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Transcriptional Regulation by Antitermination. Interaction of RNA with NusB Protein and NusB/NusE Protein Complex of Escherichia coli
Abstract
A recombinant heterodimeric NusB/NusE protein complex of Escherichia coli was expressed under the control of a synthetic mini operon. Surface plasmon resonance measurements showed that the heterodimer complex has substantially higher affinity for the boxA RNA sequence motif of the ribosomal RNA (rrn) operons of E.coli as compared to monomeric NusB protein. Single base exchanges in boxA RNA reduced the affinity of the protein complex up to 15-fold. The impact of base exchanges in the boxA RNA on the interaction with NusB protein was studied by (1)H,(15)N heterocorrelation NMR spectroscopy. Spectra obtained with modified RNA sequences were analysed by a novel generic algorithm. Replacement of bases in the terminal segments of the boxA RNA motif caused minor chemical shift changes as compared to base exchanges in the central part of the dodecameric boxA motif.
... A classical assignment would have required a set of triple resonance experiments and triple labeled Nhsp90 for each ligand complex. Due to the low sample stability, a different strategy was used (Lüttgen et al., 2002), where chemical shift perturbations of the Nhsp90 complexes were determined by the minimum deviation between each position of the free and complexed peak in the HSQC spectra (Lüttgen et al., 2002). Using the disappearance of a signal and by identifying the next neighbor the minimal induced shift due to complexation of each residue was determined. ...
... A classical assignment would have required a set of triple resonance experiments and triple labeled Nhsp90 for each ligand complex. Due to the low sample stability, a different strategy was used (Lüttgen et al., 2002), where chemical shift perturbations of the Nhsp90 complexes were determined by the minimum deviation between each position of the free and complexed peak in the HSQC spectra (Lüttgen et al., 2002). Using the disappearance of a signal and by identifying the next neighbor the minimal induced shift due to complexation of each residue was determined. ...
... Accordingly, a second signal set for the ligand-complexed Nhsp90 appeared during the titration. Chemical shift perturbations of the Nhsp90 complexes were determined by the minimum deviation between each position of the free and complexed peak in the HSQC spectra (Lüttgen et al., 2002). For the analysis of the chemical shift perturbations of 1 H and 15 N backbone resonances a weighted average chemical shift change was calculated and normalized by Δ av /Δ max = 1.0, ...
In der vorliegenden Arbeit wurden Protein-Interaktionen biochemisch und NMR-spektroskopisch, u.a. mit Hilfe von PFG-Diffusionsexperimenten, identifiziert. Mutationsstudien an p53 zeigen, dass dessen kooperative DNA-Bindung auf eine intermolekulare Salzbrücke zurückzuführen ist, die auch das unterschiedliche DNA Bindungsverhalten von p63 strukturell erklärt. Für die transkriptionsunabhängige Apoptoseinduktion von p53 durch Komplexierung von z.B. Bcl-xL, konnte aus chemischen Verschiebungsänderungen ein p53Bcl-xL-Modellkomplex durch strukturelles docking berechnet werden. Hitzeschockproteine werden in Tumorzellen stark überexprimiert und steuern Apoptose durch konformationelle Regulation. Unterschiedliche Effekte von AMP-PNP, ADP, Geldanamycin und Radicicol auf die N-terminale ATPase-Domäne von Hsp90 konnten durch NMR-Titrationsexperimente gezeigt werden. SDS-Micellen induzieren in nativ ungefaltetem Hsp12 eine helikale Sekundärstruktur, die mit NMR-Spektroskopie näher untersucht wurde.
... AutoDock3.05 [48] was used to simulate the binding modes of starch molecules at the binding sites. The carbohydrate molecules were docked to different binding sites in separate simulations. Affinity grids, 90 × 90 × 90, three-dimensionally centred on the binding sites with 0.375 Å (1 Å = 0.1 nm) spacing were calculated using the program Autogrid [48]. Th ...
... The carbohydrate molecules were docked to different binding sites in separate simulations. Affinity grids, 90 × 90 × 90, three-dimensionally centred on the binding sites with 0.375 Å (1 Å = 0.1 nm) spacing were calculated using the program Autogrid [48]. The Lamarckian genetic algorithm was used for conformational searches. ...
CBMs (carbohydrate-binding modules) function independently to assist carbohydrate-active enzymes. Family 21 CBMs contain approx. 100 amino acid residues, and some members have starchbinding functions or glycogen-binding activities. We report here the first structure of a family 21 CBM from the SBD (starch-binding domain) of Rhizopus oryzae glucoamylase (RoCBM21) determined by NMR spectroscopy. This CBM has a beta-sandwich fold with an immunoglobulin-like structure. Ligand-binding properties of RoCBM21 were analysed by chemical-shift perturbations and automated docking. Structural comparisons with previously reported SBDs revealed two types of topologies, namely type I and type II, with CBM20, CBM25, CBM26 and CBM41 showing type I topology, with CBM21 and CBM34 showing type II topology. According to the chemical-shift perturbations, RoCBM21 contains two ligand-binding sites. Residues in site II are similar to those found in the family 20 CBM from Aspergillus niger glucoamylase (AnCBM20). Site I, however, is embedded in a region with unique sequence motifs only found in some members of CBM21s. Additionally, docking of beta-cyclodextrin and malto-oligosaccharides highlights that side chains of Y83 and W47 (one-letter amino acid code) form the central part of the conserved binding platform in the SBD. The structure of RoCBM21 provides the first direct evidence of the structural features and the basis for protein-carbohydrate recognition from an SBD of CBM21.
... The S10 protein encoded by rpsJ has two roles in the cell. It is incorporated into the 30S ribosome subunit but also forms a transcription anti-termination complex with NusB (Lüttgen et al., 2002;Luo et al., 2008;Baniulyte et al., 2017). We evaluated the importance of each of the two S10 roles to flagella number by EM. ...
Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here, we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ ²⁸ ( fliA ) in Escherichia coli . Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3´ UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.
... The S10 protein encoded by rpsJ has two roles in the cell. It is incorporated into the 30S ribosome subunit but also forms a transcription anti-termination complex with NusB (Lüttgen et al., 2002, Luo et al., 2008, Baniulyte et al., 2017. We evaluated the importance of each of the two S10 roles to flagella number by EM. ...
Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ28 (fliA) in Escherichia coli. Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3’ UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.
... The S10 protein encoded by rpsJ has two roles in the cell. It is incorporated into the 30S ribosome subunit but also forms a transcription anti-termination complex with NusB (Lüttgen et al., 2002;Luo et al., 2008;Baniulyte et al., 2017). We evaluated the importance of each of the two S10 roles to flagella number by electron microscopy. ...
Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ28 (fliA) in Escherichia coli. Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3’ UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and uniquely act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.
... The protein-protein interactions (PPI) between RNAP-transcription factor or factor-factor PPIs are potential targets for antimicrobial drug discovery [10]. Amongst, the PPI between two bacterial transcription factors NusB and NusE plays an important role in the formation of antitermination complex with RNAP that prevents premature transcription termination, particularly for the synthesis of rRNA [11][12][13]. Moreover, NusB and NusE are highly conserved and exclusively existing in bacteria [14,15]. ...
Staphylococcus aureus is a common human commensal pathogen that causes a wide range of infectious diseases. Due to the generation of antimicrobial resistance, the pathogen becomes resistant to more and more antibiotics, resulting in methicillin-resistant S. aureus (MRSA) and even multidrug-resistant S. aureus (MDRSA), namely ‘superbugs’. This situation highlights the urgent need for novel antimicrobials. Bacterial transcription, which is responsible for bacterial RNA synthesis, is a valid but underutilized target for developing antimicrobials. Previously, we reported a novel class of antimicrobials, coined nusbiarylins, that inhibited bacterial transcription by interrupting the protein–protein interaction (PPI) between two transcription factors NusB and NusE. In this work, we developed a ligand-based workflow based on the chemical structures of nusbiarylins and their activity against S. aureus. The ligand-based models—including the pharmacophore model, 3D QSAR, AutoQSAR, and ADME/T calculation—were integrated and used in the following virtual screening of the ChemDiv PPI database. As a result, four compounds, including J098-0498, 1067-0401, M013-0558, and F186-026, were identified as potential antimicrobials against S. aureus, with predicted pMIC values ranging from 3.8 to 4.2. The docking study showed that these molecules bound to NusB tightly with the binding free energy ranging from −58 to −66 kcal/mol.
... For many methyl TROSY cross-peaks Euclidian distances Δν Eucl between the apo and the GCDCA-bound form of P-domains are in the range of 50-150 Hz and correspond to slow exchange on the chemical shift time scale, leading to separate sets of peaks for the apo P-domain and GCDCA-bound P-dimers (Fig. S8a). Assignment followed a nearest-neighbor approach 36,[41][42][43] , which simply grouped peaks emerging upon removal of GCDCA to an assigned cross-peak into one group belonging to the same methyl group. Other cross-peaks in the intermediate exchange regime (Δν Eucl = 20-50 Hz) and in the intermediate to fast exchange regime (Δν Eucl < 20 Hz) were fewer but easier to assign ( Fig. S8bd). ...
Norovirus capsids are icosahedral particles composed of 90 dimers of the major capsid protein VP1. The C-terminus of the VP1 proteins forms a protruding (P)-domain, mediating receptor attachment, and providing a target for neutralizing antibodies. NMR and native mass spectrometry directly detect P-domain monomers in solution for murine (MNV) but not for human norovirus (HuNoV). We report that the binding of glycochenodeoxycholic acid (GCDCA) stabilizes MNV-1 P-domain dimers (P-dimers) and induces long-range NMR chemical shift perturbations (CSPs) within loops involved in antibody and receptor binding, likely reflecting corresponding conformational changes. Global line shape analysis of monomer and dimer cross-peaks in concentration-dependent methyl TROSY NMR spectra yields a dissociation rate constant k off of about 1 s ⁻¹ for MNV-1 P-dimers. For structurally closely related HuNoV GII.4 Saga P-dimers a value of about 10 −6 s ⁻¹ is obtained from ion-exchange chromatography, suggesting essential differences in the role of GCDCA as a cofactor for MNV and HuNoV infection.
... These two small proteins are highly conserved and exclusively exist in bacteria [4,5]. They bind to each other to form a bacterial transcription complex with RNAP ( Fig. 1A) [6], which is responsible for bacterial rRNA synthesis [7,8]. Studies showed that the function of PPI between NusB and NusE highly correlated to bacterial cell viability [9,10], which may serve as an appropriate target for novel antimicrobial agent discovery (Fig. 1B) [1]. ...
Bacterial transcription is a valid but underutilized target for antimicrobial agent discovery because of its function of bacterial RNA synthesis. Bacterial transcription factors NusB and NusE form a transcription complex with RNA polymerase for bacterial ribosomal RNA synthesis. We previously identified a series of diarylimine and -amine inhibitors capable of inhibiting the interaction between NusB and NusE and exhibiting good antimicrobial activity. To further explore the structural viability of these inhibitors, coined “nusbiarylins”, 36 new derivatives containing diverse substituents at the left benzene ring of inhibitors were synthesized based upon isosteric replacement and the structure-activity relationship concluded from earlier studies. Some of the derivatives displayed good to excellent antibacterial efficacy towards a panel of clinically significant pathogens including methicillin-resistance Staphylococcus aureus (MRSA) and vancomycin-resistance S. aureus (VRSA). In particular, compound 22r exhibited the best antimicrobial activity with a minimum inhibitory concentration (MIC) of 0.5 μg/mL. Diverse mechanistic studies validated the capability of 22r inhibiting the function of NusB protein and bacterial rRNA synthesis. In silico study of drug-like properties also provided promising results. Overall, this series of derivatives showed potential antimicrobial activity and drug-likeness and provided guidance for further optimization.
... The S10 protein has two roles in the cell. It is incorporated into the 30S ribosome subunit but also forms a transcription anti-termination complex with NusB, primarily binding to the Box A sequences of rrn operons (Lüttgen et al., 2002, Luo et al., 2008. We evaluated the importance of each of two roles to flagella number by electron microscopy. ...
Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ ²⁸ ( fliA ) in Escherichia coli . Interestingly, MotR and FliX have opposing effects on flagellin protein levels, flagella number and cell motility, with MotR accelerating flagella synthesis and FliX decelerating flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs. They also uniquely act on ribosomal protein mRNAs thus coordinating flagella synthesis with ribosome production. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.
... RoCBM21 (1 mM) was titrated with individual ligands, and the 1 H-15 N HSQC spectra of RoCBM21 were recorded to monitor the interactions. Weighted averaged 1 H and 15 N chemical-shift changes were calculated using the following equation [44][45][46][47]: ...
... Therefore the same analysis of shifts can be made as for fast exchange, the main difference being that the assignment of the bound spectrum has to be done all over again, as there is no straightforward way to assign the bound spectrum from the free. This is often not a practical proposition, and a number of authors have used a 'minimum chemical shift procedure' [48,51], in which each free resonance is linked to its closest bound resonance. In this way, a chemical shift change can be ascribed to each resonance. ...
Chemical shift perturbation (CSP, chemical shift mapping or complexation-induced changes in chemical shift, CIS) follows changes in the chemical shifts of a protein when a ligand is added, and uses these to determine the location of the binding site, the affinity of the ligand, and/or possibly the structure of the complex. A key factor in determining the appearance of spectra during a titration is the exchange rate between free and bound, or more specifically the off-rate koff. When koff is greater than the chemical shift difference between free and bound, which typically equates to an affinity Kd weaker than about 3μM, then exchange is fast on the chemical shift timescale. Under these circumstances, the observed shift is the population-weighted average of free and bound, which allows Kd to be determined from measurement of peak positions, provided the measurements are made appropriately. (1)H shifts are influenced to a large extent by through-space interactions, whereas (13)Cα and (13)Cβ shifts are influenced more by through-bond effects. (15)N and (13)C' shifts are influenced both by through-bond and by through-space (hydrogen bonding) interactions. For determining the location of a bound ligand on the basis of shift change, the most appropriate method is therefore usually to measure (15)N HSQC spectra, calculate the geometrical distance moved by the peak, weighting (15)N shifts by a factor of about 0.14 compared to (1)H shifts, and select those residues for which the weighted shift change is larger than the standard deviation of the shift for all residues. Other methods are discussed, in particular the measurement of (13)CH3 signals. Slow to intermediate exchange rates lead to line broadening, and make Kd values very difficult to obtain. There is no good way to distinguish changes in chemical shift due to direct binding of the ligand from changes in chemical shift due to allosteric change. Ligand binding at multiple sites can often be characterised, by simultaneous fitting of many measured shift changes, or more simply by adding substoichiometric amounts of ligand. The chemical shift changes can be used as restraints for docking ligand onto protein. By use of quantitative calculations of ligand-induced chemical shift changes, it is becoming possible to determine not just the position but also the orientation of ligands.
... N interacts with BoxB and converts the TEC to a terminationresistant form (6,7). Binding of N to BoxB results in an indirect interaction with RNAP through NusA (8,9). NusB interacts with the nut site by binding to BoxA, an interaction that is $10-fold strengthened upon NusE:NusB heterodimer formation (10)(11)(12)(13). The NusB:NusE:RNA ternary complex is proposed to associate with RNAP through NusE (1,14,15). ...
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.
... Whereas ecNusB has been shown to bind boxA RNA (Mogridge et al., 1995; Court et al., 1995; Lüttgen et al., 2002) Gopal and coworkers have failed to detect binding of mtNusB to boxA RNA in vitro (Gopal et al., 2000). In order to test RNA binding by tmNusB, two variants of the E. coli rrn-boxA sequence (5'-UGCUCUUUA-3' and ...
Die Kristallstruktur von Chinolin-2-oxidoreduktase (Qor) aus der Molybdänhydroxylase-Familie wurde mit hoher Auflösung aufgeklärt. Eine Aufgabe bestand darin, die Natur des apikalen Molybdänliganden zu untersuchen. Dabei wurde für Qor der Sulfidoligand in äquatorialer Position, dagegen der Oxoligand in apikaler Stellung gefunden. Darüber hinaus wurden strukturelle Studien an zwei Proteinen der Nus-Familie durchgeführt. Diese führten zur Aufklärung der Kristallstrukturen des Transkriptionsfaktors NusB aus Thermotoga maritima und des Komplexes von E. coli NusA mit der Protein N-Bindungsdomäne des Phagen λ. Ein Fragment des λN-Proteins (Reste 34-47) war mit zwei Molekülen der ersten Wiederholungssequenz AR1 von NusA komplexiert. Eine Mutationsanalyse zeigte, dass nur einer der beobachteten AR1-λN-Kontakte biologische Bedeutung hat. λN führt NusA an Antiterminationskomplexe heran, indem es an dessen C-terminalen Ausläufer bindet und als Gerüst für die Anlagerung von NusA an die mRNA dient.
... S10 is an important architectural element in the 30S ribosomal subunit, as revealed by reconstitution (Mizushima and Nomura, 1970) and crystal structure analyses (Schluenzen et al., 2000; Wimberly et al., 2000). During antitermination, S10 forms a stable complex with NusB (Mason et al., 1992) that has enhanced affinity for BoxA-containing RNAs compared to NusB alone (Lüttgen et al., 2002; Mogridge et al., 1998; Nodwell and Greenblatt, 1993). Since BoxA is strictly conserved in all seven rrn operons of E. coli whereas the BoxB-like element is dispensable for rrn antitermination (Berg et al., 1989), association of NusB, S10 and BoxA is considered as a key nucleation event during processive antitermination (Greive et al., 2005). ...
Protein S10 is a component of the 30S ribosomal subunit and participates together with NusB protein in processive transcription antitermination. The molecular mechanisms by which S10 can act as a translation or a transcription factor are not understood. We used complementation assays and recombineering to delineate regions of S10 dispensable for antitermination, and determined the crystal structure of a transcriptionally active NusB-S10 complex. In this complex, S10 adopts the same fold as in the 30S subunit and is blocked from simultaneous association with the ribosome. Mass spectrometric mapping of UV-induced crosslinks revealed that the NusB-S10 complex presents an intermolecular, composite, and contiguous binding surface for RNAs containing BoxA antitermination signals. Furthermore, S10 overproduction complemented a nusB null phenotype. These data demonstrate that S10 and NusB together form a BoxA-binding module, that NusB facilitates entry of S10 into the transcription machinery, and that S10 represents a central hub in processive antitermination.
... It is possible that the E. coli NusB protein simply binds poorly to the Caulobacter boxA sequence ending with AU, or our result opens up the interesting possibility that in some systems transcription antitermination is possible in rrn operons in the absence of NusB. Swapping or adding the Caulobacter nusA and nusB genes to the E. coli test system used here and physical interaction studies such as those reported elsewhere (23,31) would address our unexpected result with the Caulobacter AT sequence. Also noteworthy in the presumptive rrn boxA sequences we chose to study is that the presence of the nucleotide U at position 3 is universal (Fig. 2). ...
Transcription antitermination in the ribosomal operons of Escherichia coli results in the modification of RNA polymerase by specific proteins, altering its basic properties. For such alterations to
occur, signal sequences in rrn operons are required as well as individual interacting proteins. In this study we tested putative rrn transcription antitermination-inducing sequences from five different bacteria for their abilities to function in E. coli. We further examined their response to the lack of one known rrn transcription antitermination protein from E. coli, NusB. We monitored antitermination activity by assessing the ability of RNA polymerase to read through a factor-dependent
terminator. We found that, in general, the closer the regulatory sequence matched that of E. coli, the more likely there was to be a successful antitermination-proficient modification of the transcription complex. The rrn leader sequences from Pseudomonas aeruginosa, Bacillus subtilis, and Caulobacter crescentus all provided various levels of, but functionally significant antitermination properties to, RNA polymerase, while those of
Mycobacterium tuberculosis and Thermotoga maritima did not. Possible RNA folding structures of presumed antitermination sequences and specific critical bases are discussed
in light of our results. An unexpected finding was that when using the Caulobacter crescentus rrn leader sequence, there was little effect on terminator readthrough in the absence of NusB. All other hybrid antitermination
system activities required this factor. Possible reasons for this finding are discussed.
... Zn-ribbons participate in various functions, in particular DNA or RNA binding and redox reactions (42). The RNA-binding protein NusB is involved in anti-termination of E.coli ribosomal RNA operons and lambdoid phage genes (43,44). Their functional relationship with the ribo¯avin biosynthesis is not clear, although is has been suggested that YbaD is the ribo¯avin repressor (45). ...
The riboflavin biosynthesis in bacteria was analyzed using comparative analysis of genes, operons and regulatory elements.
A model for regulation based on formation of alternative RNA structures involving the RFN elements is suggested. In Gram‐positive bacteria including actinomycetes, Thermotoga, Thermus and Deinococcus, the riboflavin metabolism and transport genes are predicted to be regulated by transcriptional attenuation, whereas in most
Gram‐negative bacteria, the riboflavin biosynthesis genes seem to be regulated on the level of translation initiation. Several
new candidate riboflavin transporters were identified (impX in Desulfitobacterium halfniense and Fusobacterium nucleatum; pnuX in several actinomycetes, including some Corynebacterium species and Strepto myces coelicolor; rfnT in Rhizobiaceae). Traces of a number of likely horizontal transfer events were found: the complete riboflavin operon with
the upstream regulatory element was transferred to Haemophilus influenzae and Actinobacillus pleuropneumoniae from some Gram‐positive bacterium; non‐regulated riboflavin operon in Pyrococcus furiousus was likely transferred from Thermotoga; and the RFN element was inserted into the riboflavin operon of Pseudomonas aeruginosa from some other Pseudomonas species, where it had regulated the ribH2 gene.
... In Escherichia coli, NusB alone can bind to boxA [19,22,23] , but NusE significantly enhances the association [21] by an as yet unknown mechanism. The interaction of NusB and boxA may be of particular importance, as it has been suggested that it precludes the action of an antitermination inhibitor [24]. Surprisingly, despite the prominence of the NusB–NusE interaction in λN antitermination , only NusB has so far been found to be essential in rrn antitermination [16,18]. ...
NusB is a prokaryotic transcription factor involved in antitermination processes, during which it interacts with the boxA portion of the mRNA nut site. Previous studies have shown that NusB exhibits an all-helical fold, and that the protein from Escherichia coli forms monomers, while Mycobacterium tuberculosis NusB is a dimer. The functional significance of NusB dimerization is unknown. We have determined five crystal structures of NusB from Thermotoga maritima. In three crystal forms the protein appeared monomeric, whereas the two other crystal forms contained assemblies, which resembled the M. tuberculosis dimers. In solution, T. maritima NusB could be cross-linked as dimers, but it migrated as a monomer in gel-filtration analyses, suggesting a monomer/dimer equilibrium with a preference for the monomer. Binding to boxA-like RNA sequences could be detected by gel-shift analyses and UV-induced cross-linking. An N-terminal arginine-rich sequence is a probable RNA binding site of the protein, exhibiting aromatic residues as potential stacking partners for the RNA bases. Anions located in various structures support the assignment of this RNA binding site. The proposed RNA binding region is hidden in the subunit interface of dimeric NusB proteins, such as NusB from M. tuberculosis, suggesting that such dimers have to undergo a considerable conformational change or dissociate for engagement with RNA. Therefore, in certain organisms, dimerization may be employed to package NusB in an inactive form until recruitment into antitermination complexes.
... In this model, the Rho dependent transcription terminators (nut sites) located downstream of rrn P2 can prevent RNAP from transcribing rrn operons. The antitermination complexes, composed of r-proteins S10 (=NusE), S4, L3, L4 L13 and Nus factors (A, B, G) are able to permit read-through of nut sites (Torres et al., 2001; Luttgen et al., 2002;Torres et al., 2004). In addition, r-proteins, S4 and L4, can act as regulatory elements in repressing mRNA translation by means of translational feedback, and they play key roles in restoring the stoichiometry of r-protein and rRNA. ...
The regulation of ribosome synthesis has been investigated for nearly five decades. In earlier studies, the control of rRNA synthesis in bacteria was found to be dependent on nutrient composition of the growth media or cell growth rates, and these observations led to the growth rate-dependent regulation model. Also developed were stringent control, feedback ribosome synthesis, passive regulation, and antitermination models. Current evidence indicates that upstream (UP) element, molecular effectors, ppGpp and iNTP (initiating nucleoside triphosphate), and trans-acting proteins, Fis and H-NS, play important roles in the control of rRNA synthesis in response to changing nutritional environments. The mechanisms for the ribosome feedback regulation, and growth rate-dependent controls of rRNA synthesis remain to be determined despite numerous investigations. r-protein synthesis can be controlled by translational coupling, translation repression, or premature transcription termination. In Synechococcus, a photoautotroph, ribosome synthesis occurs early in the cell cycle as programmed events under conditions that support balanced growth. Periods of r-protein synthesis occur before rRNA synthesis periods, and rRNA synthesis is stimulated by a light-activated gene regulatory protein. These observations suggest that gene regulatory proteins are involved in the coordinate regulation of ribosome assembly in Synechococcus.
