Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus.

The spatial organization of DNA within the bacterial nucleoid remains unclear. To investigate chromosome organization in Escherichia coli, we examined the relative positions of the ribosomal RNA (rRNA) operons in space. The seven rRNA operons are nearly identical and separated from each other by as much as 180° on the circular genetic map, a distance of ≥2 million base pairs. By inserting binding sites for fluorescent proteins adjacent to the rRNA operons and then examining their positions pairwise in live cells by epifluorescence microscopy, we found that all but rrnC are in close proximity. Colocalization of the rRNA operons required the rrn P1 promoter region but not the rrn P2 promoter or the rRNA structural genes and occurred with and without active transcription. Non-rRNA operon pairs did not colocalize, and the magnitude of their physical separation generally correlated with that of their genetic separation. Our results show that E. coli bacterial chromosome folding in three dimensions is not dictated entirely by genetic position but rather includes functionally related, genetically distant loci that come into close proximity, with rRNA operons forming a structure reminiscent of the eukaryotic nucleolus.

Engineered ribosomal RNA operon copy-number variants of E. coli reveal the evolutionary trade-offs shaping rRNA operon number.

Ribosomal RNA (rrn) operons, characteristically present in several copies in bacterial genomes (7 in E. coli), play a central role in cellular physiology. We investigated the factors determining the optimal number of rrn operons in E. coli by constructing isogenic variants with 5-10 operons. We found that the total RNA and protein content, as well as the size of the cells reflected the number of rrn operons. While growth parameters showed only minor differences, competition experiments revealed a clear pattern: 7-8 copies were optimal under conditions of fluctuating, occasionally rich nutrient influx and lower numbers were favored in stable, nutrient-limited environments. We found that the advantages of quick adjustment to nutrient availability, rapid growth and economic regulation of ribosome number all contribute to the selection of the optimal rrn operon number. Our results suggest that the wt rrn operon number of E. coli reflects the natural, 'feast and famine' life-style of the bacterium, however, different copy numbers might be beneficial under different environmental conditions. Understanding the impact of the copy number of rrn operons on the fitness of the cell is an important step towards the creation of functional and robust genomes, the ultimate goal of synthetic biology.

The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation.

The global regulatory nucleotide ppGpp ("magic spot") regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp's target site on RNAP, and therefore its mechanism of action, has remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and ß', and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.

An improved procedure for the purification of the Escherichia coli RNA polymerase omega subunit.

We report an improved procedure for purification of the omega subunit of Escherichia coli RNA polymerase. In contrast to the original procedure, the revised procedure: (i) allows purification of omega entirely from the soluble fraction, obviating the need for denaturation/renaturation, (ii) results in >99% pure omega in only two chromatographic steps, and (iii) improves the yield of purified omega by at least 5-fold. Reconstitution of E. coli RNAP from omega purified by this procedure, as well as purified sigma and core RNAP lacking omega, produces active holoenzyme in vitro, and co-overexpression of omega from a plasmid containing rpoZ and an additional plasmid encoding the other RNAP core subunits results in production of active core enzyme in vivo.

Escherichia coli DksA binds to Free RNA polymerase with higher affinity than to RNA polymerase in an open complex.

The transcription factor DksA binds in the secondary channel of RNA polymerase (RNAP) and alters transcriptional output without interacting with DNA. Here we present a quantitative assay for measuring DksA binding affinity and illustrate its utility by determining the relative affinities of DksA for three different forms of RNAP. Whereas the apparent affinities of DksA for RNAP core and holoenzyme are the same, the apparent affinity of DksA for RNAP decreases almost 10-fold in an open complex. These results suggest that the conformation of RNAP present in an open complex is not optimal for DksA binding and that DNA directly or indirectly alters the interface between the two proteins.

Still looking for the magic spot: the crystallographically defined binding site for ppGpp on RNA polymerase is unlikely to be responsible for rRNA transcription regulation.

Identification of the RNA polymerase (RNAP) binding site for ppGpp, a central regulator of bacterial transcription, is crucial for understanding its mechanism of action. A recent high-resolution X-ray structure defined a ppGpp binding site on Thermus thermophilus RNAP. We report here effects of ppGpp on 10 mutant Escherichia coli RNAPs with substitutions for the analogous residues within 3-4 A of the ppGpp binding site in the T. thermophilus cocrystal. None of the substitutions in E. coli RNAP significantly weakened its responses to ppGpp. This result differs from the originally reported finding of a substitution in E. coli RNAP eliminating ppGpp function. The E. coli RNAPs used in that study likely lacked stoichiometric amounts of omega, an RNAP subunit required for responses of RNAP to ppGpp, in part explaining the discrepancy. Furthermore, we found that ppGpp did not inhibit transcription initiation by T. thermophilus RNAP in vitro or shorten the lifetimes of promoter complexes containing T. thermophilus RNAP, in contrast to the conclusion in the original report. Our results suggest that the ppGpp binding pocket identified in the cocrystal is not the one responsible for regulation of E. coli ribosomal RNA transcription initiation and highlight the importance of inclusion of omega in bacterial RNAP preparations.

