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1AbstractInE. coli, one RNA polymerase (RNAP) transcribes all RNA species, and different regulons are transcribed by employing different sigma (σ) factors. RNAP containingσ38(σS) activates genes responding to stress conditions such as stationary phase. The structure ofσ38promoters has been controversial for more than two decades. To construct a model ofσ38promoters using information theory, we aligned proven transcriptional start sites to maximize the sequence information, in bits, and identified a −10 element similar toσ70promoters. We could not align any −35 sequence logo; instead we found two patterns upstream of the −35 region. These patterns have dyad symmetry sequences and correspond to the location of UP elements in ribosomal RNA (rRNA) promoters. Additionally the UP element dyad symmetry suggests that the two polymeraseαsubunits, which bind to the UPs, should have two-fold dyad axis of symmetry on the polymerase and this is indeed observed in an X-ray crystal structure. Curiously theαCTDs should compete for overlapping UP elements.In vitroexperiments confirm thatσ38recognizes therrnBP1 promoter, requires a −10, UP elements and no −35. This clarifies the long-standing paradox of howσ38promoters differ from those ofσ70.

Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The NAD+-dependent protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modification in E. coli. Here, we show that the absence of CobB in a ΔcobB mutant reduces intracellular TopA catalytic activity and increases negative DNA supercoiling. TopA expression level is elevated as topA transcription responds to the increased negative supercoiling. The slow growth phenotype of the ΔcobB mutant can be partially compensated by further increase of intracellular TopA level via overexpression of recombinant TopA. The relaxation activity of purified TopA is decreased by in vitro non-enzymatic acetyl phosphate mediated lysine acetylation, and the presence of purified CobB protects TopA from inactivation by such non-enzymatic acetylation. The specific activity of TopA expressed from His-tagged fusion construct in the chromosome is inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. These findings demonstrate that E. coli TopA catalytic activity can be modulated by lysine acetylation–deacetylation, and prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the CobB protein deacetylase.

Bundled piezoelectric motors combine several actuators to achieve high output power. However, mutual interference between the actuators leads to reduction in working efficiency. This work presents an adaptive stator that can reduce the mutual interference in bundled piezowalk motors. The stator consists of leaf springs for improving motor contact condition and proof masses that serve as an inertial body for maintaining high output force. Parameters of the proof masses and leaf springs are analyzed, and the working zone of the stator is discussed. A prototype of a linear motor with the designed adaptive stator is fabricated and tested. The maximum speed of a six-leg motor is 103 mm/s, and the stall force is approximately 1.2 N, driven with sinusoidal waveforms of 25Vpp at 30 kHz. Mutual interference between actuators is reduced remarkably with the adaptive stator. A comparison of six- and four-leg motors proves that motor performance has a linear relationship with the number of actuators, thereby indicating the potential of increasing output capability with additional actuators.

RNA polymerase (RNAP), the transcription machinery, shows dynamic binding across the genomic DNA under different growth conditions. The genomic features that selectively redistribute the limited RNAP molecules to dictate genome-wide transcription in response to environmental cues remain largely unknown. We chose the bacterial osmotic stress response model to determine genomic features that direct genome-wide redistribution of RNAP during the stress. Genomic mapping of RNAP and transcriptome profiles corresponding to the different temporal states after salt shock were determined. We found rapid redistribution of RNAP across the genome, primarily at σ70 promoters. Three subsets of genes exhibiting differential salt sensitivities were identified. Sequence analysis using an information-theory based σ70 model indicates that the intergenic regions of salt-responsive genes are enriched with a higher density of σ70 promoter-like sites than those of salt-sensitive genes. In addition, the density of promoter-like sites has a positive linear correlation with RNAP binding at different salt concentrations. The RNAP binding contributed by the non-initiating promoter-like sites is important for gene transcription at high salt concentration. Our study demonstrates that hyperdensity of σ70 promoter-like sites in the intergenic regions of salt-responsive genes drives the RNAP redistribution for reprograming the transcriptome to counter osmotic stress.

In the fast-growing Escherichia coli cells, RNA polymerase (RNAP) molecules are concentrated and form foci at clusters of ribosomal RNA (rRNA) operons resembling eukaryotic nucleolus. The bacterial nucleolus-like organization, spatially compartmentalized at the surface of the compact bacterial chromosome (nucleoid), serves as transcription factories for rRNA synthesis and ribosome biogenesis, which influences the organization of the nucleoid. Unlike wild type that has seven rRNA operons in the genome in a mutant that has six (Δ6rrn) rRNA operons deleted in the genome, there are no apparent transcription foci and the nucleoid becomes uncompacted, indicating that formation of RNAP foci requires multiple copies of rRNA operons clustered in space and is critical for nucleoid compaction. It has not been determined, however, whether a multicopy plasmid-borne rRNA operon (prrnB) could substitute the multiple chromosomal rRNA operons for the organization of the bacterial nucleolus-like structure in the mutants of Δ6rrn and Δ7rrn that has all seven rRNA operons deleted in the genome. We hypothesized that extrachromosomal nucleolus-like structures are similarly organized and functional in trans from prrnB in these mutants. In this report, using multicolor images of three-dimensional superresolution Structured Illumination Microscopy (3D-SIM), we determined the distributions of both RNAP and NusB that are a transcription factor involved in rRNA synthesis and ribosome biogenesis, prrnB clustering, and nucleoid structure in these two mutants in response to environmental cues. Our results found that the extrachromosomal nucleolus-like organization tends to be spatially located at the poles of the mutant cells. In addition, formation of RNAP foci at the extrachromosomal nucleolus-like structure condenses the nucleoid, supporting the idea that active transcription at the nucleolus-like organization is a driving force in nucleoid compaction.

Members of the Swi2/Snf2 (switch/sucrose non-fermentable) family depend on their ATPase activity to mobilize nucleic acid-protein complexes for gene expression. In bacteria, RapA is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein that mediates RNAP recycling during transcription. It is known that the ATPase activity of RapA is stimulated by its interaction with RNAP. It is not known, however, how the RapA-RNAP interaction activates the enzyme. Previously, we determined the crystal structure of RapA. The structure revealed the dynamic nature of its N-terminal domain (Ntd), which prompted us to elucidate the solution structure and activity of both the full-length protein and its Ntd-truncated mutant (RapAΔN). Here, we report the ATPase activity of RapA and RapAΔN in the absence or presence of RNAP and the solution structures of RapA and RapAΔN either ligand-free or in complex with RNAP. Determined by small-angle x-ray scattering, the solution structures reveal a new conformation of RapA, define the binding mode and binding site of RapA on RNAP, and show that the binding sites of RapA and σ(70) on the surface of RNAP largely overlap. We conclude that the ATPase activity of RapA is inhibited by its Ntd but stimulated by RNAP in an allosteric fashion and that the conformational changes of RapA and its interaction with RNAP are essential for RNAP recycling. These and previous findings outline the functional cycle of RapA, which increases our understanding of the mechanism and regulation of Swi2/Snf2 proteins in general and of RapA in particular. The new structural information also leads to a hypothetical model of RapA in complex with RNAP immobilized during transcription.

In a fast-growing Escherichia coli cell, most RNA polymerase (RNAP) is allocated to rRNA synthesis forming transcription foci at clusters of rrn operons or bacterial nucleolus, and each of the several nascent nucleoids contains multiple pairs of replication forks. The composition of transcription foci has not been determined. In addition, how the transcription machinery is three-dimensionally organized to promote cell growth in concord with replication machinery in the nucleoid remains essentially unknown. Here, we determine the spatial and functional landscapes of transcription and replication machineries in fast-growing E. coli cells using super-resolution-structured illumination microscopy. Co-images of RNAP and DNA reveal spatial compartmentation and duplication of the transcription foci at the surface of the bacterial chromosome, encompassing multiple nascent nucleoids. Transcription foci cluster with NusA and NusB, which are the rrn anti-termination system and are associated with nascent rRNAs. However, transcription foci tend to separate from SeqA and SSB foci, which track DNA replication forks and/or the replisomes, demonstrating that transcription machinery and replisome are mostly located in different chromosomal territories to maintain harmony between the two major cellular functions in fast-growing cells. Our study suggests that bacterial chromosomes are spatially and functionally organized, analogous to eukaryotes.

Overview. We have learned a great deal about RNA polymerase (RNA Pol), transcription factors, and the transcriptional regulation mechanisms in prokaryotes for specific genes, operons, or transcriptomes. However, we have only begun to understand how the transcription machinery is 3-dimensionally (3D) organized into bacterial chromosome territories to orchestrate the transcription process and to maintain harmony with the replication machinery in the cell. Much progress has been made recently in our understanding of the spatial organization of the transcription machinery in fast-growing Escherichia coli cells using state-of-the-art superresolution imaging techniques. Co-imaging of RNA polymerase (RNA Pol) with DNA and transcription elongation factors involved in ribosomal RNA (rRNA) synthesis and ribosome biogenesis has revealed similarities between bacteria and eukaryotes in the spatial organization of the transcription machinery for growth genes, most of which are rRNA genes. Evidence supports the notion that RNA Pol molecules are concentrated, forming foci at the clustering of rRNA operons resembling the eukaryotic nucleolus. RNA Pol foci are proposed to be active transcription factories for both rRNA genes expression and ribosome biogenesis to support maximal growth in optimal growing conditions. Thus, in fast-growing bacterial cells, RNA Pol foci mimic eukaryotic Pol I activity, and transcription factories resemble nucleolus-like compartmentation. In addition, the transcription and replication machineries are mostly segregated in space to avoid the conflict between the two major cellular functions in fast-growing cells.

One of the major structural differences that provides a taxonomic distinction between eukaryotic and prokaryotic organisms is that prokaryotes lack the nuclear envelope, with exceptions such as planctomycetes 1, and genomic organization mechanisms found in eukaryotes. Instead, the prokaryotic genome is partitioned into a somewhat amorphous region of the cell known as the nucleoid 2–4. In both prokaryotes and eukaryotes, the genome must be compacted to fit into its allotted space while maintaining a level of organization that allows efficient functionality. In eukaryotes, this is accomplished by well-defined histone proteins that form nucleosomal and chromatin structures and yet can be remodeled to allow for the decondensation critical for gene expression 5–9. Although the bacterial genome must also be densely compacted, more than one-thousand-fold, and also organized for optimum functionality (chromosome replication/segregation, recombination and transcription) 10–14, the mechanisms by which this is accomplished remain unclear. The lack of “beads-on-a-string” morphology indicates different and/or multiple roles for nucleoid-associated proteins. Since transcription is a major cellular function and involves protein-DNA interaction and DNA topological domain structures, the transcription machinery themselves may play a role in nucleoid organization. Conversely, the nucleoid structure and organization may influence gene expression in a manner analogous to chromatin decondensation in eukaryotes. The interactions involved in nucleoid structure and gene expression continue to be challenging issues in the post-genomic era. Escherichia coli is a preferred model system for prokaryotic research due to the extensive studies into its genetic, physiology, biochemistry, and molecular biology mechanisms 15. The study of nucleoid organization is intrinsically difficult mainly due to the small size of bacteria in comparison to the limits of optical resolution of conventional instrumentation. Until very recently, images of the nucleoid have not changed much over the last four decades and reveal no structural details 16–18. Consequently, most of the earlier studies were focused on the biochemical properties and morphologies of isolated nucleoids and electron microscopic images after fixation 3–4,19–24. However, any preparation or fixation procedure will potentially introduce different organizations of the nucleoids or even artifacts. For example, different fixation procedures produced different nucleoid shapes of electron microscopic images 3. Additionally, growing evidence indicates that the organization of the nucleoid in the cell is plastic and sensitive to changes in growth and stress. The challenge of the research is to capture the “true” organization of the nucleoid reflecting the dynamic states of the nucleoid in living cells. Extensive studies have primarily emphasized the “histone-like” proteins including FIS, HU, H-NS, and IHF for their architectural roles in bending, looping, bridging and compacting DNA 25–36. The first three and RNA polymerase (RNAP) are the major nucleoid-associated proteins (NAPs) isolated from exponentially growing cells. Compared to the “histone-like” proteins, the binding of RNAP to DNA is much stronger. A prevailing view is that bacterial “histone-like proteins” are primarily responsible for the nucleoid organization and compaction in growing cells. Recent advances in the cell biology of E. coli RNAP and the nucleoid have shown that the distribution of RNAP, which is coupled to cell growth, plays an important role in the nucleoid dynamic structure. Emerging evidence indicates that formation of the transcription foci centered at the putative nucleolus structure is critical in nucleoid remodeling and influencing global gene expression. As other recent comprehensive reviews have dealt with the roles of other factors in bacterial nucleoid organization 37–43, this review provides an alternative and/or complementary perspective to the traditional views on bacterial nucleoid compaction and expansion.

Growth rate regulation in bacteria has been an important issue in bacterial physiology for the past 50 years. This review, using Escherichia coli as a paradigm, summarizes the mechanisms for the regulation of rRNA synthesis in the context of systems biology, particularly, in the context of genome-wide competition for limited RNA polymerase (RNAP) in the cell under different growth conditions including nutrient starvation. The specific location of the seven rrn operons in the chromosome and the unique properties of the rrn promoters contribute to growth rate regulation. The length of the rrn transcripts, coupled with gene dosage effects, influence the distribution of RNAP on the chromosome in response to growth rate. Regulation of rRNA synthesis depends on multiple factors that affect the structure of the nucleoid and the allocation of RNAP for global gene expression. The magic spot ppGpp, which acts with DksA synergistically, is a key effector in both the growth rate regulation and the stringent response induced by nutrient starvation, mainly because the ppGpp level changes in response to environmental cues. It regulates rRNA synthesis via a cascade of events including both transcription initiation and elongation, and can be explained by an RNAP redistribution (allocation) model.

Helicobacter pylori persists deep in the human gastric mucus layer in a harsh, nutrient-poor environment. Survival under these conditions depends on the ability of this human pathogen to invoke starvation/stress responses when needed. Unlike many bacteria, H. pylori lacks starvation/stress-responding alternative sigma factors, suggesting an additional mechanism might have evolved in this bacterium. Helicobacter pylori produces polyphosphate; however, the role and target of polyphosphate during starvation/stress have not been identified. Here we show that polyphosphate accumulated during nutrient starvation directly targets transcriptional machinery by binding to the principal sigma factor in H. pylori, uncovering a novel mechanism in microbial stress response. A positively charged Lys-rich region at the N-terminal domain of the major sigma factor is identified as the binding region for polyphosphate (region P) in vivo and in vitro, revealing a new element in sigma 70 family proteins. This interaction is biologically significant because mutant strains defective in the interaction undergo premature cell death during starvation. We suggested that polyphosphate is a second messenger employed by H. pylori to mediate gene expression during starvation/stress. The putative 'region P' is present in sigma factors of other human pathogens, suggesting that the uncovered interaction might be a general strategy employed by other pathogens.

