptides for MSC assembly and specific functions. Human cytosolic methionyl-tRNA synthetase (MRS) has appended peptides at both termini of the catalytic main body. The N-terminal extension includes a glutathione transferase (GST) domain responsible for interacting with AIMP3, and a long linker peptide between the GST and catalytic domains. Herein, we determined crystal structures of the human MRS catalytic main body, and the complex of the GST domain and AIMP3. The structures reveal human-specific structural details of the MRS, and provide a dynamic model for MRS at the level of domain orientation. A movement of zinc knuckles inserted in the catalytic domain is required for MRS catalytic activity. Depending on the position of the GST domain relative to the catalytic main body, MRS can either block or present its tRNA binding site. Since MRS is part of a huge MSC, we propose a dynamic switching between two possible MRS conformations; a closed conformation in which the catalytic domain is compactly attached to the MSC, and an open conformation with a free catalytic domain dissociated from other MSC components.

RfaH May Oppose Silencing by H-NS and YmoA Proteins during Transcription Elongation.

Nucleoid-associated proteins (NAPs) silence xenogenes by blocking RNA polymerase binding to promoters and hindering transcript elongation. In Escherichia coli, H-NS and its homolog SptA interact with YmoA proteins Hha and YdgT to assemble nucleoprotein filaments that facilitate transcription termination by Rho, which acts in synergy with NusG. Countersilencing during initiation is facilitated by proteins that exclude NAPs from promoter regions, but auxiliary factors that alleviate silencing during elongation are not known. A specialized NusG paralog, RfaH, activates lipopolysaccharide core biosynthesis operons, enabling survival in the presence of detergents and antibiotics. RfaH strongly inhibits Rho-dependent termination by reducing RNA polymerase pausing, promoting translation, and competing with NusG. We hypothesize that RfaH also acts as a countersilencer of NAP/YmoA filaments. We show that deletions of hns and hha+ydgT suppress the growth defects of ΔrfaH by alleviating Rho-mediated polarity within the waa operon. The absence of YmoA proteins exacerbates cellular defects caused by reduced Rho levels or Rho inhibition by bicyclomycin but has negligible effects at a strong model Rho-dependent terminator. Our findings that the distribution of Hha and RfaH homologs is strongly correlated supports a model in which they comprise a silencing/countersilencing pair that controls expression of chromosomal and plasmid-encoded xenogenes. IMPORTANCE Horizontally acquired DNA drives bacterial evolution, but its unregulated expression may harm the recipient. Xenogeneic silencers recognize foreign genes and inhibit their transcription. However, some xenogenes, such as those encoding lipo- and exopolysaccharides, confer resistance to antibiotics, bile salts, and detergents, necessitating the existence of countersilencing fitness mechanisms. Here, we present evidence that Escherichia coli antiterminator RfaH alleviates silencing of the chromosomal waa operon and propose that plasmid-encoded RfaH homologs promote dissemination of antibiotic resistance genes through conjugation.

The N-terminal domain of RfaH plays an active role in protein fold-switching.

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) paused at a DNA signal. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a ß-barrel while RfaH CTD is forming an α-hairpin blocking such site. Upon recognition of the specific DNA exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a ß-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, little attention has been given to the role of the NTD in the fold-switching process, as its structure remains unchanged. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards α-folded CTD, whereas the NTD interferes with ßCTD folding, becoming trapped in a ß-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the α-folded RfaH by specifically orienting the αCTD upon interdomain binding and by favoring ß-barrel rupture into an intermediate from which fold-switching proceeds.

Structural fluctuations and mechanical stabilities of the metamorphic protein RfaH.