Effects of DksA, GreA, and GreB on transcription initiation: insights into the mechanisms of factors that bind in the secondary channel of RNA polymerase.

Escherichia coli DksA, GreA, and GreB have similar structures and bind to the same location on RNA polymerase (RNAP), the secondary channel. We show that GreB can fulfil some roles of DksA in vitro, including shifting the promoter-open complex equilibrium in the dissociation direction, thus allowing rRNA promoters to respond to changes in the concentration of ppGpp and NTPs. However, unlike deletion of the dksA gene, deletion of greB had no effect on rRNA promoters in vivo. We show that the apparent affinities of DksA and GreB for RNAP are similar, but the cellular concentration of GreB is much lower than that of DksA. When over-expressed and in the absence of competing GreA, GreB almost completely complemented the loss of dksA in control of rRNA expression, indicating its inability to regulate rRNA transcription in vivo results primarily from its low concentration. In contrast to GreB, the apparent affinity of GreA for RNAP was weaker than that of DksA, GreA affected rRNA promoters only modestly in vitro and, even when over-expressed, GreA did not affect rRNA transcription in vivo. Thus, binding in the secondary channel is necessary but insufficient to explain the effect of DksA on rRNA transcription. Neither Gre factor was capable of fulfilling two other functions of DksA in transcription initiation: co-activation of amino acid biosynthetic gene promoters with ppGpp and compensation for the loss of the omega subunit of RNAP in the response of rRNA promoters to ppGpp. Our results provide important clues to the mechanisms of both negative and positive control of transcription initiation by DksA.

Crl facilitates RNA polymerase holoenzyme formation.

The Escherichia coli Crl protein has been described as a transcriptional coactivator for the stationary-phase sigma factor sigma(S). In a transcription system with highly purified components, we demonstrate that Crl affects transcription not only by the Esigma(S) RNA polymerase holoenzyme but also by Esigma(70) and Esigma(32). Crl increased transcription dramatically but only when the sigma concentration was low and when Crl was added to sigma prior to assembly with the core enzyme. Our results suggest that Crl facilitates holoenzyme formation, the first positive regulator identified with this mechanism of action.

rRNA promoter regulation by nonoptimal binding of sigma region 1.2: an additional recognition element for RNA polymerase.

Regulation of transcription initiation is generally attributable to activator/repressor proteins that bind to specific DNA sequences. However, regulators can also achieve specificity by binding directly to RNA polymerase (RNAP) and exploiting the kinetic variation intrinsic to different RNAP-promoter complexes. We report here a previously unknown interaction with Escherichia coli RNAP that defines an additional recognition element in bacterial promoters. The strength of this sequence-specific interaction varies at different promoters and affects the lifetime of the complex with RNAP. Selection of rRNA promoter mutants forming long-lived complexes, kinetic analyses of duplex and bubble templates, dimethylsulfate footprinting, and zero-Angstrom crosslinking demonstrated that sigma subunit region 1.2 directly contacts the nontemplate strand base two positions downstream of the -10 element (within the "discriminator" region). By making a nonoptimal sigma1.2-discriminator interaction, rRNA promoters create the short-lived complex required for specific responses to the RNAP binding factors ppGpp and DksA, ultimately accounting for regulation of ribosome synthesis.

Response of RNA polymerase to ppGpp: requirement for the omega subunit and relief of this requirement by DksA.

Previous studies have come to conflicting conclusions about the requirement for the omega subunit of RNA polymerase in bacterial transcription regulation. We demonstrate here that purified RNAP lacking omega does not respond in vitro to the effector of the stringent response, ppGpp. DksA, a transcription factor that works in concert with ppGpp to regulate rRNA expression in vivo and in vitro, fully rescues the ppGpp-unresponsiveness of RNAP lacking omega, likely explaining why strains lacking omega display a stringent response in vivo. These results demonstrate that omega plays a role in RNAP function (in addition to its previously reported role in RNAP assembly) and highlight the importance of inclusion of omega in RNAP purification protocols. Furthermore, these results suggest that either one or both of two short segments in the beta' subunit that physically link omega to the ppGpp-binding region of the enzyme may play crucial roles in ppGpp and DksA function.

rRNA transcription in Escherichia coli.

