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Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of intestinal pathogens responsible for a range of illnesses, including kidney failure and neurological compromise. EHEC produce critical virulence factors, Shiga toxin (Stx) 1 or 2, and the synthesis of Stx2 is associated with worse disease manifestations. Infected patients only receive supportive treatment because some conventional antibiotics enable toxin production. Shiga toxin 2 genes (stx2) are carried in λ-like bacteriophages (stx2-phages) inserted into the EHEC genome as prophages. Factors that cause DNA damage induce the lytic cycle of stx2-phages, leading to Stx2 production. The phage Q protein is critical for transcription antitermination of stx2 and phage lytic genes. This study reports that deficiency of two endoribonucleases (RNases), E and G, significantly delayed cell lysis and impaired production of both Stx2 and stx2-phages, unlike deficiency of either enzyme alone. Moreover, scarcity of both enzymes reduced the concentrations of Q and stx2 transcripts and slowed cell growth.

Significance Bacterial cells are small and were long thought to have little to no internal structure. However, advances in microscopy have revealed that bacteria do indeed contain subcellular compartments. But how these compartments form has remained a mystery. Recent progress in larger, more complex eukaryotic cells has identified a novel mechanism for intracellular organization known as liquid–liquid phase separation. This process causes certain types of molecules to concentrate within distinct compartments inside the cell. Here, we demonstrate that the same process also occurs in bacteria. This work, together with a growing body of literature, suggests that liquid–liquid phase separation is a common mechanism for intracellular organization in both eukaryotic and prokaryotic cells., Once described as mere “bags of enzymes,” bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid–liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.

Background Respiratory syncytial virus (RSV) disease is a major cause of infant morbidity and mortality. This Phase I, randomized, observer-blind, placebo- and active-controlled study evaluated an investigational vaccine against RSV (ChAd155-RSV) using the viral vector chimpanzee-adenovirus-155, encoding RSV fusion (F), nucleocapsid, and transcription antitermination proteins. Methods Healthy 18–45-year-old adults received ChAd155-RSV, a placebo, or an active control (Bexsero) at Days (D) 0 and 30. An escalation from a low dose (5 × 109 viral particles) to a high dose (5 × 1010 viral particles) occurred after the first 16 participants. Endpoints were solicited/unsolicited and serious adverse events (SAEs), biochemical/hematological parameters, cell-mediated immunogenicity by enzyme-linked immunospot, functional neutralizing antibodies, anti RSV-F immunoglobin (Ig) G, and ChAd155 neutralizing antibodies. Results There were 7 participants who received the ChAd155-RSV low dose, 31 who received the ChAd155-RSV high dose, 19 who received the placebo, and 15 who received the active control. No dose-related toxicity or attributable SAEs at the 1-year follow-up were observed. The RSV-A neutralizing antibodies geometric mean titer ratios (post/pre-immunization) following a high dose were 2.6 (D30) and 2.3 (D60). The ratio of the fold-rise (D0 to D30) in anti-F IgG over the fold-rise in RSV-A–neutralizing antibodies was 1.01. At D7 after the high dose of the study vaccine, the median frequencies of circulating B-cells secreting anti-F antibodies were 133.3/106 (IgG) and 16.7/106 (IgA) in peripheral blood mononuclear cells (PBMCs). The median frequency of RSV-F–specific interferon γ–secreting T-cells after a ChAd155-RSV high dose was 108.3/106 PBMCs at D30, with no increase after the second dose. Conclusions In adults previously naturally exposed to RSV, ChAd155-RSV generated increases in specific humoral and cellular immune responses without raising significant safety concerns. Clinical Trials Registration NCT02491463., We investigated the safety and immunogenicity of a novel chimpanzee-adenovirus-155 vaccine against respiratory syncytial virus (RSV; ChAd155-RSV). In healthy adults with previous natural RSV exposure, ChAd155-RSV generated antigen-specific humoral and cellular immune responses without raising significant safety concerns.

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.