RfaH is a compact two-domain bacterial transcription factor that functions both as a regulator of transcription and an enhancer of translation. Underpinning the dual functional roles of RfaH is a partial but dramatic fold switch, which completely transforms the ~50-amino acid C-terminal domain (CTD) from an all-α state to an all-ß state. The fold switch of the CTD occurs when RfaH binds to RNA polymerase (RNAP), however, the details of how this structural transformation is triggered is not well understood. Here we use all-atom Monte Carlo simulations to characterize structural fluctuations and mechanical stability properties of the full-length RfaH and the CTD as an isolated fragment. In agreement with experiments, we find that interdomain contacts are crucial for maintaining a stable, all-α CTD in free RfaH. To probe mechanical properties, we use pulling simulations to measure the work required to inflict local deformations at different positions along the chain. The resulting mechanical stability profile reveals that free RfaH can be divided into a "rigid" part and a "soft" part, with a boundary that nearly coincides with the boundary between the two domains. We discuss the potential role of this feature for how fold switching may be triggered by interaction with RNAP.

EFL1 mutations impair eIF6 release to cause Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is a recessive disorder typified by bone marrow failure and predisposition to hematological malignancies. SDS is predominantly caused by deficiency of the allosteric regulator Shwachman-Bodian-Diamond syndrome that cooperates with elongation factor-like GTPase 1 (EFL1) to catalyze release of the ribosome antiassociation factor eIF6 and activate translation. Here, we report biallelic mutations in EFL1 in 3 unrelated individuals with clinical features of SDS. Cellular defects in these individuals include impaired ribosomal subunit joining and attenuated global protein translation as a consequence of defective eIF6 eviction. In mice, Efl1 deficiency recapitulates key aspects of the SDS phenotype. By identifying biallelic EFL1 mutations in SDS, we define this leukemia predisposition disorder as a ribosomopathy that is caused by corruption of a fundamental, conserved mechanism, which licenses entry of the large ribosomal subunit into translation.

Cys-pair reporters detect a constrained trigger loop in a paused RNA polymerase.

Transcriptional pausing, which regulates transcript elongation in both prokaryotes and eukaryotes, is thought to involve formation of alternative RNA polymerase conformations in which nucleotide addition is inhibited in part by restriction of trigger loop (TL) folding. The polymorphous TL must convert from a random coil to a helical hairpin that contacts the nucleotide triphosphate (NTP) substrate to allow rapid nucleotide addition. Understanding the distribution of TL conformations in different enzyme states is made difficult by the TL's small size and sensitive energetics. Here, we report a Cys-pair reporter strategy to elucidate the relative occupancies of different TL conformations in E. coli RNA polymerase based on the ability of Cys residues engineered into the TL and surrounding regions to form disulfide bonds. Our results indicate that a paused complex stabilized by a nascent RNA hairpin favors nonproductive TL conformations that persist after NTP binding but can be reversed by the elongation factor RfaH.

SuhB is an integral part of the ribosomal antitermination complex and interacts with NusA.

The synthesis of ribosomal RNA (rRNA) is a tightly regulated central process in all cells. In bacteria efficient expression of all seven rRNA operons relies on the suppression of termination signals (antitermination) and the proper maturation of the synthesized rRNA. These processes depend on N-utilization substance (Nus) factors A, B, E and G, as well as ribosomal protein S4 and inositol monophosphatase SuhB, but their structural basis is only poorly understood. Combining nuclear magnetic resonance spectroscopy and biochemical approaches we show that Escherichia coli SuhB can be integrated into a Nus factor-, and optionally S4-, containing antitermination complex halted at a ribosomal antitermination signal. We further demonstrate that SuhB specifically binds to the acidic repeat 2 (AR2) domain of the multi-domain protein NusA, an interaction that may be involved in antitermination or posttranscriptional processes. Moreover, we show that SuhB interacts with RNA and weakly associates with RNA polymerase (RNAP). We finally present evidence that SuhB, the C-terminal domain of the RNAP α-subunit, and the N-terminal domain of NusG share binding sites on NusA-AR2 and that all three can release autoinhibition of NusA, indicating that NusA-AR2 serves as versatile recruitment platform for various factors in transcription regulation.

A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure.