In Helicobacter pylori the stringent response is mediated solely by spoT . The spoT gene is known to encode (p)ppGpp synthetase activity and is required for H. pylori survival in the stationary phase. However, neither the hydrolase activity of the H. pylori SpoT protein nor the role of SpoT in the regulation of growth during serum starvation and intracellular survival of H. pylori in macrophages has been determined. In this study, we examined the effects of SpoT on these factors. Our results showed that the H. pylori spoT gene encodes a bifunctional enzyme with both a hydrolase activity and the previously described (p)ppGpp synthetase activity, as determined by introducing the gene into Escherichia coli relA and spoT defective strains. Also, we found that SpoT mediates a serum starvation response, which not only restricts the growth but also maintains the helical morphology of H. pylori. Strikingly, a spoT null mutant was able to grow to a higher density in serum-free medium than the wild-type strain, mimicking the “relaxed” growth phenotype of an E. coli relA mutant during amino acid starvation. Finally, SpoT was found to be important for intracellular survival in macrophages during phagocytosis. The unique role of (p)ppGpp in cell growth during serum starvation, in the stress response, and in the persistence of H. pylori is discussed.

RapA, as abundant as σ70 in the cell, is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein with ATPase activity. It stimulates RNAP recycling during transcription. Here, we report the first structure of RapA, which is also the first full-length structure for the entire Swi2/Snf2 family. RapA contains seven domains, two of which exhibit novel protein folds. Our model of RapA in complex with ATP and double-stranded (ds) DNA suggests that RapA may bind to and translocate on dsDNA. Our kinetic template-switching assay shows that RapA facilitates the release of sequestered RNAP from a posttranscrption/posttermination complex (PTC) for transcription reinitiation. Our in vitro competition experiment indicates that RapA binds to core RNAP only but is readily displaceable by σ70. RapA is likely another general transcription factor, the structure of which provides a framework for future studies of this bacterial Swi2/Snf2 protein and its important roles in RNAP recycling during transcription.

In Escherichia coli , stringently controlled genes are highly transcribed during rapid growth, but “turned off” under nutrient limiting conditions, a process called the stringent response. To understand how transcriptional initiation at these promoters is coordinately regulated, we analyzed the interactions between RNA polymerase (RNAP) (both wild type and mutants) and four stringently controlled promoters. Our results show that the interactions between RNAP and stringently controlled promoters are intrinsically unstable and can alternate between relatively stable and metastable states. The mutant RNAPs appear to specifically further weaken interactions with these promoters in vitro and behave like “stringent” RNAPs in the absence of the stringent response in vivo , constituting a novel class of mutant RNAPs. Consistently, these mutant RNAPs also activate the expression of other genes that normally require the response. We propose that the stability of initiation complexes is coupled to the transcription of stringently controlled promoters, and this unique feature coordinates the expression of genes positively and negatively regulated by the stringent response.

Transcription in bacteria requires the interaction of sigma factors (ς) and core RNA polymerase (RNAP), which is composed of β, β′, and α2 subunits. This interaction creates a holoenzyme which initiates transcription via direct contacts between the RNA polymerase and specific promoter DNA. At least one primary sigma factor (ς70) and six alternative sigma factors (ς32, ςE, ςF, ςS, ς54, and FecI) are present in Escherichia coli, all promoting the expression of different sets of genes (1, 15). The primary sigma factor controls the expression of the housekeeping genes and exists as the most abundant sigma factor during the exponential phase of growth. The activities and the level of the other six alternative sigma factors become more crucial to the viability of the cell in response to certain stress conditions. Depending on the stimuli, an alternative sigma factor may preferentially bind to core RNAP in lieu of the primary sigma factor to initiate transcription of genes that are under its control. This interchange of sigma factors on core RNAP causes an efficient switching of gene expression in response to the internal and external environment. A few studies of sigma-core RNAP interaction have provided information about the location of core RNAP binding regions on sigma factors. Amino acid sequence alignment of sigma factors in the ς70 family has revealed that region 2.2 is the most highly conserved region (12, 22). Some have speculated that the most conserved region is probably the core RNAP binding region, based on the idea that sigma factors contact the same surface on core RNAP (9, 27, 32). Recently, we reported that a single amino acid change in region 2.2 of ς32, the heat shock sigma factor in E. coli encoded by rpoH, reduced its affinity for core RNAP (17). This result suggests that at least one residue in this most highly conserved region is directly involved in sigma factor-core RNAP interaction. Another highly conserved region, 2.1, has also been implicated in core RNAP binding, based on the deletion analysis of ς70 (20) and, subsequently, a single amino acid substitution of ςE in Bacillus subtilis (30). The crystal structure of the proteolytically stable fragment of E. coli ς70, which includes regions 2.1 and 2.2, further implicates these two regions as important for protein-protein interaction (23). Using ς32 with 24 amino acids deleted, Zhou et al. (35) have proposed that region 3 may also be involved in core RNAP binding. This region, however, is weakly conserved, especially among alternative sigma factors. In this communication, we report our analysis of the products of nine rpoH alleles, each carrying a mutation downstream of region 2.2. These alleles suppress the temperature sensitivity of rpoD285 by preventing the proteolytic degradation of ς70 that contains a small internal deletion (10). In order to study the activities of these mutant sigma factors in vitro, we have purified the products of nine rpoH alleles. Subsequent biochemical examination of these purified products revealed that most of the mutants may exhibit reduced affinity for core RNAP. Interestingly, not all mutations were located in conserved regions, nor did they affect conserved residues. These results suggest that there are residues outside of regions 2.1 and 2.2 that may also participate in core RNAP interaction and that the mutated nonconserved residues may represent unique core RNAP binding sites for ς32.