Two strains of good fortune in my career were to stumble upon the Watson–Gilbert laboratory at Harvard when I entered graduate school in 1964, and to study gene regulation in bacteriophage λ when I was there. λ was almost entirely a genetic item a few years before, awaiting biochemical incarnation. Throughout my career I was a relentless consumer of the work of previous and current generations of λ geneticists. Empowered by this background, my laboratory made contributions in two areas. The first was regulation of early gene transcription in λ, the study of which began with the discovery of the Rho transcription termination factor, and the regulatory mechanism of transcription antitermination by the λ N protein, subjects of my thesis work. This was developed into a decades-long program during my career at Cornell, studying the mechanism of transcription termination and antitermination. The second area was the classic problem of prophage induction in response to cellular DNA damage, the study of which illuminated basic cellular processes to survive DNA damage.

The soybean symbiont Bradyrhizobium diazoefficiens grows anaerobically in the presence of nitrate using the denitrification pathway, which involves the nap, nir, nor, and nos genes. We previously showed that NasT acts as a transcription antitermination regulator for nap and nos gene expression. In the present study, we investigated the targets of NasT in B. diazoefficiens during denitrifying growth by performing transcription profiling with RNA-seq and quantitative reverse-transcription PCR. Most of the genes with altered expression in the absence of NasT were related to nitrogen metabolism, specifically several systems for branched-chain amino acid transport. The present results suggest that the reduced expression of genes involved in nitrogen acquisition leads to the induction of alternative sets of genes with similar functions. The ΔnasT mutant of B. diazoefficiens grew better than the wild type under denitrifying conditions. However, this enhanced growth was completely abolished by an additional loss of the narK or bjgb genes, which encode cytoplasmic systems for nitrite and nitric oxide detoxification, respectively. Since the expression of narK and bjgb was increased in the ΔnasT mutant, the growth of the ΔnasT mutant may be promoted by increased detoxification activity.

Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators.

In Bradyrhizobium diazoefficiens, maximal expression of the nitrous oxide reductase gene (nosZ) requires oxygen limitation and the presence of a nitrogen oxide. The putative transcription antiterminator NasT is a positive regulator of nosZ; but in the absence of nitrate, NasT is counteracted by the nitrate sensor NasS. Here, we examined the NasT-mediated mechanism of nosRZDFYLX gene cluster expression. We mapped two transcription start sites of nosR and identified two potential hairpins, H1 and H2, within the 5'-leader of nosR transcripts. Electrophoretic mobility shift assay showed that NasT specifically bound the nosR-leader RNA and deletion of H1 abolished such binding. Under aerobic nitrate-deficient conditions, deletion of H1 or H2 increased the level of nosRZD transcripts. Under denitrifying conditions (anaerobiosis with nitrate supply), the level of nosRZD transcripts was severely impaired in the nasT mutant; in the nasT background, deletions of either hairpin led to increased level of nosRZD transcripts. In contrast to nosRZD coding region, nosR-leader transcript level was not affected by nasS or nasT mutations under aerobic or denitrifying conditions respectively. These results suggest that the two-hairpin RNA structure acts for transcription termination upstream of nosR and the binding of NasT to H1 facilitates read-through transcription to induce nos expression.

Polyamines are essential metabolites that play an important role in cell growth, stress adaptation, and microbial virulence1–3. In order to survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues2. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in γ-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF4, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryo-EM structure of an Escherichia coli (E. coli) ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor RF1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.

Bacterial cellulose is a widespread biofilm component that can modulate microbial fitness and virulence both in the environment and infected hosts. Whereas its secretion generally involves an inner membrane c-di-GMP-dependent synthase tandem (BcsAB) across the bacterial domain of life, enterobacteria feature sophisticated Escherichia coli-like Bcs secretion systems, where multiple additional subunits are either required for secretion or contribute to the maximal production of the polysaccharide in vivo. Here, we demonstrate that essential-for-secretion BcsR and BcsQ regulate each other's folding and stability and are recruited to the inner membrane via c-di-GMP-sensing BcsE and its intraoperon partner, BcsF. Crystallographic and functional data reveal that BcsE features unexpected domain architecture and likely acts on multiple levels to fine-tune bacterial cellulose production, from the early stages of secretion system assembly to the maintenence of a membrane-proximal pool of dimeric c-di-GMP for processive synthase activation., Most bacteria respond to surfaces by biogenesis of intracellular c-di-GMP, which inhibits motility and induces secretion of biofilm-promoting adherence factors. Bacterial cellulose is a widespread biofilm component whose secretion in Gram-negative species requires an inner membrane, c-di-GMP-dependent synthase tandem (BcsAB), an outer membrane porin (BcsC), and various accessory subunits that regulate synthase assembly and function as well as the exopolysaccharide’s chemical composition and mechanical properties. We recently showed that in Escherichia coli, most Bcs proteins form a megadalton-sized secretory nanomachine, but the role and structure of individual regulatory components remained enigmatic. Here, we demonstrate that essential-for-secretion BcsR and BcsQ regulate each other’s folding and stability and are recruited to the inner membrane via c-di-GMP-sensing BcsE and its intraoperon partner BcsF. Crystallographic and solution-based data show that BcsE’s predicted GIL domain is a degenerate receiver-GGDEF domain tandem (BcsEREC*-GGDEF*), where the divergent diguanylate cyclase module binds both dimeric c-di-GMP and BcsQ through mutually independent interfaces. In addition, we reveal that a third N-terminal domain (BcsENTD) determines the protein’s homooligomerization and targeting of BcsERQ to the membrane as well as previously unreported interactions with transcription antitermination complex components. Together, the data suggest that BcsE acts on multiple levels to fine-tune bacterial cellulose secretion, from the early stages of secretion system assembly to the maintenance of a membrane-proximal pool of dimeric c-di-GMP for processive synthase activation.