The vectorial (5'-to-3' at varying velocity) synthesis of RNA by cellular RNA polymerases (RNAPs) creates a rugged kinetic landscape, demarcated by frequent, sometimes long-lived, pauses. In addition to myriad gene-regulatory roles, these pauses temporally and spatially program the co-transcriptional, hierarchical folding of biologically active RNAs. Conversely, these RNA structures, which form inside or near the RNA exit channel, interact with the polymerase and adjacent protein factors to influence RNA synthesis by modulating pausing, termination, antitermination, and slippage. Here, we review the evolutionary origin, mechanistic underpinnings, and regulatory consequences of this interplay between RNAP and nascent RNA structure. We categorize and rationalize the extensive linkage between the transcriptional machinery and its product, and provide a framework for future studies.

Differential Local Stability Governs the Metamorphic Fold Switch of Bacterial Virulence Factor RfaH.

RfaH, a two-domain protein from a universally conserved NusG/Spt5 family of regulators, is required for the transcription and translation of long virulence and conjugation operons in many Gram-negative bacterial pathogens. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA element. Upon recruitment, the C-terminal domain of RfaH refolds from an α-hairpin, which is bound to RNA polymerase binding site within the N-terminal domain, into an unbound ß-barrel that interacts with the ribosome. Although structures of the autoinhibited (α-hairpin) and active (ß-barrel) states and plausible refolding pathways have been reported, how this reversible switch is encoded within RfaH sequence and structure is poorly understood. Here, we combined hydrogen-deuterium exchange measurements by mass spectrometry and nuclear magnetic resonance with molecular dynamics to evaluate the differential local stability between both RfaH folds. Deuteron incorporation reveals that the tip of the C-terminal hairpin (residues 125-145) is stably folded in the autoinhibited state (∼20% deuteron incorporation), whereas the rest of this domain is highly flexible (>40% deuteron incorporation), and its flexibility only decreases in the ß-folded state. Computationally predicted ΔG agree with these results by displaying similar anisotropic stability within the tip of the α-hairpin and on neighboring N-terminal domain residues. Remarkably, the ß-folded state shows comparable structural flexibility than nonmetamorphic homologs. Our findings provide information critical for understanding the metamorphic behavior of RfaH and other chameleon proteins and for devising targeted strategies to combat bacterial infections.

NMR and crystallographic structural studies of the Elongation factor P from Staphylococcus aureus.

Elongation factor P (EF-P) is a translation protein factor that plays an important role in specialized translation of consecutive proline amino acid motifs. EF-P is an essential protein for cell fitness in native environmental conditions. It regulates synthesis of proteins involved in bacterial motility, environmental adaptation and bacterial virulence, thus making EF-P a potential drug target. In the present study, we determined the solution and crystal structure of EF-P from the pathogenic bacteria Staphylococcus aureus at 1.48 Å resolution. The structure can serve as a platform for structure-based drug design of novel antibiotics to combat the growing antibiotic resistance of S. aureus.

Dom34:hbs1 plays a general role in quality-control systems by dissociation of a stalled ribosome at the 3' end of aberrant mRNA.

Translation arrest leads to an endonucleolytic cleavage of mRNA that is termed no-go decay (NGD). It has been reported that the Dom34:Hbs1 complex stimulates this endonucleolytic cleavage of mRNA induced by translation arrest in vivo and dissociates subunits of a stalled ribosome in vitro. Here we report that Dom34:Hbs1 dissociates the subunits of a ribosome that is stalled at the 3' end of mRNA in vivo, and has a crucial role in both NGD and nonstop decay. Dom34:Hbs1-mediated dissociation of a ribosome that is stalled at the 3' end of mRNA is required for degradation of a 5'-NGD intermediate. Dom34:Hbs1 facilitates the decay of nonstop mRNAs from the 3' end by exosomes and is required for the complete degradation of nonstop mRNA decay intermediates. We propose that Dom34:Hbs1 stimulates degradation of the 5'-NGD intermediate and of nonstop mRNA by dissociating the ribosome that is stalled at the 3' end of the mRNA.

Miscoding-induced stalling of substrate translocation on the bacterial ribosome.

Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G-catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors.