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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 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.

▪ Abstract 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.

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.

Although more than 30 Escherichia coli promoters utilize the RNA polymerase holoenzyme containing sigmaS (EsigmaS), and it is known that there is some overlap between the promoters recognized by EsigmaS and by the major E. coli holoenzyme (Esigma70), the sequence elements responsible for promoter recognition by EsigmaS are not well understood. To define the DNA sequences recognized best by EsigmaS in vitro, we started with random DNA and enriched for EsigmaS promoter sequences by multiple cycles of binding and selection. Surprisingly, the sequences selected by EsigmaS contained the known consensus elements (-10 and -35 hexamers) for recognition by Esigma70. Using genetic and biochemical approaches, we show that EsigmaS and Esigma70 do not achieve specificity through 'best fit' to different consensus promoter hexamers, the way that other forms of holoenzyme limit transcription to discrete sets of promoters. Rather, we suggest that EsigmaS-specific promoters have sequences that differ significantly from the consensus in at least one of the recognition hexamers, and that promoter discrimination against Esigma70 is achieved, at least in part, by the two enzymes tolerating different deviations from consensus. DNA recognition by EsigmaS versus Esigma70 thus presents an alternative solution to the problem of promoter selectivity.

The UP element stimulates transcription from the rrnB P1 promoter through a direct interaction with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). We investigated the effect on transcription from rrnB P1 of varying both the location of the UP element and the length of the alpha subunit interdomain linker, separately and in combination. Displacement of the UP element by a single turn of the DNA helix resulted in a large decrease in transcription from rrnB P1, while displacement by half a turn or two turns totally abolished UP element-dependent transcription. Deletions of six or more amino acids from within the alpha subunit linker resulted in a decrease in UP element-dependent stimulation, which correlated with decreased binding of alphaCTD to the UP element. Increasing the alpha linker length was less deleterious to RNA polymerase function at rrnB P1 but did not compensate for the decrease in activation that resulted from displacing the UP element. Our results suggest that the location of the UP element at rrnB P1 is crucial to its function and that the natural length of the alpha subunit linker is optimal for utilisation of the UP element at this promoter.

CooA is a CO-sensing protein that activates the transcription of genes encoding the CO-oxidation (coo) regulon, whose polypeptide products are required for utilizing CO as an energy source in Rhodospirillum rubrum. CooA binds to a position overlapping the −35 element of the PcooF promoter, similar to the arrangement of class II CRP (cAMP receptor protein)- and FNR (fumarate and nitrate reductase activator protein)-dependent promoters when expressed inEscherichia coli. Gain-of-function CooA variants were isolated in E. coli following mutagenesis of the portion ofcooA encoding the effector-binding domain. Some of the mutations affect regions of CooA that are homologous to the activating regions (AR2 and AR3) previously identified in CRP and FNR, whereas others affect residues that lie in a region of CooA between AR2 and AR3. These CooA variants are comparable to wild-type (WT) CooA in DNA binding affinity in response to CO but differ in transcription activation, presumably because of altered interactions with E. coli RNA polymerase. Based on predictions of similarity to CRP and FNR, loss-of-function CooA variants were obtained in the AR2 and AR3 regions that have minimal transcriptional activity, yet have WT-like DNA binding affinities in response to CO. This study demonstrates that WT CooA contains AR2- and AR3-like surfaces that are required for optimal transcription activation.

To determine the role of ppGpp in both negative and positive regulation of transcription initiation during exponential growth in Escherichia coli, we examined transcription in vivo and in vitro from the growth-rate-dependent rRNA promoter rrnB P1 and from the inversely growth-rate-dependent amino acid biosynthesis/transport promoters PargI, PhisG, PlysC, PpheA, PthrABC, and PlivJ. rrnB P1 promoter activity was slightly higher at all growth-rates in strains unable to synthesize ppGpp (deltarelAdeltaspoT) than in wild-type strains. Consistent with this observation and with the large decrease in rRNA transcription during the stringent response (when ppGpp levels are much higher), ppGpp inhibited transcription from rrnB P1 in vitro. In contrast, amino acid promoter activity was considerably lower in deltarelAdeltaspoT strains than in wild-type strains, but ppGpp had no effect on amino acid promoter activity in vitro. Detailed kinetic analysis in vitro indicated that open complexes at amino acid promoters formed much more slowly and were much longer-lived than rrnB P1 open complexes. ppGpp did not increase the rates of association with, or escape from, amino acid promoters in vitro, consistent with its failure to stimulate transcription directly. In contrast, ppGpp decreased the half-lives of open complexes at all promoters, whether the half-life was seconds (rrnB P1) or hours (amino acid promoters). The results described here and in the accompanying paper indicate that ppGpp directly inhibits transcription, but only from promoters like rrnB P1 that make short-lived open complexes. The results indicate that stimulation of amino acid promoters occurs indirectly. The accompanying paper evaluates potential models for positive control of amino acid promoters by ppGpp that might explain the requirement of ppGpp for amino acid prototrophy.

