Structural and functional basis of the universal transcription factor NusG pro-pausing activity in Mycobacterium tuberculosis.

Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger pausing by stabilizing RNA polymerase (RNAP) swiveling and inhibiting DNA translocation. The N-terminal domain (NGN) of the only universal transcription factor, NusG/Spt5, modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs enhance pauses, whereas anti-pausing NusGs suppress pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). We report that MtbNusG is pro-pausing. MtbNusG captures paused, swiveled RNAP by contacts to the RNAP protrusion and nontemplate-DNA wedged between the NGN and RNAP gate loop. In contrast, anti-pausing Escherichia coli (Eco) NGN contacts the MtbRNAP gate loop, inhibiting swiveling and pausing. Using CRISPR-mediated genetics, we show that pro-pausing NGN is required for mycobacterial fitness. Our results define an essential function of mycobacterial NusG and the structural basis of pro- versus anti-pausing NusG activity, with broad implications for the function of all NusG orthologs.

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.

Chemical-genetic interrogation of RNA polymerase mutants reveals structure-function relationships and physiological tradeoffs.

The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated RNAP mechanism, and RNAP mutations drive Escherichia coli adaptation to many health- and industry-relevant environments, yet a paucity of systematic analyses hampers our understanding of the fitness trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We discover phenotypes for non-essential insertions, show that clustering mutant phenotypes increases their predictive power for drawing functional inferences, and demonstrate that some RNA polymerase mutants both decrease average cell length and prevent killing by cell-wall targeting antibiotics. Our findings demonstrate that RNAP chemical-genetic interactions provide a general platform for interrogating structure-function relationships in vivo and for identifying physiological trade-offs of mutations, including those relevant for disease and biotechnology. This strategy should have broad utility for illuminating the role of other important protein complexes.

Trigger loop dynamics can explain stimulation of intrinsic termination by bacterial RNA polymerase without terminator hairpin contact.

In bacteria, intrinsic termination signals cause disassembly of the highly stable elongating transcription complex (EC) over windows of two to three nucleotides after kilobases of RNA synthesis. Intrinsic termination is caused by the formation of a nascent RNA hairpin adjacent to a weak RNA-DNA hybrid within RNA polymerase (RNAP). Although the contributions of RNA and DNA sequences to termination are largely understood, the roles of conformational changes in RNAP are less well described. The polymorphous trigger loop (TL), which folds into the trigger helices to promote nucleotide addition, also is proposed to drive termination by folding into the trigger helices and contacting the terminator hairpin after invasion of the hairpin in the RNAP main cleft [Epshtein V, Cardinale CJ, Ruckenstein AE, Borukhov S, Nudler E (2007) Mol Cell 28:991-1001]. To investigate the contribution of the TL to intrinsic termination, we developed a kinetic assay that distinguishes effects of TL alterations on the rate at which ECs terminate from effects of the TL on the nucleotide addition rate that indirectly affect termination efficiency by altering the time window in which termination can occur. We confirmed that the TL stimulates termination rate, but found that stabilizing either the folded or unfolded TL conformation decreased termination rate. We propose that conformational fluctuations of the TL (TL dynamics), not TL-hairpin contact, aid termination by increasing EC conformational diversity and thus access to favorable termination pathways. We also report that the TL and the TL sequence insertion (SI3) increase overall termination efficiency by stimulating pausing, which increases the flux of ECs into the termination pathway.

Trigger loop of RNA polymerase is a positional, not acid-base, catalyst for both transcription and proofreading.

The active site of multisubunit RNA polymerases (RNAPs) is highly conserved from humans to bacteria. This single site catalyzes both nucleotide addition required for RNA transcript synthesis and excision of incorrect nucleotides after misincorporation as a proofreading mechanism. Phosphoryl transfer and proofreading hydrolysis are controlled in part by a dynamic RNAP component called the trigger loop (TL), which cycles between an unfolded loop and an α-helical hairpin [trigger helices (TH)] required for rapid nucleotide addition. The precise roles of the TL/TH in RNA synthesis and hydrolysis remain unclear. An invariant histidine residue has been proposed to function in the TH form as a general acid in RNA synthesis and as a general base in RNA hydrolysis. The effects of conservative, nonionizable substitutions of the TL histidine (or a neighboring TL arginine conserved in bacteria) have not yet been rigorously tested. Here, we report that glutamine substitutions of these residues, which preserve polar interactions but are incapable of acid-base chemistry, had little effect on either phosphoryl transfer or proofreading hydrolysis by Escherichia coli RNAP. The TL substitutions did, however, affect the backtracking of RNAP necessary for proofreading and potentially the reactivity of the backtracked nucleotide. We describe a unifying model for the function of the RNAP TL, which reconciles available data and our results for representative RNAPs. This model explains diverse effects of the TL basic residues on catalysis through their effects on positioning reactants for phosphoryl transfer and easing barriers to transcript backtracking, rather than as acid-base catalysts.