Bacteriophage Q protein engages σ-dependent paused RNA polymerase (RNAP) by binding to a DNA site embedded in late gene promoter and renders RNAP resistant to termination signals. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact Q-engaged arrested complex. The structure reveals key interactions responsible for σ-dependent pause, Q engagement, and Q-mediated transcription antitermination. The structure shows that two Q protomers (QI and QII) bind to a direct-repeat DNA site and contact distinct elements of the RNA exit channel. Notably, QI forms a narrow ring inside the RNA exit channel and renders RNAP resistant to termination signals by prohibiting RNA hairpin formation in the RNA exit channel. Because the RNA exit channel is conserved among all multisubunit RNAPs, it is likely to serve as an important contact site for regulators that modify the elongation properties of RNAP in other organisms, as well., Bacteriophage Q protein serves as a model regulator for the study of transcription elongation. Here the authors report a cryo-EM structure of an intact Q-engaged arrested complex, revealing the interactions responsible for σ-dependent pause, Q engagement, and Q-mediated transcription antitermination.

Bacteriophage O276 is a laboratory-generated hybrid that carries the immunity region of bacteriophage HK022 and all remaining genes from phage λ. Its construction was instrumental in the discovery of RNA-mediated antitermination, an intriguing alternative to the protein-mediated mechanism of transcription antitermination found in most lambdoid phages.

The T-box riboswitch is a unique, RNA-based regulatory mechanism that modulates expression of a wide variety of amino acid-related genes, predominantly in Firmicutes . RNAs of this class selectively bind a specific cognate tRNA, utilizing recognition of the tRNA anticodon and other tRNA features. The riboswitch monitors the aminoacylation status of the tRNA to induce expression of the regulated downstream gene(s) at the level of transcription antitermination or derepression of translation initiation in response to reduced tRNA charging via stabilization of an antiterminator or antisequestrator. Recent biochemical and structural studies have revealed new features of tRNA recognition that extend beyond the initially identified Watson-Crick base-pairing of a codon-like sequence in the riboswitch with the tRNA anticodon, and residues in the antiterminator or antisequestrator with the tRNA acceptor end. These studies have revealed new tRNA contacts and new modes of riboswitch function and ligand recognition that expand our understanding of RNA-RNA recognition and the biological roles of tRNA.

Interaction of the lambdoid phage N protein with the bacterial transcription elongation factor NusA is the key component in the process of transcription antitermination. A convex surface of E. coli NusA-NTD, located opposite to its RNA polymerase-binding domain (the β-flap domain), directly interacts with N in the antitermination complex. We hypothesized that this N-NusA interaction induces allosteric effects on the NusA-RNAP interaction leading to transformation of NusA into a facilitator of the antitermination process. Here we showed that mutations in β-flap domain specifically defective for N antitermination exhibited altered NusA-nascent RNA interaction and have widened RNA exit channel indicating an intricate role of flap domain in the antitermination. The presence of N reoriented the RNAP binding surface of NusA-NTD, which changed its interaction pattern with the flap domain. These changes caused significant spatial rearrangement of the β-flap as well as the β' dock domains to form a more constricted RNA exit channel in the N-modified elongation complex (EC), which might play key role in converting NusA into a facilitator of the N antitermination. We propose that in addition to affecting the RNA exit channel and the active center of the EC, β-flap domain rearrangement is also a mechanistic component in the N antitermination process.

Until recently, only two small regulatory RNAs encoded by lambdoid bacteriophages were known. These transcripts are derived from paQ and pO promoters. The former one is supposed to act as an antisense RNA for expression of the Q gene, encoding a transcription antitermination protein. The latter transcript, called oop RNA, was initially proposed to have a double role, in establishing expression of the cI gene and in providing a primer for DNA replication. Although the initially proposed mechanisms by which oop RNA could influence the choice between two alternative developmental pathways of the phage and the initiation of phage DNA replication were found not true, the pO promoter has been demonstrated to be important for both regulation of phage development and control of DNA replication. Namely, the pO-derived transcript is an antisense RNA for expression of the cII gene, and pO is a part of a dual promoter system responsible for regulation of initiation of DNA synthesis from the oriλ region. Very recent studies identified a battery of small RNAs encoded by lambdoid bacteriophages existing as prophages in chromosomes of enterohemorrhagic Escherichia coli strains. Some of them have very interesting functions, like anti-small RNAs.

RNA transcription of mononegavirales decreases gradually from the 3′ leader promoter toward the 5′ end of the genome, due to a decay in polymerase processivity. In the respiratory syncytial virus and metapneumovirus, the M 2–1 protein ensures transcription anti-termination. Despite being a homotetramer, respiratory syncytial virus M 2–1 binds two molecules of RNA of 13mer or longer per tetramer, and temperature-sensitive secondary structure in the RNA ligand is unfolded by stoichiometric interaction with M 2–1 . Fine quantitative analysis shows positive cooperativity, indicative of conformational asymmetry in the tetramer. RNA binds to M 2–1 through a fast bimolecular association followed by slow rearrangements corresponding to an induced-fit mechanism, providing a sequential description of the time events of cooperativity. The first binding event of half of the RNA molecule to one of the sites increases the affinity of the second binding event on the adjacent contacting protomer by 15-fold, product of increased effective concentration caused by the entropic link. This mechanism allows for high-affinity binding with an otherwise relaxed sequence specificity, and instead suggests a yet undefined structural recognition signature in the RNA for modulating gene transcription. This work provides a basis for an essential event for understanding transcription antitermination in pneumoviruses and its counterpart Ebola virus VP30. Fil: Molina, Ivana Gisele. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Esperante, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Marino Buslje, Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Chemes, Lucia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

λN-mediated processive antitermination constitutes a paradigmatic transcription regulatory event, during which phage protein λN, host factors NusA, NusB, NusE and NusG, and an RNA nut site render elongating RNA polymerase termination-resistant. The structural basis of the process has so far remained elusive. Here we describe a crystal structure of a λN–NusA–NusB–NusE–nut site complex and an electron cryo-microscopic structure of a complete transcription antitermination complex, comprising RNA polymerase, DNA, nut site RNA, all Nus factors and λN, validated by crosslinking/mass spectrometry. Due to intrinsic disorder, λN can act as a multiprotein/RNA interaction hub, which, together with nut site RNA, arranges NusA, NusB and NusE into a triangular complex. This complex docks via the NusA N-terminal domain and the λN C-terminus next to the RNA exit channel on RNA polymerase. Based on the structures, comparative crosslinking analyses and structure-guided mutagenesis, we hypothesize that λN mounts a multipronged strategy to reprogram the transcriptional machinery, which may include (1) the λN C terminus clamping the RNA exit channel, thus stabilizing the DNA:RNA hybrid; (2) repositioning of NusA and RNAP elements, thus redirecting nascent RNA and sequestering the upstream branch of a terminator hairpin; and (3) hindering RNA engagement of termination factor ρ and/or obstructing ρ translocation on the transcript. Structural characterization sheds mechanistic insights behind λN-dependent processive antitermination.