Ribosomal RNA transcription is the rate-limiting step in ribosome synthesis in bacteria and has been investigated intensely for over half a century. Multiple mechanisms ensure that rRNA synthesis rates are appropriate for the cell's particular growth condition. Recently, important advances have been made in our understanding of rRNA transcription initiation in Escherichia coli. These include (a) a model at the atomic level of the network of protein-DNA and protein-protein interactions that recruit RNA polymerase to rRNA promoters, accounting for their extraordinary strength; (b) discovery of the nonredundant roles of two small molecule effectors, ppGpp and the initiating NTP, in regulation of rRNA transcription initiation; and (c) identification of a new component of the transcription machinery, DksA, that is absolutely required for regulation of rRNA promoter activity. Together, these advances provide clues important for our molecular understanding not only of rRNA transcription, but also of transcription in general.

NTP-sensing by rRNA promoters in Escherichia coli is direct.

We showed previously that rrn P1 promoters require unusually high concentrations of the initiating nucleoside triphosphates (ATP or GTP, depending on the promoter) for maximal transcription in vitro. We proposed that this requirement for high initiating NTP concentrations contributes to control of the rrn P1 promoters from the seven Escherichia coli rRNA operons. However, the previous studies did not prove that variation in NTP concentration affects rrn P1 promoter activity directly in vivo. Here, we create conditions in vivo in which ATP and GTP concentrations are altered in opposite directions relative to one another, and we show that transcription from rrn P1 promoters that initiate with either ATP or GTP follows the concentration of the initiating NTP for that promoter. These results demonstrate that the effect of initiating NTP concentration on rrn P1 promoter activity in vivo is direct. As predicted by a model in which homeostatic control of rRNA transcription results, at least in part, from sensing of NTP concentrations by rrn P1 promoters, we show that inhibition of protein synthesis results in an increase in ATP concentration and a corresponding increase in transcription from rrnB P1. We conclude that translation is a major consumer of purine NTPs, and that NTP-sensing by rrn P1 promoters serves as a direct regulatory link between translation and ribosome synthesis.

The RNA polymerase alpha subunit from Sinorhizobium meliloti can assemble with RNA polymerase subunits from Escherichia coli and function in basal and activated transcription both in vivo and in vitro.

Sinorhizobium meliloti, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family. To facilitate our studies of transcription in S. meliloti, we cloned and characterized the gene for the alpha subunit of RNA polymerase (RNAP). S. meliloti rpoA encodes a 336-amino-acid, 37-kDa protein. Sequence analysis of the region surrounding rpoA identified six open reading frames that are found in the conserved gene order secY (SecY)-adk (Adk)-rpsM (S13)-rpsK (S11)-rpoA (alpha)-rplQ (L17) found in the alpha-proteobacteria. In vivo, S. meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E. coli rpoA, demonstrating that S. meliloti alpha supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of E. coli. In vitro, we reconstituted RNAP holoenzyme from S. meliloti alpha and E. coli beta, beta', and sigma subunits. Similar to E. coli RNAP, the hybrid RNAP supported transcription from an E. coli core promoter and responded to both upstream (UP) element- and Fis-dependent transcription activation. We obtained similar results using purified RNAP from S. meliloti. Our results demonstrate that S. meliloti alpha functions are conserved in heterologous host E. coli even though the two alpha subunits are only 51% identical. The ability to utilize E. coli as a heterologous system in which to study the regulation of S. meliloti genes could provide an important tool for our understanding and manipulation of these processes.

rRNA promoter activity in the fast-growing bacterium Vibrio natriegens.

The bacterium Vibrio natriegens can double with a generation time of less than 10 min (R. G. Eagon, J. Bacteriol. 83:736-737, 1962), a growth rate that requires an extremely high rate of protein synthesis. We show here that V. natriegens' high potential for protein synthesis results from an increase in ribosome numbers with increasing growth rate, as has been found for other bacteria. We show that V. natriegens contains a large number of rRNA operons, and its rRNA promoters are extremely strong. The V. natriegens rRNA core promoters are at least as active in vitro as Escherichia coli rRNA core promoters with either E. coli RNA polymerase (RNAP) or V. natriegens RNAP, and they are activated by UP elements, as in E. coli. In addition, the E. coli transcription factor Fis activated V. natriegens rrn P1 promoters in vitro. We conclude that the high capacity for ribosome synthesis in V. natriegens results from a high capacity for rRNA transcription, and the high capacity for rRNA transcription results, at least in part, from the same factors that contribute most to high rates of rRNA transcription in E. coli, i.e., high gene dose and strong activation by UP elements and Fis.