This work describes a mutational analysis of the interaction between the beta and sigma subunits of Escherichia coli RNA polymerase. The rpoD800 mutant has a temperature-sensitive growth phenotype because the mutant sigma70 polypeptide is not stable at a high temperature. Some rpoB mutations, including rpoB114, enhanced the temperature sensitivity of the rpoD800 mutant. We determined the mechanism by which the rpoB114 rpoD800 double mutant becomes hyper-temperature sensitive for growth. We found that the levels of the mutant sigma70 in the rpoB114 rpoD800 mutant were dramatically reduced compared to that in the rpoD800 mutant after temperature shift-up. The rate of synthesis of the sigma70 polypeptide was reduced in the rpoB114 rpoD800 double mutant compared to the rpoD800 mutant, whereas the half-life of the mutant sigma70 polypeptide after temperature shift-up was the same in both strains. We conclude that because of the reduction of expression of rpoD800 by rpoB114, in concert with the intrinsic instability of the mutant sigma70 polypeptide, the amount of holoenzyme containing sigma70 becomes limiting upon temperature shift-up. This results in the hyper-temperature sensitivity of the rpoB114 rpoD800 double mutant. Furthermore, the effect of rpoB114 on the expression of sigma70 is independent of the rpoD800 allele and is at the transcriptional level. In vitro transcription assays showed that the mutant RNA polymerase RpoB114 was defective in transcribing the two major promoters of the rpoD operon specifically. The effects of these rpoB mutations on gene expression are discussed.

Gal repressor inhibits transcription from the gal promoter ( P 1) when it binds to the cognate operator ( O E ). The repression is relieved by the presence of the inducer d -galactose. Compared with its interaction with free repressor, d -galactose binds to the repressor–operator complex with 10-fold reduced affinity as determined by fluorescence enhancement measurements. Thermodynamic analysis and fluorescence anisotropy showed that the stability of the repressor–operator complex is reduced by only 7-fold by the presence of the inducer in the complex. The formation of the inducer–repressor–operator ternary complex has been confirmed by CD spectral analysis. Fluorescence spectroscopy and energy transfer experiments suggest that individual allosteric effects of the two ligands, inducer and operator, on Gal repressor are responsible for the slightly weakened stability of the ternary complex compared with the stability of the inducer–repressor and repressor–operator complexes. In vitro transcription results demonstrated full derepression of transcription of the P 1 promoter under conditions in which the concentrations of the inducer–repressor binary complex are severalfold higher than the dissociation constant of the inducer–repressor–operator ternary complex into inducer–repressor and free DNA. These results strongly suggest that the inducer binding to the repressor–operator complex does not lead to dissociation of the repressor from the operator during transcription induction. Because Gal repressor inhibits transcription by modulating the α subunit of the P 1-bound RNA polymerase, we conclude that the inducer binding to the operator-bound repressor only allosterically relieves the inhibitory effect of repressor on RNA polymerase without dissociating the repressor from DNA.

Transcription fidelity is critical for maintaining the accurate flow of genetic information. The study of transcription fidelity has been limited because the intrinsic error rate of transcription is obscured by the higher error rate of translation, making identification of phenotypes associated with transcription infidelity challenging. Slippage of elongating RNA polymerase (RNAP) on homopolymeric A/T tracts in DNA represents a special type of transcription error leading to disruption of open reading frames in Escherichia coli mRNA. However, the regions in RNAP involved in elongation slippage and its molecular mechanism are unknown. We constructed an A/T tract that is out of frame relative to a downstream lacZ gene on the chromosome to examine transcriptional slippage during elongation. Further, we developed a genetic system that enabled us for the first time to isolate and characterize E. coli RNAP mutants with altered transcriptional slippage in vivo. We identified several amino acid residues in the β subunit of RNAP that affect slippage in vivo and in vitro. Interestingly, these highly clustered residues are located near the RNA strand of the RNA-DNA hybrid in the elongation complex. Our E. coli study complements an accompanying study of slippage by yeast RNAP II and provides the basis for future studies on the mechanism of transcription fidelity. Background: The domains in RNA polymerase involved in elongation slippage are unknown. Results: We isolated E. coli RNA polymerase rpoB mutants with altered transcriptional slippage. Conclusion: The fork domain of RNA polymerase controls slippage. Biochemical analysis of the mutants validates the genetic schemes. Significance: Our work sheds light on the mechanism for maintenance of RNA-DNA register during transcription.