The bacterial transcription elongation factor, NusA, functions as an antiterminator when it is bound to the lambdoid phage derived antiterminator protein, N. The mode of N-NusA interaction is unknown, knowledge of which is essential to understand the antitermination process. It was reported earlier that in the absence of the transcription elongation complex (EC), N interacts with the C-terminal AR1 domain of NusA. However, the functional significance of this interaction is obscure. Here we identified mutations in NusA N terminus (NTD) specifically defective for N-mediated antitermination. These are located at a convex surface of the NusA-NTD, situated opposite its concave RNA polymerase (RNAP) binding surface. These NusA mutants disrupt the N-nut site interactions on the nascent RNA emerging out of a stalled EC. In the N/NusA-modified EC, a Cys-53 (S53C) from the convex surface of the NusA-NTD forms a specific disulfide (S-S) bridge with a Cys-39 (S39C) of the NusA binding region of the N protein. We conclude that when bound to the EC, the N interaction surface of NusA shifts from the AR1 domain to its NTD domain. This occurred due to a massive away-movement of the adjacent AR2 domain of NusA upon binding to the EC. We propose that the close proximity of this altered N-interaction site of NusA to its RNAP binding surface, enables N to influence the NusA-RNAP interaction during transcription antitermination that in turn facilitates the conversion of NusA into an antiterminator.

Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34 °C/35 °C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates.

The control of several catabolic operons in bacteria by transcription antitermination is mediated by RNA-binding proteins that consist of an RNA-binding domain and two reiterated phosphotransferase system regulation domains (PRDs). The Bacillus subtilis GlcT antitermination protein regulates the expression of the ptsG gene, encoding the glucose-specific enzyme II of the phosphotransferase system. In the absence of glucose, GlcT becomes inactivated by enzyme II-dependent phosphorylation at its PRD1, whereas the phosphotransferase HPr phosphorylates PRD2. However, here we demonstrate by NMR analysis and mass spectrometry that HPr also phosphorylates PRD1 in vitro but with low efficiency. Size exclusion chromatography revealed that non-phosphorylated PRD1 forms dimers that dissociate upon phosphorylation. The effect of HPr on PRD1 was also investigated in vivo. For this purpose, we used GlcT variants with altered domain arrangements or domain deletions. Our results demonstrate that HPr can target PRD1 when this domain is placed at the C terminus of the protein. In agreement with the in vitro data, HPr exerts a negative control on PRD1. This work provides the first insights into how specificity is achieved in a regulator that contains duplicated regulatory domains with distinct dimerization properties that are controlled by phosphorylation by different phosphate donors. Moreover, the results suggest that the domain arrangement of the PRD-containing antitermination proteins is under selective pressure to ensure the proper regulatory output, i.e. transcription antitermination of the target genes specifically in the presence of the corresponding sugar.

In Escherichia coli, the essential motor protein Rho promotes transcription termination in a tightly controlled manner that is not fully understood. Here, we show that the general post-transcriptional regulatory protein Hfq associates with Rho to regulate Rho function. The Hfq:Rho complex can be further stabilized by RNA bridging both factors in a configuration that inhibits the ATP hydrolysis and duplex unwinding activities of Rho and that mediates transcription antitermination at Rho-dependent terminators in vitro and in vivo. Antitermination at a prototypical terminator (λtR1) requires Hfq binding to an A/U-rich transcript region directly upstream from the terminator. Antitermination is modulated by trans-acting factors (NusG or nucleic acid competitors) that affect Hfq association with Rho or RNA. These data unveil a new Hfq function and a novel transcription regulatory mechanism with potentially important implications for bacterial RNA metabolism, gene silencing, and pathogenicity.

Processive transcription antitermination requires the assembly of the complete antitermination complex, which is initiated by the formation of the ternary NusB–NusE–BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that the BoxA RNA is composed of 8 nt that are recognized by the NusB–NusE heterodimer. Functional biologic and biophysical data support the structural observations and establish the relative significance of key protein–protein and protein–RNA interactions. Further crystallographic investigation of a NusB–NusE–dsRNA complex reveals a heretofore unobserved dsRNA binding site contiguous with the BoxA binding site. We propose that the observed dsRNA represents BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex.