... BoxB forms a stem-loop, required for l N-mediated antitermination but dispensable for rrn antitermination (Gourse et al, 1986). BoxA is a highly conserved sequence with consensus UGCUCUUUAACA and has been demonstrated to bind to NusB (Luttgen et al, 2002) and to a NusB-NusE complex (Nodwell and Greenblatt, 1993;Luttgen et al, 2002). The BoxC region is less well characterised but, in the rrn of M. tb, a specific binding site for NusA that includes BoxC has been identified ( Arnvig et al, 2004). ...
NusA is a key regulator of bacterial transcriptional elongation, pausing, termination and antitermination, yet relatively little is known about the molecular basis of its activity in these fundamental processes. In Mycobacterium tuberculosis, NusA has been shown to bind with high affinity and specificity to BoxB-BoxA-BoxC antitermination sequences within the leader region of the single ribosomal RNA (rRNA) operon. We have determined high-resolution X-ray structures of a complex of NusA with two short oligo-ribonucleotides derived from the BoxC stem-loop motif and have characterised the interaction of NusA with a variety of RNAs derived from the antitermination region. These structures reveal the RNA bound in an extended conformation to a large interacting surface on both KH domains. Combining structural data with observed spectral and calorimetric changes, we now show that NusA binding destabilises secondary structure within rRNA antitermination sequences and propose a model where NusA functions as a chaperone for nascently forming RNA structures.
Structured noncoding RNAs (ncRNAs) contribute to many important cellular processes involving chemical catalysis, molecular recognition and gene regulation. Few ncRNA classes are broadly distributed among organisms from all three domains of life, but the list of rarer classes that exhibit surprisingly diverse functions is growing. We previously developed a computational pipeline that enables the near-comprehensive identification of structured ncRNAs expressed from individual bacterial genomes. The regions between protein coding genes are first sorted based on length and the fraction of guanosine and cytidine nucleotides. Long, GC-rich intergenic regions are then examined for sequence and structural similarity to other bacterial genomes. Herein, we describe the implementation of this pipeline on 50 bacterial genomes from varied phyla. More than 4700 candidate intergenic regions with the desired characteristics were identified, which yielded 44 novel riboswitch candidates and numerous other putative ncRNA motifs. Although experimental validation studies have yet to be conducted, this rate of riboswitch candidate discovery is consistent with predictions that many hundreds of novel riboswitch classes remain to be discovered among the bacterial species whose genomes have already been sequenced. Thus, many thousands of additional novel ncRNA classes likely remain to be discovered in the bacterial domain of life.
Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ28 (fliA) in Escherichia coli. Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3’ UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and uniquely act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.
Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase/polyketide synthase (NRPS/PKS) followed by a number of `tailoring' steps to form the two ring systems that are present in the mature product. It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels–Alder reaction. Here, the crystal structure of a selenomethionine-labelled truncated form of IdmH (IdmH-Δ99–107) was solved using single-wavelength anomalous dispersion (SAD) phasing. This truncated variant allows consistent and easy crystallization, but importantly the structure was used as a search model in molecular replacement, allowing the full-length IdmH structure to be determined to 2.7 Å resolution. IdmH is a homodimer, with the individual protomers consisting of an α+β barrel. Each protomer contains a deep hydrophobic pocket which is proposed to constitute the active site of the enzyme. To investigate the reaction catalysed by IdmH, 88% of the backbone NMR resonances were assigned, and using chemical shift perturbation of [ ¹⁵ N]-labelled IdmH it was demonstrated that indanomycin binds in the active-site pocket. Finally, combined quantum mechanical/molecular mechanical (QM/MM) modelling of the IdmH reaction shows that the active site of the enzyme provides an appropriate environment to promote indane-ring formation, supporting the assignment of IdmH as the key Diels–Alderase catalysing the final step in the biosynthesis of indanomycin through a similar mechanism to other recently characterized Diels–Alderases involved in polyketide-tailoring reactions. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at https://proteopedia.org/w/Journal:IUCrJ:S2052252519012399.
Chemical shift perturbation (CSP, also known as chemical shift mapping and complexation-induced changes in shift, CIS) is a common technique for demonstrating ligand binding to proteins, locating the binding site, and measuring ligand affinity. The most common application is to use changes in the ¹H, ¹⁵N HSQC spectrum. In favorable cases it can be used to provide structural details of the complex. The technique is most simply applied when binding is weak (dissociation constant μM or weaker), where the dissociation rate is fast in comparison with shift changes, but can be applied with stronger binding. In the fast exchange limit, signals from regions of the protein adjacent to the ligand shift smoothly from free to bound positions during the titration. The method is simple to use and suffers from few complications, which include slower exchange, more than one binding site, multiple bound states, and protein precipitation during the titration. Some recent developments are discussed, including improving the data analysis using statistical methods and attempts to separate direct chemical shift effects of binding from indirect effects such as allosteric change. © Springer International Publishing AG, part of Springer Nature 2018.
Chemical shift perturbation (CSP, also known as chemical shift mapping and complexation-induced changes in shift, CIS) is a common technique for demonstrating ligand binding to proteins, locating the binding site, and measuring ligand affinity. The most common application is to use changes in the 1H, 15N HSQC spectrum. In favorable cases it can be used to provide structural details of the complex. The technique is most simply applied when binding is weak (dissociation constant μM or weaker), where the dissociation rate is fast in comparison with shift changes, but can be applied with stronger binding. In the fast exchange limit, signals from regions of the protein adjacent to the ligand shift smoothly from free to bound positions during the titration. The method is simple to use and suffers from few complications, which include slower exchange, more than one binding site, multiple bound states, and protein precipitation during the titration. Some recent developments are discussed, including improving the data analysis using statistical methods and attempts to separate direct chemical shift effects of binding from indirect effects such as allosteric change.
Nus Factors of Escherichia coli, Page 1 of 2
Abstract
The highly conserved Nus factors of bacteria were discovered as essential host proteins for the growth of temperate phage λ in Escherichia coli. Later, their essentiality and functions in transcription, translation, and, more recently, in DNA repair have been elucidated. Close involvement of these factors in various gene networks and circuits is also emerging from recent genomic studies. We have described a detailed overview of their biochemistry, structures, and various cellular functions, as well as their interactions with other macromolecules. Towards the end, we have envisaged different uncharted areas of studies with these factors, including their participation in pathogenicity.
RAHMOUNI Rachid, Directeur de thèse BOSSI Lionello, Rapporteur KOLB Annie, Rapporteur HEVOR Tobias, Examinateur PAOLETTI Jacques, Examinateur
Nus Factors of Escherichia coli, Page 1 of 2
Abstract
The Nus factors—NusA, NusB, NusE, and NusG—area set of well-conserved proteins in bacteria and are involved in transcription elongation, termination, antitermination, and translation processes. Originally, Escherichia coli host mutations defective for supporting bacteriophage λ N-mediated antitermination were mapped to the nusA (nusA1), nusB (nusB5, nusB101), and nusE (nusE71) genes, and hence, these genes were named nus for Nutilization substances (Nus). Subsequently,the Nus factors were purified and their roles in different host functions were elucidated. Except for NusB, deletion of which is conditionally lethal, all the other Nus factors are essential for E. coli. Among the Nus factors, NusA has the most varied functions. It specifically binds to RNA polymerase (RNAP), nascent RNA, and antiterminator proteins like N and Q and hence takes part in modulating transcription elongation, termination, and antitermination. It is also involved in DNA repair pathways. NusG interacts with RNAP and the transcription termination factor Rho and therefore is involved in both factor-dependent termination and transcription elongation processes. NusB and NusE are mostly important in antitermination at the ribosomal operon-transcription. NusE is a component of ribosome and may take part in facilitating the coupling between transcription and translation. This chapter emphasizes the structure-function relationship of these factors and their involvement in different fundamental cellular processes from a mechanistic angle.