Dynamics of GreB-RNA polymerase interaction allow a proofreading accessory protein to patrol for transcription complexes needing rescue.

The secondary channel (SC) of multisubunit RNA polymerases (RNAPs) allows access to the active site and is a nexus for the regulation of transcription. Multiple regulatory proteins bind in the SC and reprogram the catalytic activity of RNAP, but the dynamics of these factors' interactions with RNAP and how they function without cross-interference are unclear. In Escherichia coli, GreB is an SC protein that promotes proofreading by transcript cleavage in elongation complexes backtracked by nucleotide misincorporation. Using multiwavelength single-molecule fluorescence microscopy, we observed the dynamics of GreB interactions with elongation complexes. GreB binds to actively elongating complexes at nearly diffusion-limited rates but remains bound for only 0.3-0.5 s, longer than the duration of the nucleotide addition cycle but far shorter than the time needed to synthesize a complete mRNA. Bound GreB inhibits transcript elongation only partially. To test whether GreB preferentially binds backtracked complexes, we reconstituted complexes stabilized in backtracked and nonbacktracked configurations. By verifying the functional state of each molecular complex studied, we could exclude models in which GreB is selectively recruited to backtracked complexes or is ejected from RNAP by catalytic turnover. Instead, GreB binds rapidly and randomly to elongation complexes, patrolling for those requiring nucleolytic rescue, and its short residence time minimizes RNAP inhibition. The results suggest a general mechanism by which SC factors may cooperate to regulate RNAP while minimizing mutual interference.

RNA polymerase pausing and nascent-RNA structure formation are linked through clamp-domain movement.

The rates of RNA synthesis and the folding of nascent RNA into biologically active structures are linked via pausing by RNA polymerase (RNAP). Structures that form within the RNA-exit channel can either increase pausing by interacting with RNAP or decrease pausing by preventing backtracking. Conversely, pausing is required for proper folding of some RNAs. Opening of the RNAP clamp domain has been proposed to mediate some effects of nascent-RNA structures. However, the connections among RNA structure formation and RNAP clamp movement and catalytic activity remain uncertain. Here, we assayed exit-channel structure formation in Escherichia coli RNAP with disulfide cross-links that favor closed- or open-clamp conformations and found that clamp position directly influences RNA structure formation and RNAP catalytic activity. We report that exit-channel RNA structures slow pause escape by favoring clamp opening through interactions with the flap that slow translocation.

A pause sequence enriched at translation start sites drives transcription dynamics in vivo.

Transcription by RNA polymerase (RNAP) is interrupted by pauses that play diverse regulatory roles. Although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. Using nascent transcript sequencing, we identified a 16-nucleotide consensus pause sequence in Escherichia coli that accounts for known regulatory pause sites as well as ~20,000 new in vivo pause sites. In vitro single-molecule and ensemble analyses demonstrate that these pauses result from RNAP-nucleic acid interactions that inhibit next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from diverse lineages and is enriched at translation start sites in both E. coli and Bacillus subtilis. Our results thus reveal a conserved mechanism unifying known and newly identified pause events.

Mycobacterial RNA polymerase requires a U-tract at intrinsic terminators and is aided by NusG at suboptimal terminators.

Intrinsic terminators, which encode GC-rich RNA hairpins followed immediately by a 7-to-9-nucleotide (nt) U-rich "U-tract," play principal roles of punctuating and regulating transcription in most bacteria. However, canonical intrinsic terminators with strong U-tracts are underrepresented in some bacterial lineages, notably mycobacteria, leading to proposals that their RNA polymerases stop at noncanonical intrinsic terminators encoding various RNA structures lacking U-tracts. We generated recombinant forms of mycobacterial RNA polymerase and its major elongation factors NusA and NusG to characterize mycobacterial intrinsic termination. Using in vitro transcription assays devoid of possible mycobacterial contaminants, we established that mycobacterial RNA polymerase terminates more efficiently than Escherichia coli RNA polymerase at canonical terminators with imperfect U-tracts but does not terminate at putative terminators lacking U-tracts even in the presence of mycobacterial NusA and NusG. However, mycobacterial NusG exhibits a novel termination-stimulating activity that may allow intrinsic terminators with suboptimal U-tracts to function efficiently. IMPORTANCE Bacteria rely on transcription termination to define and regulate units of gene expression. In most bacteria, precise termination and much regulation by attenuation are accomplished by intrinsic terminators that encode GC-rich hairpins and U-tracts necessary to disrupt stable transcription elongation complexes. Thus, the apparent dearth of canonical intrinsic terminators with recognizable U-tracts in mycobacteria is of significant interest both because noncanonical intrinsic terminators could reveal novel routes to destabilize transcription complexes and because accurate understanding of termination is crucial for strategies to combat mycobacterial diseases and for computational bioinformatics generally. Our finding that mycobacterial RNA polymerase requires U-tracts for intrinsic termination, which can be aided by NusG, will guide future study of mycobacterial transcription and aid improvement of predictive algorithms to annotate bacterial genome sequences.