Strains containing ppGpp, a nucleotide whose synthesis is dependent on the RelA and SpoT proteins of Escherichia coli, display slightly lower rRNA promoter activity and much higher amino acid biosynthesis/transport promoter activity than deltarelAdeltaspoT strains. In the accompanying paper, we show that ppGpp directly inhibits rRNA promoter activity in vitro by decreasing the lifetime of the rrn P1 open complex. However, ppGpp does not stimulate amino acid promoter activity in vitro. We show here that RNA polymerase (RNAP) mutants, selected to confer prototrophy to deltarelAdeltaspoT strains, mimic the effects of ppGpp on wild-type RNAP. Based on the positions of the mutant residues that confer prototrophy in the structure of core RNAP, we suggest molecular models for how the mutants, and by analogy ppGpp, generally decrease the lifetime of open complexes. We show that amino acid promoters require higher concentrations of RNAP for function in vitro and in vivo than control promoters, and are more sensitive to competition for RNAP in vivo than control promoters. Furthermore, we show that the requirement of an amino acid promoter for ppGpp in vivo can be alleviated by increasing its rate-limiting RNAP-binding step. Our data are consistent with a previously proposed passive model in which ppGpp inhibits stable RNA synthesis directly by reducing the lifetime of the rrn P1 open complex, liberating enough RNAP to stimulate transcription from amino acid promoters. Our data also place considerable constraints on models invoking hypothetical factors that might increase amino acid promoter activity in a ppGpp-dependent fashion.

We recently identified Escherichia coli RNA polymerase (RNAP) mutants (RNAP β′ Δ215–220 and β RH454) that form extremely unstable complexes with rRNA P1 ( rrn P1) core promoters. The mutant RNAPs reduce transcription and alter growth rate-dependent regulation of rrn P1 core promoters, because the mutant RNAPs require higher concentrations of the initiating nucleoside triphosphate (NTP) for efficient transcription from these promoters than are present in vivo. Nevertheless, the mutants grow almost as well as wild-type cells, suggesting that rRNA synthesis is not greatly perturbed. We report here that the rrn transcription factor FIS activates the mutant RNAPs more strongly than wild-type RNAP, thereby compensating for the altered properties of the mutant RNAPs. FIS activates the mutant RNAPs, at least in part, by reducing the apparent K ATP for the initiating NTP. This and other results suggest that FIS affects a step in transcription initiation after closed-complex formation in addition to its stimulatory effect on initial RNAP binding. FIS and NTP levels increase with growth rate, suggesting that changing FIS concentrations, in conjunction with changing NTP concentrations, are responsible for growth rate-dependent regulation of rrn P1 transcription in the mutant strains. These results provide a dramatic demonstration of the interplay between regulatory mechanisms in rRNA transcription.

CooA, a member of the cAMP receptor protein (CRP) family, is a CO-sensing transcription activator from Rhodospirillum rubrum that binds specific DNA sequences in response to CO. The location of the CooA-binding sites relative to the start sites of transcription suggested that the CooA-dependent promoters are analogous to class II CRP-dependent promoters. In this study, we developed an in vivo CooA reporter system in Escherichia coli and an in vitro transcription assay using RNA polymerases (RNAP) from E. coli and from Rhodobacter sphaeroides to study the transcription properties of CooA and the protein-protein interaction between CooA and RNAP. The ability of CooA to activate CO-dependent transcription in vivo in heterologous backgrounds suggested that CooA is sufficient to direct RNAP to initiate transcription and that no other factors are required. This hypothesis was confirmed in vitro with purified CooA and purified RNAP. Use of a mutant form of E. coli RNAP with alpha subunits lacking their C-terminal domain (alpha-CTD) dramatically decreased CooA-dependent transcription of the CooA-regulated R. rubrum promoter PcooF in vitro, which indicates that alpha-CTD plays an important role in this activation. DNase I footprinting analysis showed that CooA facilitates binding of wild-type RNAP, but not alpha-CTD-truncated RNAP, to PcooF. This facilitated binding provides evidence for a direct contact between CooA and alpha-CTD of RNAP during activation of transcription. Mapping the CooA-contact site in alpha-CTD suggests that CooA is similar but not identical to CRP in terms of its contact sites to the alpha-CTD at class II promoters.

We have characterized the Chlamydia trachomatis ribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purified C. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous to Escherichia coli promoters recognized by E-ς 70 . We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

The sequence of a promoter determines not only the efficiency with which it forms a complex with RNA polymerase, but also the concentration of nucleoside triphosphate (NTP) required for initiating transcription. Escherichia coli ribosomal RNA ( rrn P1) promoters require high initiating NTP concentrations for efficient transcription because they form unusually short-lived complexes with RNA polymerase; high initiating NTP concentrations [adenosine or guanosine triphosphate (ATP or GTP), depending on the rrn P1 promoter] are needed to bind to and stabilize the open complex. ATP and GTP concentrations, and therefore rrn P1 promoter activity, increase with growth rate. Because ribosomal RNA transcription determines the rate of ribosome synthesis, the control of ribosomal RNA transcription by NTP concentration provides a molecular explanation for the growth rate–dependent control and homeostatic regulation of ribosome synthesis.

Expression of the Escherichia coli adaptive response genes (ada, aidB, and alkA) is regulated by the transcriptional activator, Ada. However, the interactions of RNA polymerase and Ada with these promoters differ. In this report we characterize the interactions of Ada, methylated Ada (meAda), and RNA polymerase at the alkA promoter and contrast these interactions with those characterized previously for the ada and aidB promoters. At the alkA promoter, we do not detect the RNA polymerase alpha subunit-mediated binary complex detected at the ada and aidB promoters. In the presence of either of these two activators, RNA polymerase protects the alkA core promoter, including the elements at -35 and -10, and is more efficient in transcription initiation in vitro. RNA polymerase holoenzyme containing the alpha subunit mutation R265A is severely impaired in Ada-independent basal alkA transcription, shows no activation by Ada or meAda, and fails to bind the alkA promoter in vitro. Binding of the purified wild type alpha subunit to alkA was not detected, but a complex of promoter DNA, Ada or meAda, and alpha was observed in gel shift assays. These observations suggest that both forms of Ada protein activate alkA transcription by enhancing RNA polymerase holoenzyme and alpha subunit binding.

The extraordinary strength of the Escherichia coli rRNA promoter rrnB P1 derives primarily from sequences upstream of the core (-10, -35) region. We find that sequences between -40 and -60 increase the activity of this promoter at least 30-fold in vitro and in vivo. This region, which we refer to as the upstream (UP) element, is located between the -35 consensus hexamer and the previously characterized binding sites for the rRNA transcription factor Fis. The effect of the UP element is independent of Fis in vivo, and independent of any other proteins besides RNA polymerase (RNAP) in vitro. The UP element increases the overall second-order rate constant for association of RNAP with the promoter (ka) and probably the apparent overall first-order isomerization constant (ki). Together with the previously reported protection of the UP element region by RNAP in footprinting experiments, these results indicate that rrnB P1 has an "extended" promoter structure, consisting of the UP element and the core promoter region. We find that the UP element is a separable promoter module that can function to increase the activity of the lac core promoter in an rrnB P1-lac hybrid promoter construct. A functional UP element is not absolutely essential for stimulation of rrnB P1 by the Fis protein.

The P1 promoters of the seven Escherichia coli rRNA operons contain recognition sequences for the RNA polymerase (RNAP) holoenzyme containing sigma 70 (E sigma 70), which has been shown to interact with and initiate transcription from rrn P1 promoters in vivo and in vitro. The rrn P1 promoters also contain putative recognition elements for E sigma 32, the RNAP holoenzyme responsible for the transcription of heat shock genes. Using in vitro transcription assays with purified RNAP holoenzyme, we show that E sigma 32 is able to transcribe from the rrnB P1 promoter. Antibodies specific to sigma 70 eliminate transcription of rrnB P1 by E sigma 70 but have no effect on E sigma 32-directed transcription. Physical characterization of the E sigma 32-rrnB P1 complex shows that there are differences in the interactions made by E sigma 70 and E sigma 32 with the promoter. E sigma 32 responds to both Fis-mediated and factor-independent upstream activation, two systems shown previously to stimulate rrnB P1 transcription by E sigma 70. We find that E sigma 32 is not required for two major control systems known to regulate rRNA transcription initiation at normal temperatures in vivo, stringent control and growth rate-dependent control. On the basis of the well-characterized role of E sigma 32 in transcription from heat shock promoters in vivo, we suggest that E sigma 32-directed transcription of rRNA promoters might play a role in ribosome synthesis at high temperatures.

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.

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.

rRNA synthesis in Escherichia coli is subject to at least two regulation systems, growth rate-dependent control and stringent control. The inverse correlation between rRNA synthesis rates and guanosine 3'-diphosphate 5'-diphosphate (ppGpp) levels under various physiological conditions has led to the supposition that ppGpp is the mediator of both control mechanisms by inhibiting transcription from rrn P1 promoters. Recently, relA- spoT- strains have been constructed in which both ppGpp synthesis pathways most likely have been removed (M. Cashel, personal communication). We have confirmed that such strains produce no detectable ppGpp and therefore offer a direct means for testing the involvement of ppGpp in the regulation of rRNA synthesis in vivo. Stringent control was determined by measurement of rRNA synthesis after amino acid starvation, while growth rate control was determined by measurement of rRNA synthesis under different nutritional conditions. As expected, the relA- spoT- strain is relaxed for stringent control. However, growth rate-dependent regulation is unimpaired. These results indicate that growth rate regulation can occur in the absence of ppGpp and imply that ppGpp is not the mediator, or at least is not the sole mediator, of growth rate-dependent control. Therefore, growth rate-dependent control and stringent control may utilize different mechanisms for regulating stable RNA synthesis.

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 ω 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.

The Escherichia coli Crl protein has been described as a transcriptional coactivator for the stationary-phase sigma factor σ S . In a transcription system with highly purified components, we demonstrate that Crl affects transcription not only by the Eσ S RNA polymerase holoenzyme but also by Eσ 70 and Eσ 32 . Crl increased transcription dramatically but only when the σ concentration was low and when Crl was added to σ 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.

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.

The α-proteobacterium Sinorhizobium meliloti is able to live either as a soil saprophyte or in a symbiotic relationship with members of the legume family, such as alfalfa. Recent studies have focused on understanding how rhizobia adapt to these unique environments, especially at the level of gene expression (6, 13, 62). For the symbiosis to occur, expression of a subset of genes, such as the nod and nif genes, must be tightly regulated (reviewed in reference 22). As is the case with other bacteria, much of the gene regulation occurs at the level of initiation of transcription (28). To facilitate our studies of transcription and its regulation in S. meliloti, we must understand RNA polymerase (RNAP) structure and function. Previous work demonstrated that RNAP from S. meliloti displays the characteristic α2ββ′ core subunit structure found in most bacteria (23, 45). In addition σ70, σ54, and σ 72 homologs have been cloned from S. meliloti (47, 48, 52, 55). These results are consistent with the evidence that bacterial RNAPs display overall sequence and functional similarities, although they can exhibit some differences in individual steps during transcription such as promoter recognition and promoter escape (4). Since only a limited number of S. meliloti promoters have been characterized, the cis-acting elements are not yet as well defined as in Escherichia coli promoters (7, 23, 55). Nevertheless, S. meliloti RNAP can initiate transcription at typical E. coli promoters (19, 23). However, most S. meliloti promoters that have been characterized are not transcribed by E. coli RNAP in vivo or in vitro (5, 19), perhaps because the S. meliloti Eσ70 homolog recognizes these promoters slightly differently from E. coli Eσ70 or because these promoters utilize σ factors or transcription activators not found in E. coli. In the past decade, based primarily on work with E. coli, RNAP α has emerged as a key player in both basal transcription and in transcriptional activation (reviewed in references 20 and 29). RNAP α consists of two independently folded domains connected by a flexible linker (9, 64). The amino-terminal domain (αNTD) is required for α dimerization, for RNAP assembly, and for interaction with a subset of transcription factors (32, 51); the carboxy-terminal domain (αCTD) is required for binding to the upstream (UP) element, an A+T-rich sequence found upstream of the −35 hexamer, and for interaction with a number of transcription factors (35, 53). Several screens for α mutants have identified residues required for the activation of transcription. These α-activator contacts help recruit RNAP to promoters and/or stimulate the isomerization of the RNAP-promoter complex from the closed to the open state (46). Furthermore, the αCTD may also interact with the σCTD during transcription initiation at some promoters (29). In some cases, α-activator contacts appear be species specific, suggesting that α and activators have coevolved. For example, Agrobacterium tumefaciens α is required for VirG-activated transcription of the virB promoter in E. coli (39). Similarly, transcription activation from the Bacillus subtilis phage A3 promoter requires RNAP containing B. subtilis α and is not supported by E. coli RNAP (43). In both cases, the species specificity of the α-activator contact was mapped to the αCTD (40, 43). Interestingly, Bordetella pertussis α reconstituted into E. coli RNAP does not support transcription at the E. coli CAP-dependent lac promoter (58), suggesting that different activator-α specificities may exist, despite striking sequence homologies in the αCTDs of B. pertussis and E. coli. Our ultimate goal is to understand how α interacts with transcription factors to initiate transcription at S. meliloti promoters. In this paper we describe the cloning and characterization of the S. meliloti RNAP α subunit. Furthermore, we establish that S. meliloti α can functionally replace E. coli α in vivo and that S. meliloti α reconstituted into E. coli RNAP holoenzyme can support both UP element- and Fis-dependent transcription in vitro. These results suggest that the study of transcription activation in S. meliloti may be facilitated by utilizing tools developed for E. coli RNAP.