The ability of bacteria to utilize ethanolamine (EA) as a carbon and nitrogen source may confer an advantage for survival, colonization, and pathogenicity in the human intestinal tract. Enterococcus faecalis , a Gram-positive human commensal organism, depends on a two-component signaling system (TCS-17) for sensing EA and regulating the expression of the ethanolamine utilization genes. Multiple promoters participate in eut gene expression in the presence of EA as the sole carbon source and cobalamin (CoB12), an essential cofactor in the enzymatic degradation process. By means of in vivo and in vitro approaches, this study characterized the transcriptional activity identified in the eutT-eutG intergenic region of the E. faecalis eut cluster. Two novel promoters in this region were shown to be active in vivo . The distal P2-1 promoter was associated with a B12 riboswitch that terminated transcription in the presence of CoB12. Transcription elongation from the proximal P2-2 promoter was regulated by antitermination mediated by the phosphorylated form of the response regulator of TCS-17 (RR17). 3′-Rapid amplification of cDNA ends (RACE) analyses of the terminated RNA products allowed precise identification of the hairpin loop structures involved in termination/antitermination. The results uncovered the role of the B12 riboswitch and RR17 in eut gene expression, adding to the complexity of this regulatory pathway and extending the knowledge of possible means of transcription regulation in Gram-positive organisms.

Gram-positive bacteria utilize a tRNA-responsive transcription antitermination mechanism, designated the T box system, to regulate expression of many amino acid biosynthetic and aminoacyl-tRNA synthetase genes. The RNA transcripts of genes controlled by this mechanism contain 5' untranslated regions, or leader RNAs, that specifically bind cognate tRNA molecules through pairing of nucleotides in the tRNA anticodon loop with nucleotides in the Specifier Loop domain of the leader RNA. We have determined the solution structure of the Specifier Loop domain of the tyrS leader RNA from Bacillus subtilis. Fifty percent of the nucleotides in the Specifier Loop domain adopt a loop E motif. The Specifier Sequence nucleotides, which pair with the tRNA anticodon, stack with their Watson-Crick edges rotated toward the minor groove and exhibit only modest flexibility. We also show that a Specifier Loop domain mutation that impairs the function of the B. subtilis glyQS T box RNA disrupts the tyrS loop E motif. Our results suggest a mechanism for tRNA-Specifier Loop binding in which the phosphate backbone kink created by the loop E motif causes the Specifier Sequence bases to rotate toward the minor groove, which increases accessibility for pairing with bases in the anticodon loop of tRNA.

The T box transcription antitermination riboswitch is one of the main regulatory mechanisms utilized by Gram-positive bacteria to regulate genes that are involved in amino acid metabolism. The details of the antitermination event, including the role that Mg2+ plays, in this riboswitch have not been completely elucidated. In these studies, details of the antitermination event were investigated utilizing 2-aminopurine to monitor structural changes of a model antiterminator RNA when it was bound to model tRNA. Based on the results of these fluorescence studies, the model tRNA binds the model antiterminator RNA via an induced-fit. This binding is enhanced by the presence of Mg2+, facilitating the complete base pairing of the model tRNA acceptor end with the complementary bases in the model antiterminator bulge.

Phage lambda propagation in Escherichia coli host cells requires transcription antitermination on the lambda chromosome mediated by lambdaN protein and four host Nus factors, NusA, B, E (ribosomal S10) and G. Interaction of E. coli NusB:NusE heterodimer with the single stranded BoxA motif of lambdanutL or lambdanutR RNA is crucial for this reaction. Similarly, binding of NusB:NusE to a BoxA motif is essential to suppress transcription termination in the ribosomal RNA (rrn) operons. We used fluorescence anisotropy to measure the binding properties of NusB and of NusB:NusE heterodimer to BoxA-containing RNAs differing in length and sequence. Our results demonstrate that BoxA is necessary and sufficient for binding. We also studied the gain-of-function D118N NusB mutant that allows lambda growth in nusA1 or nusE71 mutants. In vivo lambda burst-size determinations, CD thermal unfolding measurements and X-ray crystallography of this as well as various other NusB D118 mutants showed the importance of size and polarity of amino acid 118 for RNA binding and other interactions. Our work suggests that the affinity of the NusB:NusE complex to BoxA RNA is precisely tuned to maximize control of transcription termination.

