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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.

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.

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.

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.

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.

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.