We isolated and characterized mutant repressors (GalR) of the gal operon in Escherichia coli. These repressors (super-repressors), called GalRs, have a non-inducible phenotype. Repression of the gal operon by super-repressors cannot be lifted by inducer. The mutant galR genes, galRs, have been cloned and the mutational changes determined. Two of them, galRuv7s and galR78s, were located in the proposed sugar binding domains of the repressor. The repressor from wild-type (galR+), as well as from mutant galRuv7s, was purified and characterized biochemically. The results showed that, like wild-type GalR+, GalRuv7s binds to DNA normally and represses transcription from the P1 promoter and stimulates that from the P2 promoter of the gal operon. Nevertheless, compared to GalR+, GalRuv7s is much less sensitive to the presence of the inducer, D-galactose. The affinity of D-galactose to GalRuv7s is 10 to 30-fold lower, as measured by the effect of the inducer on GalR tryptophan fluorescence; GalR complexes with DNA and on GalR repression of transcription. Our results suggest that the super-repressor phenotype of GalRuv7s is because of a defect in D-galactose binding rather than a defect in the ligand-induced allosteric change or increased affinity for the operator.

Chloride Intracellular Channel (CLIC) proteins are non-canonical ion channels without a signal sequence targeting them to a membrane or transmembrane domain. CLICs exist insoluble and membranous forms, and are structurally similar to the omega class of Glutathione S transferases (GST). We have earlier shown that CLICs can auto-insert into planar bilayers to form trans-redox regulated ion channels. To identify a prokaryotic CLIC homolog, insilico and electrophysiological analysis were performed. Here we report a class of proteins called Stringent Starvation Protein A (SspA) in prokaryotes, which have a sequence and structural homology to CLIC proteins. Analysis of crystal structures of soluble forms of SspA and CLICs show omega folds and homologues α-helices and β-sheets. We further tested whether SspA can autoinsert to form functional ion channels. Recombinant SspA from E.coli was reconstituted in a planar bilayer made of POPE: POPS: Cholesterol with 4:1:1 molar ratios. SspA insert into planar bilayers and form functional ion channels within 5 minutes of addition of protein to the cis-side. These ion channels demonstrated single-channel conductance of 9.0±1.5 pS (n=5) and were more selective for cations (K+) than anions (Cl-), a property similar to CLIC. When SspA was recorded in Tris-Cl, single-channel conductance was lowered to 5.5±0.5 pS, with increased selectivity for anions over cations. IAA-94, a known CLIC blocker, decreased open probability of SspA-mediated currents (n=3). We have identified and characterized the first bacterial homolog of mammalian CLICs and shown it shares similar biophysical properties with CLIC proteins.

One of the hallmarks of the Swi2/Snf2 family members is their ability to modify the interaction between DNA-binding protein and DNA in controlling gene expression. The studies of Swi2/Snf2 have been mostly focused on their roles in chromatin and/or nucleosome remodeling in eukaryotes. A bacterial Swi2/Snf2 protein named RapA from Escherichia coli is a unique addition to these studies. RapA is an RNA polymerase (RNAP)-associated protein and an ATPase. It binds nucleic acids including RNA and DNA. The ATPase activity of RapA is stimulated by its interaction with RNAP, but not with nucleic acids. RapA and the major sigma factor σ70 compete for binding to core RNAP. After one transcription cycle in vitro, RNAP is immobilized in an undefined posttranscription/posttermination complex (PTC), thus becoming unavailable for reuse. RapA stimulates RNAP recycling by ATPase-dependent remodeling of PTC, leading to the release of sequestered RNAP, which then becomes available for reuse in another cycle of transcription. Recently, the crystal structure of RapA that is also the first full-length structure for the entire Swi2/Snf2 family was determined. The structure provides a framework for future studies of the mechanism of RNAP recycling in transcription. This article is part of a Special Issue entitled: Snf2/Swi2 ATPase structure and function.