Transcription antitermination in phages λ and P22 uses N proteins that bind to similar boxB RNA hairpins in regulated transcripts. In contrast to the λ N-boxB interaction, the P22 N-boxB interaction has not been extensively studied. A nuclear magnetic resonance structure of the P22 N peptide boxB left complex and limited mutagenesis have been reported but do not reveal a consensus sequence for boxB. We have used a plasmid-based antitermination system to screen boxBs with random loops and to test boxB mutants. We find that P22 N requires boxB to have a GNRA-like loop with no simple requirements on the remaining sequences in the loop or stem. U:A or A:U base pairs are strongly preferred adjacent to the loop and appear to modulate N binding in cooperation with the loop and distal stem. A few GNRA-like hexaloops have moderate activity. Some boxB mutants bind P22 and λ N, indicating that the requirements imposed on boxB by P22 N overlap those imposed by λ N. Point mutations can dramatically alter boxB specificity between P22 and λ N. A boxB specific for P22 N can be mutated to λ N specificity by a series of single mutations via a bifunctional intermediate, as predicted by neutral theories of evolution.

Transcription antitermination by N proteins of lambdoid phages involves specific interactions of the C-terminal domain of N with the elongation complex (EC). The interacting surface of N on the EC is unknown, knowledge of which is essential to understand the mechanism of antitermination. Specific cleavage patterns were generated near the active site Mg2+ of the RNA polymerase of an N-modified stalled EC using iron-(S)-1-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate conjugated to the only cysteine residue in the C-terminal domain of N from a lambdoid phage H-19B. Modification of EC by N also induced conformational changes around the same region as revealed from the limited trypsin digestion and in situ Fe-dithiothreitol cleavage pattern of the same EC. These data, together with the previously obtained H-19B N-specific mutations in RNA polymerase, beta (G1045D), and beta' (P251S, P254L, G336S, and R270C) subunits, suggest that the active center cleft of the EC could be the site of action of this antiterminator. H-19B N induced altered interactions in this region of EC, prevented the backtracking of the stalled EC at the ops pause site and destabilized RNA hairpin-beta subunit flap domain interactions at the his pause site. We propose that the physical proximity of the C-terminal domain of H-19B N to the active center cleft of the EC is required for the process of transcription antitermination and that it involves both stabilization of the weak RNA-DNA hybrid at a terminator and destabilization of the interactions of terminator hairpin in the RNA exit channel.

Bacterial cold shock proteins (CSPs) regulate cellular adaptation to cold stress. Functions ascribed to CSP include roles as RNA chaperones and in transcription antitermination. We present the crystal structure of the Bacillus caldolyticus CSP (Bc-Csp) in complex with hexathymidine (dT(6)) at a resolution of 1.29 A. Bound to dT(6), crystalline Bc-Csp forms a domain-swapped dimer in which beta strands 1-3 associate with strands 4 and 5 from the other subunit to form a closed beta barrel and vice versa. The globular units of dimeric Bc-Csp closely resemble the well-known structure of monomeric CSP. Structural reorganization from the monomer to the domain-swapped dimer involves a strictly localized change in the peptide bond linking Glu36 and Gly37 of Bc-Csp. Similar structural reorganizations have not been found in any other CSP or oligonucleotide/oligosaccharide-binding fold structures. Each dT(6) ligand is bound to one globular unit of Bc-Csp via an amphipathic protein surface. Individual binding subsites interact with the DNA bases through stacking and hydrogen bonding. The sugar-phosphate backbone remains solvent exposed. Based on crystallographic and biochemical studies of deoxyoligonucleotide binding to CSP, we suggest a common mode of binding of single-stranded heptanucleotide motifs to proteins containing cold shock domains, including the eukaryotic Y-box factors.

The T-box transcription antitermination riboswitch controls bacterial gene expression by structurally responding to uncharged, cognate tRNA. Previous studies indicated that cofactors, such as the polyamine spermidine, might serve a specific functional role in enhancing riboswitch efficacy. As riboswitch function depends on key RNA structural changes involving the antiterminator element, the interaction of spermidine with the T-box riboswitch antiterminator element was investigated. Spermidine binds antiterminator model RNA with high affinity (micromolar Kd ) based on isothermal titration calorimetry and fluorescence-monitored binding assays. NMR titration studies, molecular modeling, and inline and enzymatic probing studies indicate that spermidine binds at the 3' portion of the highly conserved seven-nucleotide bulge in the antiterminator. Together, these results support the conclusion that spermidine binds the T-box antiterminator RNA preferentially in a location important for antiterminator function. The implications of these findings are significant both for better understanding of the T-box riboswitch mechanism and for antiterminator-targeted drug discovery efforts.