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.

In recent years, it has become clear that promoter recognition by bacterial RNA polymerase involves interactions not only between core promoter elements and the sigma subunit, but also between a DNA element upstream of the core promoter and the alpha subunit. DNA binding by alpha can increase transcription dramatically. Here we review the current state of our understanding of the alpha interaction with DNA during basal transcription initiation (i.e. in the absence of proteins other than RNA polymerase) and activated transcription initiation (i.e. when stimulated by transcription factors).

ProP is an integral membrane transporter of proline, glycine betaine, and several other osmoprotecting compounds. Fis plus RpoS collaborate to promote a burst of proP transcription in late exponential growth phase. This brief period of ProP synthesis enables stationary phase cells to cope with a potential hyperosmotic shock. Fis activates the RpoS (σ 38 )-dependent proP P2 promoter by binding to a site within the promoter region centered at −41 and thus functions as a class II activator. We show here that activation by Fis at this promoter is completely dependent upon the α-CTD of RNA polymerase and that the activation domain on Fis is localized to a four amino acid ridge on the surface of Fis adjacent to the helix-turn-helix DNA binding domain in only one subunit of the homodimer. Fis mutants containing amino acid substitutions within this region are defective in cooperative binding interactions with the σ 38 -form of RNA polymerase. Some of these substitutions also alter interactions with DNA sequences flanking the core binding site, but we show that changes in Fis-mediated curvature do not affect promoter activity. We conclude that the same amino acids are used by Fis to activate transcription from a class I (−71, rrnB P1) and class II (−41, proP P2) location, but this region is distinct from that required to regulate the Hin site-specific DNA inversion reaction.

We demonstrate here that the previously described bacterial promoter upstream element (UP element) consists of two distinct subsites, each of which, by itself, can bind the RNA polymerase holoenzyme alpha subunit carboxy-terminal domain (RNAP alphaCTD) and stimulate transcription. Using binding-site-selection experiments, we identify the consensus sequence for each subsite. The selected proximal subsites (positions -46 to -38; consensus 5'-AAAAAARNR-3') stimulate transcription up to 170-fold, and the selected distal subsites (positions -57 to -47; consensus 5'-AWWWWWTTTTT-3') stimulate transcription up to 16-fold. RNAP has subunit composition alpha(2)betabeta'sigma and thus contains two copies of alphaCTD. Experiments with RNAP derivatives containing only one copy of alphaCTD indicate, in contrast to a previous report, that the two alphaCTDs function interchangeably with respect to UP element recognition. Furthermore, function of the consensus proximal subsite requires only one copy of alphaCTD, whereas function of the consensus distal subsite requires both copies of alphaCTD. We propose that each subsite constitutes a binding site for a copy of alphaCTD, and that binding of an alphaCTD to the proximal subsite region (through specific interactions with a consensus proximal subsite or through nonspecific interactions with a nonconsensus proximal subsite) is a prerequisite for binding of the other alphaCTD to the distal subsite.

