The synthesis of ribosomal RNA is the rate-limiting step in ribosome synthesis in bacteria. There are multiple mechanisms that determine the rate of rRNA synthesis. Ribosomal RNA promoter sequences have evolved for exceptional strength and for regulation in response to nutritional conditions and amino acid availability. Strength derives in part from an extended RNA polymerase (RNAP) recognition region involving at least two RNAP subunits, in part from activation by a transcription factor and in part from modification of the transcript by a system that prevents premature termination. Regulation derives from at least two mechanistically distinct systems, growth rate-dependent control and stringent control. The mechanisms contributing to rRNA transcription work together and compensate for one another when individual systems are rendered inoperative.

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The HAMP linker, a predicted structural element observed in sensor proteins from all domains of life, is proposed to transmit signals between extracellular sensory input domains and cytoplasmic output domains. HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) linkers are located just inside the cytoplasmic membrane and are projected to form two short amphipathic ␣-helices (AS-1 and AS-2) joined by an unstructured connector. The presumed helices are comprised of hydrophobic residues in heptad repeats, with only three positions exhibiting strong conservation. We generated missense mutations at these three positions and throughout the HAMP linker in the Escherichia coli nitrate sensor kinase NarX and screened the resulting mutants for defective responses to nitrate. Most missense mutations in this region resulted in a constitutive phenotype mimicking the ligand-bound state, and only one residue (a conserved Glu before AS-2) was essential for HAMP linker function. We also scanned the narX HAMP linker with an overlapping set of seven-residue deletions. Deletions in AS-1 and the connector resulted in constitutive phenotypes. Two deletions in AS-2 resulted in a novel reversed response phenotype in which the response to ligand was the opposite of that seen for the narX ؉ strain. These observations are consistent with the proposed HAMP linker structure, show that the HAMP linker plays an active role in transmembrane signal transduction, and indicate that the two amphipathic ␣-helices have different roles in signal transduction.Two-component regulatory systems are used by all domains of life to sense and respond to environmental changes (37). These systems stereotypically consist of a sensor kinase and a cytoplasmic response regulator. One prototypical class of sensor kinases shares the structure of methyl-accepting chemotaxis proteins (MCPs), which are homodimers located in the cytoplasmic membrane. This structure consists of a short amino-terminal cytoplasmic segment, a transmembrane ␣-helix (TM-1), an external ligand-binding sensory input domain, a second transmembrane ␣-helix (TM-2), and a cytoplasmic output module (13).Sequence comparisons and mutational analysis of sensor kinases and MCPs identified a common structural element hypothesized to transmit signals between external input domains and cytoplasmic output modules (2,17,42). This element is called the HAMP (histidine kinase, adenylyl cyclase, MCP, and phosphatase) linker, or the P-type linker. More than 95% of HAMP linkers identified by similarity to the Escherichia coli EnvZ HAMP linker are from sensor kinases or MCPs; all five MCPs and 15 of the 30 known sensor kinases of E. coli contain HAMP linkers (Fig. 1) (2).The HAMP linker comprises the approximately 50 amino acyl residues distal to TM-2. This region includes two sequences of hydrophobic amino acids in a heptameric arrangement, with phased spacing of hydrophobic residues characteristic of amphipathic ␣-helices. These sequences have been named amphipathic se...