The tumor suppressor protein p53 is often referred to as the guardian of the genome. In the past, controversial findings have been presented for the role of the C-terminal regulatory domain (RD) of p53 as both a negative regulator and a positive regulator of p53 activity. However, the underlying mechanism remained enigmatic. To understand the function of the RD and of a dominant phosphorylation site within the RD, we analyzed p53 variants in vivo and in vitro. Our experiments revealed, surprisingly, that the p53 RD of one subunit interacts with the DNA binding domain of an adjacent subunit in the tetramer. This leads to the formation of intersubunit contacts that stabilize the tetrameric state of p53 and enhance its transcriptional activity in a cooperative manner. These effects are further modulated by phosphorylation of a conserved serine within the RD.
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 regulation of ribosomal RNA biosynthesis in Escherichia coli by antitermination requires binding of NusB protein to a dodecamer sequence designated boxA on the nascent RNA. The affinity of NusB protein for boxA RNA exceeds that for the homologous DNA segment by more than three orders of magnitude as shown by surface plasmon resonance measurements. DNA RNA discrimination by NusB protein was shown to involve methyl groups (i.e. discrimination of uracil versus thymine) and 2' hydroxyl groups (i.e. discrimination of ribose versus deoxyribose side-chains) in the RNA motif. Ligand perturbation experiments monitored by 1H15N correlation NMR experiments identified amide NH groups whose chemical shifts are affected selectively by ribose/deoxyribose exchange in the 5' and the central part of the dodecameric boxA motif respectively. The impact of structural modification of the boxA motif on the affinity for NusB protein as observed by 1H15N heterocorrelation was analysed by a generic algorithm.
Hsp90 is one of the most abundant chaperone proteins in the cytosol. In an ATP-dependent manner it plays an essential role in the folding and activation of a range of client proteins involved in signal transduction and cell cycle regulation. We used NMR shift perturbation experiments to obtain information on the structural implications of the binding of AMP-PNP (adenylyl-imidodiphosphate-a non-hydrolysable ATP analogue), ADP and the inhibitors radicicol and geldanamycin. Analysis of (1)H,(15)N correlation spectra showed a specific pattern of chemical shift perturbations at N210 (ATP binding domain of Hsp90, residues 1-210) upon ligand binding. This can be interpreted qualitatively either as a consequence of direct ligand interactions or of ligand-induced conformational changes within the protein. All ligands show specific interactions in the binding site, which is known from the crystal structure of the N-terminal domain of Hsp90. For AMP-PNP and ADP, additional shift perturbations of residues outside the binding pocket were observed and can be regarded as a result of conformational rearrangement upon binding. According to the crystal structures, these regions are the first alpha-helix and the "ATP-lid" ranging from amino acids 85 to 110. The N-terminal domain is therefore not a passive nucleotide-binding site, as suggested by X-ray crystallography, but responds to the binding of ATP in a dynamic way with specific structural changes required for the progression of the ATPase cycle.
This chapter discusses the genetic and biochemical strategies to elucidate the architecture and targets of a processive transcription antiterminator from bacteriophage lambda. Antitermination of transcription is a gene activation mechanism by which cells regulate genes in response to environmental and developmental cues. This chapter discusses the strategies involved and the genetic and biochemical methods used to elucidate the architecture of N protein and its interaction with the various targets. A remarkable feature of antitermination in different lambdoid phages is their demonstrated genome specificity: N and Q proteins act preferentially on the cognate genomes to turn on the early and the late operons, respectively. Although the lambda N protein has served as an important model for understanding the basic mechanisms of processive antitermination, the homologous proteins from lambda's cousins show considerable variation in primary structures. Like the studies of different repressors and activators, a molecular genetic analysis of some of these N protein homologs (from P21 and Phi-80, for example) promises to reveal interesting variations in terms of the basic mechanisms by which RNA polymerase is transformed to a termination- resistant state. At the same time, these studies would provide a better understanding of the structure-function of RNA polymerase itself.
There are four rRNA operons rrnA, rrnB, rrnC and rrnD on the genome of Finegoldia magna (formerly Peptostreptococcus magnus) ATCC29328, which, in contrast to those of Clostridia, are dispersed around the chromosome. Using a BAC library we determined the nucleotide sequences and structures of all four operons, including their flanking regions, and performed comparative analyses. We identified putative boxA sequences in the operons, which should be required for rRNA transcription antitermination, as well as their respective tandem promoters, AT-rich UP elements in the upstream region and Rho-independent terminators in the downstream region. The mosaic features of the operons were revealed. Multiple tRNAs were identified in the downstream region of two operons, 18 in rrnC and 11 in rrnD. They were presumed to form transcription units together with rRNAs. rrnA and rrnB had repeat units with Rho-independent terminators instead of tRNAs in the downstream region. rrnB and rrnC were the most similar in rrn upstream promoter region. Focusing on the sequence variations of rRNA genes, rrnB alone was heterogeneous. In light of previous reports, we also assessed the correlation between intercistronic rRNA sequence differences and distances between the operons, but no positive correlation was seen in this strain.
We have compiled 819 articles published in the year 2002 that involved commercial optical biosensor technology. The literature demonstrates that the technology's application continues to increase as biosensors are contributing to diverse scientific fields and are used to examine interactions ranging in size from small molecules to whole cells. Also, the variety of available commercial biosensor platforms is increasing and the expertise of users is improving. In this review, we use the literature to focus on the basic types of biosensor experiments, including kinetics, equilibrium analysis, solution competition, active concentration determination and screening. In addition, using examples of particularly well-performed analyses, we illustrate the high information content available in the primary response data and emphasize the impact of including figures in publications to support the results of biosensor analyses.
Analytical ultracentrifugation and fluorescence anisotropy methods have been used to measure the equilibrium parameters that control the formation of the core subcomplex of NusB and NusE proteins and boxA RNA. This subcomplex, in turn, nucleates the assembly of the antitermination complex that is involved in controlling the synthesis of ribosomal RNA in Escherichia coli and that also participates in forming the N protein-dependent antitermination complex in lambdoid phage synthesis. In this study we determined the dissociation constants (K(d) values) for the individual binary interactions that participate in the assembly of the ternary NusB-NusE-boxA RNA subassembly, and we showed that multiple equilibria, involving both specific and nonspecific binding, are involved in the assembly pathway of this protein-RNA complex. The measured K(d) values were used to model the in vitro assembly reaction and combined with in vivo concentration data to simulate the overall control of the assembly of this complex in E. coli at two different cellular growth rates. The results showed that at both growth rates assembly proceeds via the initial formation of a weak but specific NusB-boxA complex, which is then stabilized by NusE binding. We showed that NusE also binds nonspecifically to available single-stranded RNA sequences and that such nonspecific protein binding to RNA can help to regulate crucial interactions in the assembly of the various macromolecular machines of gene expression.
In prokaryotic transcription regulation, several host factors form a complex with RNA polymerase and the nascent mRNA. As part of a process known as antitermination, two of these host factors, NusB and NusE, bind to form a heterodimer, which interacts with a specific boxA site on the RNA. The NusB/NusE/boxA RNA ternary complex interacts with the RNA polymerase transcription complex, stabilizing it and allowing transcription past premature termination points. The NusB protein also binds boxA RNA individually and retains all specificity for boxA. However, NusE increases the affinity of RNA to NusB in the ternary complex, which contributes to efficient antitermination. To understand the molecular mechanism of the process, we have determined the structure of NusB from the thermophilic bacterium Aquifex aeolicus and studied the interaction of NusB and NusE. We characterize this binding interaction using NMR, isothermal titration calorimetry, gel filtration, and analytical ultracentrifugation. The binding site of NusE on NusB was determined using NMR chemical shift perturbation studies. We have also determined the NusE binding site in the ternary Escherichia coli NusB/NusE/boxA RNA complex and show that it is very similar to that in the NusB/NusE complex. There is one loop of residues (from 113 to 118 in NusB) affected by NusE binding in the ternary complex but not in the binary complex. This difference may be correlated to an increase in binding affinity of RNA for the NusB/NusE complex.