The UP element, a component of bacterial promoters located upstream of the −35 hexamer, increases transcription by interacting with the RNA polymerase α-subunit. By using a modification of the SELEX procedure for identification of protein-binding sites, we selected in vitro and subsequently screened in vivo for sequences that greatly increased promoter activity when situated upstream of the Escherichia coli rrnB P1 core promoter. A set of 31 of these upstream sequences increased transcription from 136- to 326-fold in vivo , considerably more than the natural rrnB P1 UP element, and was used to derive a consensus sequence: −59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn −38. The most active selected sequence contained the derived consensus, displayed all of the properties of an UP element, and the interaction of this sequence with the α C-terminal domain was similar to that of previously characterized UP elements. The identification of the UP element consensus should facilitate a detailed understanding of the α–DNA interaction. Based on the evolutionary conservation of the residues in α responsible for interaction with UP elements, we suggest that the UP element consensus sequence should be applicable throughout eubacteria, should generally facilitate promoter prediction, and may be of use for biotechnological applications.

Mutations in Escherichia coli rpoB or rpoC, selected for the ability to confer prototrophy on relA spoT strains, were found to affect transcription from rrn P1 promoters. Two mutant strains (beta RH454 and beta' delta 215-220) reduced transcription of rrn P1 core promoter-lacZ fusions but not of control promoter-lacZ fusions. Purified mutant RNAPs formed complexes with rrn P1 promoters that were much less stable than those formed by wild-type RNAP and required high concentrations of the initiating NTP for efficient rrn P1 transcription. The instability of the rrn P1 core promoter complexes with the mutant RNAPs and their altered regulatory properties support a recently proposed model for the control of rRNA transcription by changing concentrations of the initiating NTPs. We further suggest that destabilization of promoter complexes by the mutant RNAPs mimics effects of ppGpp, decreasing or increasing transcription depending on the kinetic properties of the specific promoter.

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRP-dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase alpha subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within alphaCTD for transcription activation at a Class II CRP-dependent promoter. Our results indicate that Class II CRP-dependent transcription requires the side chains of residues 265, 271, 285-288 and 317. Residues 285-288 and 317 comprise a discrete 20x10 A surface on alphaCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between alpha subunits and CRP, but do not affect DNA binding by alpha subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in alphaCTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for alphaCTD-CRP interaction in Class I CRP-dependent transcription.

FIS, a site-specific DNA binding and bending protein, is a global regulator of gene expression in Escherichia coli. The ribosomal RNA promoter rrnB P1 is activated 3- to 7-fold in vivo by a FIS dimer that binds a DNA site immediately upstream of the DNA binding site for the C-terminal domain (CTD) of the alpha subunit of RNA polymerase (RNAP). In this report, we identify several FIS side chains important specifically for activation of transcription at rrnB P1. These side chains map to positions 68, 71 and 74, in and flanking a surface-exposed loop adjacent to the helix-turn-helix DNA binding motif of the protein. We also present evidence suggesting that FIS activates transcription at rrnB P1 by interacting with the RNAP alphaCTD. Our results suggest a model for FIS-mediated activation of transcription at rrnB P1 that involves interactions between FIS and the RNAP alphaCTD near the DNA surface. Although FIS and the transcription activator protein CAP have little structural similarity, they both bend DNA, use a similarly disposed activation loop and target the same region of the RNAP alphaCTD, suggesting that this is a common architecture at bacterial promoters.

In rapidly growing bacterial cultures, cells contain about 70000 ribosomes, each of which is constructed from more than 50 ribosomal proteins (r-proteins) and three ribosomal RNAs (rRNAs) (reviewed by Condon et al. 1995; Gourse et al. 1996; Keener and Nomura 1996). Under these growth conditions, the largest single expenditure of the cell’s biosynthetic energy is the synthesis of ribosomes. The ribosomal protein synthesis rate is ultimately controlled by the level of rRNA, primarily by a translational feedback regulation system. Therefore, the rate-limiting step in ribosome synthesis is the synthesis of rRNA which in turn is limited by the rate of transcription initiation. In order to make the large number of ribosomes present at high growth rates, the promoters for the seven E. coli rRNA operons transcribe well over half the cell’s total RNA (Bremer and Dennis 1987). The special mechanisms that make rRNA promoters so active will be a major focus of this chapter.

