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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.

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.

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 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.

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.

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.

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.

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.