This chapter discusses a T5 promoter-based transcription–translation system for analyzing proteins in vitro and in vivo. The chapter describes a simple method that permits the expression of cloned sequences in Escherichia coli as well as in cell-free in vitro systems of eukaryotic (wheat germ, reticulocyte, and HeLa cells) or prokaryotic (E. coli) origin, using a single expression unit. The expression of cloned genes in heterologous systems in vitro and in vivo has been instrumental in the identification and analysis of gene products and their derivatives. The essential element of the unit is a promoter derived from coliphage T5 that is utilized by E. coli ribonucleic acid (RNA) polymerase. It efficiently directs the synthesis of capped or uncapped mitochondrial RNA (mRNA) in vitro. Translation of such RNAs in the presence of [35S] methionine yields single proteins of such high specific activity and purity that they can be directly analyzed by gel electrophoresis without the necessity of prior immunoprecipitation. The chapter also describes the application of the method for the study of structure/function relationships of proteins, including (1) protein synthesis in vivo and in vitro, (2) the translocation of proteins into and through membranes, and (3) the interruption of translation at predetermined sites for the generation and characterization of truncated proteins. Plasmid pDS5 and its derivatives 5 are members of a plasmid family developed for the study of transcriptional signals. The chapter also summarizes the essential properties of the pDS5 system.
Two genes, secE and nusG, situated between the tufB and ribosomal protein rplKAJL operons in the rif region at 90 min on the Escherichia coli chromosome, have been sequenced and characterized. The secE gene encodes a 127-amino-acid-long polypeptide, which is an integral membrane protein essential for protein export (P. J. Schatz, P. D. Riggs, A. Jacq, M. J. Fath, and J. Beckwith, Genes Dev. 3:1035-1044, 1989). The nusG gene encodes a 181-amino-acid-long polypeptide and is involved in transcription antitermination. The protein product of nusG is essential for bacterial viability. The secE-nusG genes are cotranscribed, with transcripts initiated at the PEG promoter and terminated at the Rho-independent terminator in the region of the rplK promoter. The majority of transcripts are processed at a number of sites in the 5' untranslated leader region by RNase III and are possibly also processed by a second unidentified nuclease. The role of transcript processing in the regulation of secE and nusG has not yet been established. The juxtaposition and coregulation of a protein export factor and a transcriptional factor raise questions concerning a functional connection between the two processes.
Operator sequence and repressor protein regulate the activity of the lac promoter over a greater than 1000-fold range. Combinations of the lac operator with other promoter sequences, however, differ vastly in the level of repression. The data presented show that the extent of repression is determined largely by the rates of complex formation of the competing systems operator-repressor and promoter-RNA polymerase and by the rate at which RNA polymerase clears the promoter. Moreover, up to 70-fold differences in the level of repression were found when the operator was placed in different positions within the promoter sequence. A kinetic model is proposed that explains the observed effects and that allows predictions on promoters controlled by negatively acting elements.
In vivo alpha-complementation of beta-galactosidase was demonstrated in 16 Z gene terminator (nonsense) mutant strains of Escherichia coli upon introduction of the episome F'M15 which specifies production of a mutant Z gene polypeptide containing a small deletion in the N-terminal region of the enzyme monomer. Genetic and biochemical analyses of the merodiploids showed that restoration of enzyme activity was due to their terminator/F'M15 genetic constitution resulting in the production of two enzymatically inactive polypeptides which associate in vivo to reconstitute active, stable beta-galactosidase. The prematurely terminated polypeptide fragments known to be rapidly degraded in haploid cells were shown by phenotypic and biochemical studies to be stabilized (i.e., protected) in merodiploids by formation of complemented enzyme complexes with the M15 protein. Phenotypic properties of complementing diploids are described and are discussed in relation to in vitro determination of beta-galactosidase activity.
We determined the rates of mRNA and protein chain elongation on the lacZ gene during exponential growth on different carbon sources. The RNA chain elongation rate was calculated from measurements of the time elapsing between induction of lacZ expression and detection of specific hybridization with a probe near the 3' end of the mRNA. The elongation rate for the transcripts decreased 40% when the growth rate decreased by a factor of 4, and it always correlated with the rate of translation elongation. A similar growth rate dependency was seen for transcription on the infB gene and on a part of the rrnB gene fused to a synthetic, inducible promoter. However, the untranslated RNA chain specified by the rrnB gene was elongated nearly twice as fast as the two mRNA species encoded by infB and lacZ.
The ribG gene at the 5' end of the riboflavin operon of Bacillus subtilis and a reading frame at 442 kb on the Escherichia coli chromosome (subsequently designated ribD) show similarity with deoxycytidylate deaminase and with the RIB7 gene of Saccharomyces cerevisiae. The ribG gene of B. subtilis and the ribD gene of E. coli were expressed in recombinant E. coli strains and were shown to code for bifunctional proteins catalyzing the second and third steps in the biosynthesis of riboflavin, i.e., the deamination of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (deaminase) and the subsequent reduction of the ribosyl side chain (reductase). The recombinant proteins specified by the ribD gene of E. coli and the ribG gene of B. subtilis were purified to homogeneity. NADH as well as NADPH can be used as a cosubstrate for the reductase of both microorganisms under study. Expression of the N-terminal or C-terminal part of the RibG protein yielded proteins with deaminase or reductase activity, respectively; however, the truncated proteins were rather unstable.
The NusB protein of Escherichia coli is involved in the regulation of rRNA biosynthesis by transcriptional antitermination. In cooperation with several other proteins, it binds to a dodecamer motif designated rrn boxA on the nascent rRNA. The antitermination proteins of E.coli are recruited in the replication cycle of bacteriophage lambda, where they play an important role in switching from the lysogenic to the lytic cycle. Multidimensional heteronuclear NMR experiments were performed with recombinant NusB protein labelled with 13C, 15N and 2H. The three-dimensional structure of the protein was solved from 1926 NMR-derived distances and 80 torsion angle restraints. The protein folds into an alpha/alpha-helical topology consisting of six helices; the arginine-rich N-terminus appears to be disordered. Complexation of the protein with an RNA dodecamer equivalent to the rrn boxA site results in chemical shift changes of numerous amide signals. The overall packing of the protein appears to be conserved, but the flexible N-terminus adopts a more rigid structure upon RNA binding, indicating that the N-terminus functions as an arginine-rich RNA-binding motif (ARM).
We have determined the solution structure of NusB, a transcription antitermination protein from Escherichia coli. The structure reveals a novel, all alpha-helical protein fold. NusB mutations that cause a loss of function (NusB5) or alter specificity for RNA targets (NusB101) are localized to surface residues and likely affect RNA-protein or protein-protein interactions. Residues that are highly conserved among homologs stabilize the protein core. The solution structure of E. coli NusB presented here resembles that of Mycobacterium tuberculosis NusB determined by X-ray diffraction, but differs substantially from a solution structure of E. coli NusB reported earlier.
This chapter presents an application of the system for high-level production in Escherichia coli and rapid purification of recombinant proteins. The advent of gene cloning, the engineering of vectors for efficient expression, and the application of fast and high-flux methods for protein purification made available many recombinant proteins of biological interest. This represented a breakthrough for the structure–function analysis of bioactive proteins and cell receptors and facilitated X-ray crystallographic studies for definition of the three-dimensional structures of these proteins. The E. coli expression system allows the high-level production of recombinant proteins in authentic form, as fusion proteins with the [His]6 affinity tail and with mouse DHFR and the [His]6 tail. Because of the presence of the affinity tail, proteins that are produced in a soluble form or can be solubilized with GuHCl or urea can be purified almost to homogeneity in one step by nickel chelate affinity chromatography. The purified recombinant proteins simplify the production of monoclonal and polyclonal antibodies directed against defined regions of the native proteins.
Coliphage λ employs systems of transcription termination and antitermination to regulate gene expression. Early gene expression is regulated by the phage-encoded N protein working with a series of Escherichia coli proteins, Nus, at RNA sites, NUT, to modify RNA polymerase to a termination-resistant form. Expression of λ late genes is regulated by the phage-encoded Q antitermination protein. Q, which appears to use only one host factor, acts at a DNA site, qut, to modify RNA polymerase to a termination-resistant form. This review focuses on recent studies which show that: (i) N can mediate antitermination in vitro, independent of Nus proteins, (ii) Early genes in another lambdoid phage HK022 are also regulated by antitermination, where only an RNA signal appears necessary and sufficient to create a termination-resistant RNA polymerase. (iii) A part of the qut signal appears to be read from the non-template DNA strand. (iv) A host-encoded inhibitor of N antitermination appears to act through the NUT site as well as with the α subunit of RNA polymerase, and is antagonized by NusB protein.