The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli. We have identified mutants of the FIS protein resulting in reduced rrnB P1 transcription activation that nevertheless retain the ability to bind DNA in vivo. The mutations map to amino acid 74, the N-terminal amino acid of the protein's helix-turn-helix DNA binding motif, and to amino acids 71 and 72 in the adjoining surface-exposed loop. In vitro analyses of one of the activation-defective mutants (with a G-to-S mutation at position 72) indicates that it binds to and bends rrnB P1 FIS site I DNA the same as wild-type FIS. These data suggest that amino acids in this region of FIS are required for transcription activation by contacting RNA polymerase directly, independent of any other role(s) this region may play in DNA binding or protein-induced bending.

The Escherichia coli RNA polymerase alpha-subunit binds through its carboxy-terminal domain (alpha CTD) to a recognition element, the upstream (UP) element, in certain promoters. We used genetic and biochemical techniques to identify the residues in alpha CTD important for UP-element-dependent transcription and DNA binding. These residues occur in two regions of alpha CTD, close to but distinct from, residues important for interactions with certain transcription activators. We used NMR spectroscopy to determine the secondary structure of alpha CTD, alpha CTD contains a nonstandard helix followed by four alpha-helices. The two regions of alpha CTD important for DNA binding correspond to the first alpha-helix and the loop between the third and fourth alpha-helices. The alpha CTD DNA-binding domain architecture is unlike any DNA-binding architecture identified to date, and we propose that alpha CTD has a novel mode of interaction with DNA. Our results suggest models for alpha CTD-DNA and alpha CTD-DNA-activator interactions during transcription initiation.

Escherichia coli uses at least two regulatory systems, stringent control and growth-rate-dependent control, to adjust rRNA output to amino acid availability and the steady-state growth rate, respectively. We examined transcription from rrnB P1 promoters containing or lacking the cis-acting UP element and FIS protein binding sites after amino acid starvation. The "core promoter" responds to amino acid starvation like the full-length wild-type promoter; thus, neither the UP element nor FIS plays a role in stringent control. To clarify the relationship between growth-rate-dependent regulation and stringent control, we measured transcription from growth-rate-independent promoters during amino acid starvation. Four rrnB P1 mutants defective for growth-rate control and two other growth-rate-independent promoters (rrnB P2 and pS10) still displayed stringent regulation. Thus, the two systems have different promoter determinants, consistent with the idea that they function by different mechanisms. Two mutations disrupted stringent control of rrnB P1: (i) a multiple base change in the "discriminator" region between the -10 hexamer and the transcription start site and (ii) a double substitution making the promoter resemble the E sigma 70 consensus promoter. These results have important implications for the mechanisms of both stringent control and growth-rate-dependent control of rRNA transcription.

DNA sequences upstream of the rrnB P1 core promoter (-10, -35 region) increase transcription more than 300-fold in vivo and in vitro. This stimulation results from a cis-acting DNA sequence, the UP element, which interacts directly with the alpha subunit of RNA polymerase, increasing transcription about 30-fold, and from a positively acting transcription factor, FIS, which increases expression another 10-fold. A DNA region exhibiting a high degree of intrinsic curvature has been observed upstream of the rrnB P1 core promoter and has thus been often cited as an example of the effect of bending on transcription. However, the precise position of the curvature has not been determined. We address here whether this bend is in fact related to activation of rRNA transcription. Electrophoretic analyses were used to localize the major bend in the rrnB P1 upstream region to position approximately -100 with respect to the transcription initiation site. Since most of the effect of upstream sequences on transcription results from DNA between the -35 hexamer and position -88, i.e. downstream of the bend center, these studies indicate that the curvature leading to the unusual electrophoretic behavior of the upstream region does not play a major role in activation of rRNA transcription. Minor deviations from normal electrophoretic behavior were associated with the region just upstream of the -35 hexamer and could conceivably influence interactions between the UP element and the alpha subunit of RNA polymerase.

The role of naturally occurring D in living organisms has been examined by using deuterium-depleted water (30–40 ppm D) instead of water containing the natural abundance of D (150 ppm). The deuterium-depleted water significantly decreased the growth rate of the L929 fibroblast cell line, and also inhibited the tumor growth in xenotransplanted mice. Eighty days after transplantation in 10 (59%) out of 17 tumorous mice the tumor, after having grown, regressed and then disappeared. We suggest that the naturally occurring D has a central role in signal transduction involved in cell cycle regulation.