The HAMP linker, a predicted structural element observed in many sensor kinases and methyl-accepting chemotaxis proteins, transmits signals between sensory input modules and output modules. HAMP linkers are located immediately inside the cytoplasmic membrane and are predicted to form two short amphipathic ␣-helices (AS-1 and AS-2) joined by an unstructured connector. HAMP linkers are found in the Escherichia coli nitrate-and nitrite-responsive sensor kinases NarX and NarQ (which respond to ligand by increasing kinase activity) and the sensor kinase CpxA (which responds to ligand by decreasing kinase activity). We constructed a series of hybrids with fusion points throughout the HAMP linker, in which the sensory modules of NarX or NarQ are fused to the transmitter modules of NarX, NarQ, or CpxA. A hybrid of the NarX sensor module and the CpxA HAMP linker and transmitter module (NarX-CpxA-1) responded to nitrate by decreasing kinase activity, whereas a hybrid in which the HAMP linker of NarX was replaced by that of CpxA (NarX-CpxANarX-1) responded to nitrate by increasing kinase activity. However, sequence variations between HAMP linkers do not allow free exchange of HAMP linkers or their components. Certain deletions in the NarX HAMP linker resulted in characteristic abnormal responses to ligand; similar deletions in the NarQ and NarX-CpxA-1 HAMP linkers resulted in responses to ligand generally similar to those seen in NarX. We conclude that the structure and action of the HAMP linker are conserved and that the HAMP linker transmits a signal to the output domain that ligand is bound.Two-component regulatory systems are used by all domains of life to sense and respond to environmental changes (48). Many of the sensor components of two-component regulatory systems and most methyl-accepting chemotaxis proteins (MCPs) share a common molecular architecture. These proteins are dimers in the cytoplasmic membrane and have a modular structure. The sensory module of each monomer consists of a short, amino-terminal cytoplasmic tail, a transmembrane ␣-helix (TM-1), a periplasmic domain which may interact with a specific ligand, a second transmembrane ␣-helix (TM-2), and, in many but not all sensor proteins, a HAMP linker. The cytoplasmic output module interacts with a response regulator (14). Signal transduction requires that information about ligand binding in the exterior sensory input module of the protein cross the cytoplasmic membrane and regulate the activity of the output module in the interior of the cell. The output modules of MCPs interact with an associated soluble histidine kinase, CheA, whereas those of the two-component sensors are histidine kinases in equilibrium between kinase and phosphatase activities.Many of these sensors appear to use a common structural element for signal transduction between external inputs and cytoplasmic outputs. The HAMP (histidine kinase, adenylyl cyclase, MCP, and phosphatase) linker or P-type linker is predicted to be a structure of approximately 50 aminoacyl residues connecting ...

The seven rRNA operons in Escherichia coli each contain two promoters, rrn P1 and rrn P2. Most previous studies have focused on the rrn P1 promoters. Here we report a systematic analysis of the activity and regulation of the rrnB P2 promoter in order to define the intrinsic properties of rrn P2 promoters and to understand better their contributions to rRNA synthesis when they are in their natural setting downstream of rrn P1 promoters. In contrast to the conclusions reached in some previous studies, we find that rrnB P2 is regulated: it displays clear responses to amino acid availability (stringent control), rRNA gene dose (feedback control), and changes in growth rate (growth rate-dependent control). Stringent control of rrnB P2 requires the alarmone ppGpp, but growth rate-dependent control of rrnB P2 does not require ppGpp. The rrnB P2 core promoter sequence (؊37 to ؉7) is sufficient to serve as the target for growth rate-dependent regulation.To meet the requirement for protein synthesis as nutritional conditions change, bacteria modulate ribosome synthesis primarily by controlling transcription initiation from rRNA promoters. Escherichia coli contains seven rRNA operons (rrnA, -B, -C, -D, -E, -G, and -H), each of which has two promoters, rrn P1 and rrn P2, separated by ϳ120 bp (16). The rrn P1 and rrn P2 promoters have many sequence characteristics in common: near consensus Ϫ10 and Ϫ35 hexamers, separated by 16 bp, that bind the subunit of RNA polymerase (RNAP) (16); an AϩT-rich region upstream of the Ϫ35 hexamer (UP element) that increases transcription by binding the C-terminal domains of the ␣ subunits of RNAP (27, 28); and a GϩC-rich region (the discriminator) (38) between the Ϫ10 hexamer and the transcription start site that is required for proper regulation (4, 8, 15, 25) (see Fig. 1 for the rrnB P2 sequence).Regulation of rRNA transcription initiation is often analyzed in terms of the responses of rRNA promoters to three experimental situations. First, rrn P1 activity changes in proportion to the steady-state growth rate (growth rate-dependent control [12, 22; reviewed in reference 13]), coordinating the number of ribosomes with the need for protein synthesis. Second, rrn P1 activity changes in inverse proportion to changes in rRNA gene dose (feedback regulation [14; reviewed in reference 13]), maintaining ribosome synthesis homeostatically. Third, relA-dependent inhibition of rrn P1 activity is observed following amino acid starvation (stringent control [reviewed in reference 6]), preventing an overinvestment of energy in ribosome synthesis when the substrates for protein synthesis are unavailable.The literature is less clear about the regulation of the rrn P2 promoters. Early studies indicated that rrn P2 promoters are growth rate dependent (but less so than rrn P1 promoters) and not stringently controlled (31-33), but subsequent studies suggested the opposite, that rrn P2 promoters are not growth rate dependent (12,40) and are stringently controlled (11,15,19). Studies employing rrnB P2-lacZ or rrn...