A new plasmid cloning vector (pHE3) is described carrying dominant p-fluorophenylalanine-sensitivity (pheS) and chloramphenicol-resistance markers. This vector is used in combination with a p-fluorophenylalanine-resistantEscherichia coli recipient (strain RR28). Foreign DNA can be cloned into pHE3 leading to insertional inactivation of pheS. Transformation of RR28, and plating on minimal medium with chloramphenicol plus p-fluorophenylalanine (pfp) directly selects for colonies containing recombinant DNA.
München, Techn. Universiẗat, Diss., 1995.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
A special procedure was developed to isolate a new class of plaque-forming (Spi−) mutants of phage λ that are unable to express the leftward N-dependent red and gam gene functions by reason of cis-dominant defects located between the pL promoter and the N gene. The pL promoter is intact in these mutants, as the rate of transcription in the pL-N-tL1 region is high. Also, their gene N does not appear to be altered, as shown by genetic complementation. The mutations map near the center of the transcribed interval (about 124 base-pairs long) defined by the left ends of the λdv1 plasmid and the imm434 substitution. Thus, they map near the promoter, but interfere with antitermination at terminators located farther downstream. We conclude that the mutations define a new kind of recognition site (which we designate nut, for Nutilization) that controls an early step in the N-mediated antitermination mode of transcription.
We report the isolation and characterization of an Escherichia coli mutant which limits the growth of phage [lambda] by inhibiting the expression of the N gene regulatory function. The mutation involved maps near minute 11 of the E. coli chromosome and dominance tests show that the mutant allele is recessive to the wild one. Therefore, we conclude that the locus involved normally codes for a function necessary for N expression. Another mutant which exhibits a similar phenotype has previously been reported and the mutation involved, in that case, maps at minute 61. This mutant is called Nus (N utilization substance); we have named the locus at minute 61 nusA, and the locus at minute 11, nusB. Although the nusA allele is not found in Salmonella typhosa, our studies demonstrate that the nusA allele is found in this closely related enterobacteriaciae.A nusA-1 nusB-5 double mutant was constructed and exhibited a far more restrictive effect on [lambda] growth than either of the single nus mutants. Further, we have constructed a [lambda] variant which carries the nusB+ allele. This phage plates on nusB-5 mutants under restrictive conditions, but not on the nusA-1 mutants. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/21877/1/0000283.pdf
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A system for real-time biospecific interaction analysis using biosensor technology based on the optical phenomenon surface plasmon resonance is described. The biospecific interface is a sensor chip covered with a hydrogel matrix. One component of the interaction to be studied is immobilized covalently to the hydrogel and other interactants are passed over the chip in solution. The mass change at the sensor surface, reflecting the progress of the interaction studied, is monitored in real time. The technique, which does not require molecular labels for detection, can measure mass changes down to 10 pg/mm2. Repeated analyses can be performed on the same sensor chip. Applications shown include kinetic measurements, binding site analysis and concentration determination.
The Escherichia coli proteins NusB and ribosomal protein S10 are important for transcription antitermination by the bacteriophage lambda N protein. We have used sucrose gradient co-sedimentation and affinity chromatography with immobilized ribosomal protein S10, a glutathione S-transferase-S10 fusion protein, and NusB to show that NusB binds directly and very selectively to S10. The interaction is non-ionic and has an estimated Kd value of 10(-7) M. We hypothesize that NusB binds to N-modified transcription complexes primarily by interacting with S10.
The N gene product of Escherichia coli phage lambda is a transcriptional activator that captures the host RNA polymerase and modifies it to a termination-resistant form, permitting gene expression in two large polycistronic operons of the phage genome. Antitermination in vitro requires at least one host factor called NusA, which directly binds the N protein as well as RNA polymerase, and also a transcribed cis-acting site known as nut, within which lies the hypothesized N-recognition signal, boxB. BoxB is an interrupted palindrome capable of forming a hairpin in the mRNA. Inhibition studies with complementary DNA oligonucleotides provide evidence for a direct role of the boxB hairpin in antitermination. Kinetic studies of transcript elongation reveal that the boxB hairpin does not induce an appreciable pause to hold polymerase captive for engagement by N and NusA. Moreover, the efficiency of antitermination remains virtually the same whether N and NusA are added early, prior to nut site transcription, or added later, after the polymerase has already transcribed past the nut site. After transcription of the nut site, RNA polymerase remains susceptible to modification by N and NusA for an appreciable amount of time and distance, and the nut site DNA becomes dispensable for this modification. These results lead to the hypothesis that the boxB RNA hairpin acts in a manner analogous to the DNA enhancers, binding N and mediating a productive polymerase-NusA-N interaction by mRNA looping.
We have dissected the protein and nucleic acid determinants that direct a group of transcriptional antiterminators to their specific target operons. These antiterminators, the N gene products of phages lambda, 21, and P22, function solely with their respective recognition sites, nut, to modify RNA polymerase to a termination-resistant form. We demonstrate that a unique hairpin sequence within each nut site, called boxB, confers genome specificity by interacting with a small amino-terminal domain of the cognate N protein. This interaction is dependent upon an arginine-rich subdomain, which is conserved not only among the N proteins but also in many RNA binding proteins from ribosomes and RNA virus capsids. Notably, this motif constitutes an essential domain of the HIV protein Tat whose function as a trans-activator requires a specific hairpin sequence.
The ’N‘ antitermination proteins of lambdoid bacteriophages are essential for overcoming multiple transcription terminators
located within the major early operons of these phages (1). In order for N proteins to function, a genome sequence specifying
N utilization, nut, must belocated within an operon, between the promoter and the terminators (2). Two components have been identified within
nut: 8-base baxA, conserved among different phages and implicated in the recognition of host NusA protein, required for N function (3); 15-base
boxB, an interrupted palindrome (4), diverged in sequence among different lambdoid phages and hypothesized to be the site of recognition
for different N proteins, also diverged in sequence (5). Here we apply a plasmid for testing termination and antitermination
of transcription (6) to identify mutations at all positions in the 5–7 base loop of λ's boxB. Almost every base change at any position within the 5–7 base boxB loop was found to constrain antitermination of transcription
by the N protein of bacteriophage λ. Theseobservations extend previous mutational knowledge of nut(7) and are consistant with the hypothesisthat the boxB loop is the direct site of recognition for N protein. Variations among
the effects of different base changes suggest differential contacts between N protein and bases of the boxB loop, whether in DNA or RNA.
We report the genetic mapping of a locus of the Escherichia coli chromosome involved in the expression of the N gene function of phage λ. This phage specified function regulates the subsequent transcription of most of the λ genome. The bacterial locus involved in N expression, called nus for N utilization substance, maps between aspB at minute 62 and argG at minute 61 of the E. coli chromosome.Two different bacterial variants in which λ N function is not active have been used in mapping the nus locus, a mutant of E. coli K12, Nus, and a hybrid bacterium formed by genetic transfer between E. coli and S. typhosa. Although these two bacterial variants exhibit slightly different phenotypes, chromosome transfer studies demonstrate that the same genetic region is involved in the observed N-ineffective phenotype.Dominance studies show that in the case of the Nus mutant, the nus+ allele is dominant. This suggests that the nus+ allele is responsible for the expression of a function necessary for N product activity. In the case of transfer of the nus region from a Nus mutant to an E. coli-S. typhosa hybrid, the resulting hybrid assumes the phenotype of the Nus mutant. Genetic studies using P1 transduction demonstrate that the same genetic region is involved in the N-ineffective phenotype of the two bacterial variants.
We have searched among E. coli M72 (D, bio11cI857H1) temperature resistant survivors and have found two bacterial mutants, gro100 and gro101 which block λiλ and λi434 phage development but allow growth of their N-independent derivatives λiλ nin and λi434nin. It is not known yet whether these two mutants interfere with the production of the N gene product or with its function. At least part of the gro genotype maps at 12′ of the E. coli genetic map and is co-transductible by Pl with the lac locus.RésuméNous avons cherché des survivants à haute température de la souche d'E. coli M 72 (i) bio11cI857H1). Nous avons trouvé 2 mutants bactériens gro100 et gro101 chez lesquels la croissance de λiλ et λi434 est bloquée, mais non celle de leurs dérivés N-indépendants λiλnin et λi434nin. Nous ne savons pas encore si ces deux mutants interfèrent avec la production de la protéine N ou avec sa fonction. Pour partie au moins, le génotype gro est localisé à la 12e minute de la carte génétique d'E. coli, et est cotransductible par P1 avec le locus lac.