Transcription from Escherichia coli ribosomal RNA promoters is increased about 20-fold in vivo by a DNA sequence (the Upstream Activation Region, UAR) located upstream of the -35 conserved hexamer. The UAR stimulates transcription through two mechanisms: one which involves binding of the Fis protein to the UAR, and another mechanisms which functions in the absence of additional protein factors. We have previously constructed a collection of mutations in the region upstream of the -35 hexamer of rrnB P1. Most of these mutations have either no effect on promoter activity or decrease activity 2-5-fold in vivo (Gaal, T., Barkei, J., Dickson, R.R., De Boer, H.A., De Haseth, P.L., Alavi, H. and Gourse, R.L.(1989) J. Bacteriol. 171, 4852-4861). Two mutations leave both the -35 consensus hexamer and the Fis binding consensus sequence intact, yet have larger (14-50-fold) effects on transcription. One substitution just upstream of the -35 hexamer (a C to T change at position -37) primarily affects intrinsic promoter strength, leaving the UAR functional. On the other hand, a three base pair deletion (bases -38 through -40) severely reduces UAR-mediated activity. A substitution covering the three base pair deletion was constructed and found to be activated normally. UAR function appears dependent on its position relative to the RNA polymerase binding site, suggesting that a particular spatial geometry may be necessary for Fis-dependent and/or factor-independent activation to occur.

We measured the activities of 50 operon fusions from a collection of mutant and wild-type rrnB P1 (rrnB1p in the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoters under different nutritional conditions in order to analyze the DNA sequence determinants of growth rate-dependent regulation of rRNA transcription in Escherichia coli. Mutants which deviated from the wild-type -10 or -35 hexamers or from the wild-type 16-base-pair spacer length between the hexamers were unregulated, regardless of whether the mutations brought the promoters closer to the E. coli promoter consensus sequence and increased activity or whether the changes took the promoters further away from the consensus and reduced activity. These data suggest that rRNA promoters have evolved to maintain their regulatory abilities rather than to maximize promoter strength. Some double substitutions outside the consensus hexamers were almost completely unregulated, while single substitutions at several positions outside the -10 and -35 consensus hexamers exerted smaller but significant effects on regulation. These studies suggest roles for specific promoter sequences and/or structures in interactions with regulatory molecules and suggest experimental tests for models of rRNA regulation.

Ribosomal RNA promoters of Escherichia coli are probably the strongest promoters in vivo and they can be used on plasmid vectors to express protein-coding sequences at a high rate. In fact, the P2 promoter of the rrnB gene is stronger (in vivo) than the tac promoter, which has a perfect consensus sequence. Conversion of the rrnBP2 promoter sequence to consensus significantly increases in vivo promoter strength. The removal of four nucleotides downstream of the -10 region also increases the strength of this promoter. On the other hand, shifting of the A + T-rich region upstream of this promoter by an 11-bp insertion drastically decreases in vivo activity. It is concluded that the two functionally important hexanucleotide sequences, -35 and -10, are necessary but not sufficient factors for the optimalization of in vivo promoter strength.

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.

Using oligonucleotide synthesis techniques, we generated Escherichia coli rrnB P1 (rrnB1p according to the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoter fragments containing single base substitutions, insertions, deletions, and multiple mutations, covering the whole length of the promoter including the upstream activation sequence (UAS). The activities of 112 mutant promoters were assayed as operon fusions to lacZ in lambda lysogens. The activities of most mutants with changes in the core promoter recognition region (i.e., substitutions, insertions, or deletions in the region of the promoter spanning the -10 and -35 E. coli consensus hexamers) correlated with changes toward or away from the consensus in the hexamer sequences or in the spacing between them. However, changes at some positions in the core promoter region not normally associated with transcriptional activity in other systems also had significant effects on rrnB P1. Since rRNA promoter activity varies with cellular growth rate, changes in activity can be the result of changes in promoter strength or of alterations in the regulation of the promoter. The accompanying paper (R. R. Dickson, T. Gaal, H. A. deBoer, P. L. deHaseth, and R. L. Gourse, J. Bacteriol. 171:4862-4870, 1989) distinguishes between these two alternatives. Several mutations in the UAS resulted in two- to fivefold reductions in activity. However, two mutants with changes just upstream of the -35 hexamer in constructs containing the UAS had activities 20- to 100-fold lower than the wild-type level. This collection of mutant rRNA promoters should serve as an important resource in the characterization of the mechanisms responsible for upstream activation and growth rate-dependent regulation of rRNA transcription.