DksA guards elongating RNA polymerase against ribosome-stalling-induced arrest.

In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo.

Rho and NusG suppress pervasive antisense transcription in Escherichia coli.

Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its cofactor, NusG, is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global colocalization of the histone-like nucleoid-structuring protein (H-NS) with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes.

Binding and translocation of termination factor rho studied at the single-molecule level.

Rho termination factor is an essential hexameric helicase responsible for terminating 20-50% of all mRNA synthesis in Escherichia coli. We used single-molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho utilization site of the λtR1 terminator. Our results are consistent with Rho complexes adopting two states: one that binds 57 ± 2nt of RNA across all six of the Rho primary binding sites, and another that binds 85 ± 2nt at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5'→3' toward RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the Rho utilization site and RNAP. These findings lead to a general model for Rho binding and translocation and establish a novel experimental approach that should facilitate additional single-molecule studies of RNA-binding proteins.

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:

Spatial distribution and diffusive motion of RNA polymerase in live Escherichia coli.

By labeling the ß' subunit of RNA polymerase (RNAP), we used fluorescence microscopy to study the spatial distribution and diffusive motion of RNAP in live Escherichia coli cells for the first time. With a 40-ms time resolution, the spatial distribution exhibits two or three narrow peaks of 300- to 600-nm full width at half-maximum that maintain their positions within 60 nm over 1 s. The intensity in these features is 20 to 30% of the total. Fluorescence recovery after photobleaching (FRAP) measures the diffusive motion of RNAP on the 1-µm length scale. Averaged over many cells, 53%±19% of the RNAP molecules were mobile on the 3-s timescale, with a mean apparent diffusion constant of 0.22±0.16 µm2-s(-1). The remaining 47% were immobile even on the 30-s timescale. We interpret the immobile fraction as arising from RNAP specifically bound to DNA, either actively transcribing or not. The diffusive motion of the mobile fraction (fmobile) probably involves both one-dimensional sliding during nonspecific binding to DNA and three-dimensional hopping between DNA strands. There is significant cell-to-cell heterogeneity in both DRNAP and fmobile.

The ß subunit gate loop is required for RNA polymerase modification by RfaH and NusG.

In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the ß gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the ß' clamp helices. We hypothesize that upon binding to the clamp helices and the gate loop RfaH bridges the gap across the DNA channel, stabilizing RNAP contacts with nucleic acid and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity.

Mapping E. coli RNA polymerase and associated transcription factors and identifying promoters genome-wide.

The ability to examine gene regulation in living cells has been greatly enabled by the development of chromatin immunoprecipitation (ChIP) methodology. ChIP captures a snapshot of protein-DNA interactions in vivo and has been used to study interactions in bacteria, yeast, and mammalian cell culture. ChIP conditions vary depending upon the organism and the nature of the DNA-binding proteins under study. Here, we describe a customized ChIP protocol to examine the genome-wide distribution of a mobile DNA-binding enzyme, Escherichia coli RNA Polymerase (RNAP) as well as the factors that dynamically associate with RNAP during different stages of transcription. We describe new data analysis methods for determining the association of a broadly distributed DNA-binding complex. Further, we describe our approach of combining small molecules and antibiotics that perturb specific cellular events with ChIP and genomic platforms to dissect mechanisms of gene regulation in vivo. The chemical genomic methods can be leveraged to map natural and cryptic promoters and transcription units, annotate genomes, and reveal coupling between different processes in regulation of genes. This approach provides the framework for engineering gene networks and controlling biological output in a desired manner.

Roles for the transcription elongation factor NusA in both DNA repair and damage tolerance pathways in Escherichia coli.