Helicobacter pylori persists deep in the human gastric mucus layer in a harsh, nutrient-poor environment. Survival under these conditions depends on the ability of this human pathogen to invoke starvation/stress responses when needed. Unlike many bacteria, H. pylori lacks starvation/stress-responding alternative sigma factors, suggesting an additional mechanism might have evolved in this bacterium. Helicobacter pylori produces polyphosphate; however, the role and target of polyphosphate during starvation/stress have not been identified. Here we show that polyphosphate accumulated during nutrient starvation directly targets transcriptional machinery by binding to the principal sigma factor in H. pylori, uncovering a novel mechanism in microbial stress response. A positively charged Lys-rich region at the N-terminal domain of the major sigma factor is identified as the binding region for polyphosphate (region P) in vivo and in vitro, revealing a new element in sigma 70 family proteins. This interaction is biologically significant because mutant strains defective in the interaction undergo premature cell death during starvation. We suggested that polyphosphate is a second messenger employed by H. pylori to mediate gene expression during starvation/stress. The putative 'region P' is present in sigma factors of other human pathogens, suggesting that the uncovered interaction might be a general strategy employed by other pathogens.

the aim of the study was to observe the change of NADPH oxidase p22phox expression in the induced sputum cells for patients with obstructive sleep apnea hypopnea syndrome (OSAHS), and its relationship with the severity of OSAHS.thirty OSAHS patients and 23 healthy controls were recruited in the study. Before sleep and in the next morning sputum induction was performed twice for each subject, cell numbers and differentials in induced sputum were counted. NADPH oxidase p22phox mRNA expression was measured in induced sputum cells by RT-PCR and the protein was measured by immunocytochemistry.the OSAHS group showed a significant higher percentage of neutrophils and a lower percentage of macrophages in the induced sputum compared to the control group (P0.05). Macrophages and neutrophils represented the main cell types expressing NADPH oxidase p22phox in induced sputum cells by immunocytochemistry. In the control group, level of NADPH oxidase p22phox mRNA and percentages of NADPH oxidase p22phox positive neutrophils and macrophages in sputum samples collected both in the morning and before sleep was significantly lower than in OSAHS group (P0.05). In OSAHS group, higher levels of NADPH oxidase p22phox mRNA and percentages of NADPH oxidase p22phox positive neutrophils and macrophages were found in the morning than at pre-sleep. A negative correlation was found between levels of NADPH oxidase p22phox mRNA and percentage of NADPH oxidase p22phox positive neutrophils and macrophages in the morning sputum were negatively associated with LspO(2), but positively associated with AHI.these findings suggest that there are changes of NADPH oxidase p22phox expression in induced sputum cells of OSAHS patients, and these changes are related to the severity of OSAHS.

In Escherichia coli, transcription of the heat shock genes is regulated by sigma 32, the alternative sigma factor directing RNA polymerase to heat shock promoters. sigma 32, encoded by rpoH (htpR), is normally present in limiting amounts in cells. Upon temperature upshift, the amount of sigma 32 transiently increases, resulting in the transient increase in transcription of the heat shock genes known as the heat shock response. Strains carrying the rpoH165 nonsense mutation and supC(Ts), a temperature-sensitive suppressor tRNA, do not exhibit a heat shock response. This defect is suppressed by rpoD800, a mutation in the gene encoding sigma 70. We have determined the mechanism of suppression. In contrast to wild-type strains, the level of sigma 32 and the level of transcription of heat shock genes remain relatively constant in an rpoH165 rpoD800 strain after a temperature upshift. Instead, the heat shock response in this strain results from an approximately fivefold decrease in the cellular transcription carried out by the RNA polymerase holoenzyme containing mutant RpoD800 sigma 70 coupled with an overall increase in the translational efficiency of all mRNA species.

sigma 70, encoded by rpoD, is the major sigma factor in Escherichia coli. rpoD285 (rpoD800) is a small deletion mutation in rpoD that confers a temperature-sensitive growth phenotype because the mutant sigma 70 is rapidly degraded at high temperature. Extragenic mutations which reduce the rate of degradation of RpoD285 sigma 70 permit growth at high temperature. One class of such suppressors is located in rpoH, the gene encoding sigma 32, an alternative sigma factor required for transcription of the heat shock genes. One of these, rpoH113, is incompatible with rpoD+. We determined the mechanism of incompatibility. Although RpoH113 sigma 32 continues to be made when wild-type sigma 70 is present, cells show reduced ability to express heat shock genes and to transcribe from heat shock promoters. Glycerol gradient fractionation of sigma 32 into the holoenzyme and free sigma suggests that RpoH113 sigma 32 has a lower binding affinity for core RNA polymerase than does wild-type sigma 32. The presence of wild-type sigma 70 exacerbates this defect. We suggest that the reduced ability of RpoH113 sigma 32 to compete with wild-type sigma 70 for core RNA polymerase explains the incompatibility between rpoH113 and rpoD+. The rpoH113 cells would have reduced amounts of sigma 32 holoenzyme and thus be unable to express sufficient amounts of the essential heat shock proteins to maintain viability.