The most significant recent development in techniques for the sequence analysis of proteins and peptides is the advent of automated procedures for degradation by the phenylisothiocyanate (PITC) method. The chapter describes the automated procedures in some detail from a practical point of view. In the Edman procedure, the reagent PITC couples with the terminal alpha amino group of a peptide or protein to form a phenylthiocarbamyl (PTC) adduct. Under anhydrous acidic conditions, the N-terminal amino acid residue is selectively cleaved from the peptide chain as a heterocyclic derivative through the attack of the sulfur of the PTC adduct on the carbonyl component of the first peptide bond. The cleaved amino acid derivative is separated from the residual peptide by extraction with an organic solvent and then converted to a more stable isomer prior to identification by one of several possible procedures. The automated Edman procedure is particularly suitable for obtaining long degradations on polypeptides of 60–150 residues. With longer polypeptides, the increased background limits the length of the degradation.
The N gene product of coliphage lambda acts with host factors (Nus) through sites (nut) to render subsequent downstream transcription resistant to a variety of termination signals. These sites, nutR and nutL, are downstream, respectively, from the early promoters PR and PL. Thus a complicated set of molecular interactions are likely to occur at the nut sites. We have selected mutations in the nutR region that reduce the effectiveness of pN in altering transcription initiating at the PR promoter. DNA sequence analysis of three independently selected mutations revealed, in each case, a deletion of a single base pair in the cro gene. Consideration of the effect of such mutations on the extension of translation of cro message into the adjacent downstream nut region led to the identification of a consensus sequence CGCTCT(T)TAA that appears to play a role in the recognition of a host factor, possibly the NusA protein.
We report the isolation of an Escherichia coli K-12 strain with a mutation, nusE71, that results in a change in ribosomal protein S10. Phage lambda fails to grow in hosts carrying the nusE71 mutation because the lambda N gene product is not active. The N product regulates phage gene expression by altering transcription complexes so that they can overcome termination barriers. This suggests that a ribosomal protein is involved in antitermination of transcription.
The boxA sequences of the E. coli ribosomal RNA (rrn) operons are sufficient to cause RNA polymerase to read through Rho-dependent transcriptional terminators. We show that a complex of the transcription antitermination factors NusB and ribosomal protein S10 interacts specifically with boxA RNA. Neither NusB nor S10 binds boxA RNA on its own, and neither NusA nor NusG affects the interaction of the NusB-S10 complex with boxA RNA. Mutations in boxA that impair its antitermination activity compromise its interaction with NusB and S10, suggesting that ribosomal protein S10 regulates the synthesis of ribosomal RNA in bacteria. RNA containing the closely related boxA sequence from the bacteriophage lambda nutR site is not stably bound by NusB and S10. This probably explains why antitermination in phage lambda depends on the phage lambda N protein and the boxB component of the nut site, in addition to boxA.
Antiterminator proteins control gene expression by recognizing control signals near the promoter and preventing transcriptional termination which would otherwise occur at sites that may be a long way downstream. The N protein of bacteriophage lambda recognizes a sequence in the nascent RNA, and modifies RNA polymerase by catalysing the formation of a stable ribonucleoprotein complex on its surface, whereas the lambda Q protein recognizes a sequence in the DNA. These mechanisms of antitermination in lambda provide models for analysing antitermination in viruses such as HIV-1 and in eukaryotic genes.
Using an in vitro transcription assay, we have successfully demonstrated read through of a Rho-dependent terminator by the ribosomal RNA antitermination system. The assay used a DNA template containing a promoter-antiterminator-terminator arrangement, RNA polymerase, termination factor Rho, antitermination factors NusA, NusB, NusE, and NusG, and a cellular extract depleted of NusB. Terminator read-through was highly efficient only in the presence of the extract and Nus factors, suggesting that an as yet uncharacterized cellular component is required for ribosomal antitermination. The NusB-depleted extract had no activity in the absence of NusB, confirming an absolute requirement for this protein in ribosomal RNA antitermination. The DNA template requirements were the same as those previously established in vivo; transcription of a wild-type boxA sequence is both necessary and sufficient to promote RNA polymerase modification into a terminator-resistant form.
The N gene product of coliphage gamma, with a number of host proteins (Nus factors), regulates phage gene expression by modifying RNA polymerase to a form that overrides transcription-termination signals. Mutations in host nus genes diminish this N-mediated antitermination. Here, we report the isolation and characterization of the rpoAD305E mutation, a single amino acid change in the carboxy terminal domain (CTD) of the alpha subunit of RNA polymerase, that enhances N-mediated antitermination. A deletion of the 3' terminus of rpoA, resulting in the expression of an alpha subunit missing the CTD, also enhances N-mediated antitermination and, similar to rpoAD305E, suppresses the effect of nus mutations. Thus, the N-Nus complex may be affected through contacts with the CTD of the alpha subunit of RNA polymerase, as is a group of regulatory proteins that influences initiation of transcription. What distinguishes our findings on the N-Nus complex from those of previous studies with transcription proteins is that all of the regulators characterized in those studies bind DNA and influence transcription initiation; whereas the N-Nus complex binds RNA and affects transcription elongation. A screen of some previously identified rpoA mutations that influence transcription activators revealed only one other amino acid change, L290H, in the CTD of the alpha subunit, that influences antitermination. Although our results provide evidence that interactions of the alpha subunit of RNA polymerase must be considered in forming models of transcription antitermination, they do not provide information as to whether the interactions of alpha that ultimately influence antitermination occur during initiation or during elongation of transcription.
The product of the nusB gene of Escherichia coli modulates the efficiency of transcription termination at nut (N utilization) sites of various bacterial and bacteriophage lambda genes. Similar control mechanisms operate in eukaryotic viruses (e.g. human immunodeficiency virus). A recombinant strain of E. coli producing relatively large amounts of NusB protein (about 10% of cell protein) was constructed. The protein could be purified with high yield by anion-exchange chromatography followed by gel-permeation chromatography. The protein is a monomer of 15.6 kDa as shown by analytical ultracentrifugation. Structural studies were performed using protein samples labelled with 15N, 13C and 2H in various combinations. Heteronuclear three-dimensional triple-resonance NMR experiments combined with a semi-automatic assignment procedure yielded the sequential assignment of the 1H, 13C and 15N backbone resonances. Based on experimentally derived scalar couplings, chemical-shift values, amide-exchange data, and a semiquantitative interpretation of NOE data, the secondary structure of NusB has classified as alpha helical, comprising seven alpha helices.
Escherichia coli ribosomal RNA (rRNA) operons contain antitermination motifs necessary for forming terminator-resistant transcription complexes. In preliminary work, we isolated 'antiterminating' transcription complexes and identified four new proteins potentially involved in rRNA transcription antitermination: ribosomal (r-) proteins S4, L3, L4 and L13. We show here that these r-proteins and Nus factors lead to an 11-fold increase in terminator read-through in in vitro transcription reactions. A significant portion of the effect was a result of r-protein S4. We show that S4 acted as a general antitermination factor, with properties very similar to NusA. It retarded termination and increased read-through at Rho-dependent terminators, even in the absence of the rRNA antiterminator motif. High concentrations of NusG showed reduced antitermination by S4. Like rrn antitermination, S4 selectively antiterminated at Rho-dependent terminators. Lastly, S4 tightly bound RNA polymerase in vivo. Our results suggest that, like NusA, S4 is a general transcription antitermination factor that associates with RNA polymerase during normal transcription and is also involved in rRNA operon antitermination. A model for key r-proteins playing a regulatory role in rRNA synthesis is presented.
Lytic mode of lambda development
- Friedman
Friedman, D. I. & Gottesman, M. E. (1983). Lytic
mode of lambda development. In Lambda II
(Hendrix, R. W., Roberts, J. W., Sthal, F. W. &
Weisberg, R. A., eds), pp. 21-51, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY.
Transcriptional antitermination
- Greenblatt