We report observations suggesting that the transcription elongation factor NusA promotes a previously unrecognized class of transcription-coupled repair (TCR) in addition to its previously proposed role in recruiting translesion synthesis (TLS) DNA polymerases to gaps encountered during transcription. Earlier, we reported that NusA physically and genetically interacts with the TLS DNA polymerase DinB (DNA pol IV). We find that Escherichia coli nusA11(ts) mutant strains, at the permissive temperature, are highly sensitive to nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide but not to UV radiation. Gene expression profiling suggests that this sensitivity is unlikely to be due to an indirect effect on gene expression affecting a known DNA repair or damage tolerance pathway. We demonstrate that an N(2)-furfuryl-dG (N(2)-f-dG) lesion, a structural analog of the principal lesion generated by NFZ, blocks transcription by E. coli RNA polymerase (RNAP) when present in the transcribed strand, but not when present in the nontranscribed strand. Our genetic analysis suggests that NusA participates in a nucleotide excision repair (NER)-dependent process to promote NFZ resistance. We provide evidence that transcription plays a role in the repair of NFZ-induced lesions through the isolation of RNAP mutants that display altered ability to survive NFZ exposure. We propose that NusA participates in an alternative class of TCR involved in the identification and removal of a class of lesion, such as the N(2)-f-dG lesion, which are accurately and efficiently bypassed by DinB in addition to recruiting DinB for TLS at gaps encountered by RNAP.

E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNA polymerase.

NusG is an essential transcription factor in Escherichia coli that is capable of increasing the overall rate of transcription. Transcript elongation by RNA polymerase (RNAP) is frequently interrupted by pauses of varying durations, and NusG is known to decrease the occupancy of at least some paused states. However, it has not been established whether NusG enhances transcription chiefly by (1) increasing the rate of elongation between pauses, (2) reducing the lifetimes of pauses, or (3) reducing the rate of entry into paused states. Here, we studied transcription by single molecules of RNAP under various conditions of ribonucleoside triphosphate concentration, applied load, and temperature, using an optical trapping assay capable of distinguishing pauses as brief as 1 s. We found that NusG increases the rate of elongation, that is, the pause-free velocity along the template. Because pauses are off-pathway states that compete with elongation, we observed a concomitant decrease in the rate of entry into short-lifetime, paused states. The effects on short pauses and elongation were comparatively modest, however. More dramatic was the effect of NusG on suppressing entry into long-lifetime ("stabilized") pauses. Because a significant fraction of the time required for the transcription of a typical gene may be occupied by long pauses, NusG is capable of exerting a significant modulatory effect on the rates of RNA synthesis. The observed properties of NusG were consistent with a unified model where the function of this accessory factor is to promote transcriptionally downstream motion of the enzyme along the DNA template, which has the effect of forward-biasing RNAP from the pre-translocated state toward the post-translocated state.

Rho directs widespread termination of intragenic and stable RNA transcription.

The transcription termination factor Rho is a global regulator of RNA polymerase (RNAP). Although individual Rho-dependent terminators have been studied extensively, less is known about the sites of RNAP regulation by Rho on a genome-wide scale. Using chromatin immunoprecipitation and microarrays (ChIP-chip), we examined changes in the distribution of Escherichia coli RNAP in response to the Rho-specific inhibitor bicyclomycin (BCM). We found approximately 200 Rho-terminated loci that were divided evenly into 2 classes: intergenic (at the ends of genes) and intragenic (within genes). The intergenic class contained noncoding RNAs such as small RNAs (sRNAs) and transfer RNAs (tRNAs), establishing a previously unappreciated role of Rho in termination of stable RNA synthesis. The intragenic class of terminators included a previously uncharacterized set of short antisense transcripts, as judged by a shift in the distribution of RNAP in BCM-treated cells that was opposite to the direction of the corresponding gene. These Rho-terminated antisense transcripts point to a role of noncoding transcription in E. coli gene regulation that may resemble the ubiquitous noncoding transcription recently found to play myriad roles in eukaryotic gene regulation.

Regulator trafficking on bacterial transcription units in vivo.

The trafficking patterns of the bacterial regulators of transcript elongation sigma(70), rho, NusA, and NusG on genes in vivo and the explanation for promoter-proximal peaks of RNA polymerase (RNAP) are unknown. Genome-wide, E. coli ChIP-chip revealed distinct association patterns of regulators as RNAP transcribes away from promoters (rho first, then NusA, then NusG). However, the interactions of elongating complexes with these regulators did not differ significantly among most transcription units. A modest variation of NusG signal among genes reflected increased NusG interaction as transcription progresses, rather than functional specialization of elongating complexes. Promoter-proximal RNAP peaks were offset from sigma(70) peaks in the direction of transcription and co-occurred with NusA and rho peaks, suggesting that the RNAP peaks reflected elongating, rather than initiating, complexes. However, inhibition of rho did not increase RNAP levels within genes downstream from the RNAP peaks, suggesting the peaks are caused by a mechanism other than rho-dependent attenuation.