Increased production of reactive oxygen species (ROS) is thought to play a major role in the pathogenesis of obstructive sleep apnea-hypopnea syndrome (OSAHS). The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex is an important source of ROS. The p22phox subunit is polymorphic with a C242T variant that changes histidine-72 for a tyrosine in the potential heme binding site. This study aimed to investigate the relationship between NADPH oxidase subunit p22phox gene polymorphism and OSAHS.The genotypes of p22phox polymorphism were determined by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) assay in 176 unrelated subjects of the Han population in southern region of China (including 107 OSAHS subjects and 69 non-OSAHS subjects), while the plasma concentration of superoxide dismutase (SOD) was detected in the two groups, and p22phox mRNA expression in peripheral blood mononuclear cell (PBMC) was determined with reverse transcription polymerase chain reaction (RT-PCR).The phagocyte NADPH oxidase subunit p22phox mRNA expression was significantly increased in the OSAHS group than that in the non-OSAHS group (P0.01). Compared with the non-OSAHS control group ((85.31 +/- 9.23) U/ml), the levels of SOD were lower in patients with OSAHS ((59.65 +/- 11.61) U/ml (P0.01). There were significant differences in genotypes distribution in p22phox polymorphism between the two groups (P = 0.02). Compared with the non-OSAHS control group, the OSAHS group had a significantly higher T allele frequency in p22phox polymorphism (P = 0.03). There were independent effects of p22phox polymorphism on body mass index (BMI), neck circumference (NC), waist-to-hip ratio (WHR) in the OSAHS group, and the carriers of the T allele of p22phox polymorphism had greater NC, WHR, systolic blood pressure (SBP), diastolic blood pressure (DBP) and apnea-hypopnea index (AHI) (P0.05), but the carriers of the T allele had lower SOD (P0.01) and lowest SaO(2) (P = 0.04). There was no significant difference in p22phox mRNA expression between the OSAHS groups with or without T allele (P = 0.45).The NADPH oxidase subunit p22phox gene polymorphism may be associated with susceptibility to OSAHS, and it may be an important candidate gene for OSAHS.

To investigate the effect of NADPH oxidase activity inhibitor apocynin on the blood pressure of rats exposed to chronic intermittent hypoxia (CIH).Thirty healthy male SD rats were randomly divided into 3 groups of 10 each, a CIH group, an apocynin treatment group and a control group. The systolic blood pressure (SBP) was measured with tail-cuff method. RT-PCR and Western blot were used to examine mRNA and protein expression of NADPH oxidase subunit p22phox. The level of MDA, SOD, NO and .O(2)(-) were detected by colorimetric method.Compared to the normal group, the expression of p22phox mRNA and protein were significantly increased in CIH group (q = 3.202, 6.522, P0.05). There were no statistic difference of p22phox mRNA and protein expression between CIH group and apocynin treatment group (P0.05). Compared with those of CIH group, the levels of MDA, .O(2)(-) and the systolic blood pressure decreased significantly (P0.05), while SOD and NO activity increased significantly (q = 6.960, 4.385, P0.05) in apocynin treatment group.These results indicate that NADPH oxidase up-expression is closely associated with hypertension in OSAHS. Inhibition of NADPH oxidase activity may be hopefully served as a useful strategy for prevention and treatment of OSAHS-induced hypertension.

Regulation of transcription is a key step in controlling gene expression in all cells. Many diseases and cancers result from alterations of gene expression caused by defects in transcription machinery. The basic structure and function of RNA polymerase (RNAP) and RNAP-associated proteins are conserved throughout evolution. Sophisticated genetics and advanced biochemistry make single-cell organisms, such as E. coli, an ideal model system to study the role of RNAP and transcription factors in gene expression and regulation. Studies of the simple model system have contributed greatly to our knowledge of transcription in principle, which underpin our understanding of gene regulation in much more complex eukaryotic organisms. In addition, studies of E. coli and other microorganisms have laid the foundation for the biotechnology industry. This chapter describes transcription machinery including RNAP and RNAP-associated proteins and regulation of transcription cycle in E. coli, with a focus on the unique feature of global regulation by RNAP (re)distribution in response to environment cues.

The structure and function relationships of RNAP can be studied by correlating the change(s) in the rpo genes encoding different subunits of RNAP and the altered function(s) of the mutant RNAP both in vivo and in vitro. Mutational analysis has proven to be a powerful approach to the study of RNAP structure-function and to the elucidation of the mechanisms underlying different steps of transcription. Rapid progress has been made in isolating and characterizing RNAP mutants with altered step(s) of transcription. Analysis of the steps altered in these mutant RNAPs in turn has helped in studying the normal reaction mechanisms.

Cells respond to a sudden increase in temperature by increasing their rate of synthesis of a small number of proteins (reviewed in Neidhardt et al., 1984; Schlesinger et al., 1982). This response is called the heat-shock response, and the proteins synthesized in response to heat stress are called the heat-shock proteins (HSPs). Both prokaryotes and eukaryotes have been shown to have a heat-shock response (Schlesinger et al., 1982). At least two of the HSPs are strongly conserved between prokaryotes and eukaryotes (Bardwell and Craig, 1984), suggesting that the response serves a similar function in all organisms.