The dual role of DksA protein in the regulation of Escherichia coli pArgX promoter.

Gene expression regulation by the stringent response effector, ppGpp, is facilitated by DksA protein; however DksA and ppGpp can play independent roles in transcription. In Escherichia coli, the pArgX promoter which initiates the transcription of four tRNA genes was shown to be inhibited by ppGpp. Our studies on the role of DksA in pArgX regulation revealed that it can stimulate transcription by increasing the binding of RNA polymerase to the promoter and the productive transcription complex formation. However, when DksA is present together with ppGpp a severe down-regulation of promoter activity is observed. Our results indicate that DksA facilitates the effects of ppGpp to drive formation of inactive dead-end complexes formed by RNA polymerase at the ArgX promoter. In vivo, ppGpp-mediated regulation of pArgX transcription is dependent on DksA activity. The potential mechanisms of opposing pArgX regulation by ppGpp and DksA are discussed. pArgX is the first reported example of the promoter stimulated by DksA and inhibited by ppGpp in vitro when an overall inhibition occurs in the presence of both regulators. A dual role is thus proposed for DksA in the regulation of the pArgX promoter activity.

Putrescine catabolism is a metabolic response to several stresses in Escherichia coli.

Genes whose products degrade arginine and ornithine, precursors of putrescine synthesis, are activated by either regulators of the nitrogen-regulated (Ntr) response or σ(S) -RNA polymerase. To determine if dual control regulates a complete putrescine catabolic pathway, we examined expression of patA and patD, which specify the first two enzymes of one putrescine catabolic pathway. Assays of PatA (putrescine transaminase) activity and ß-galactosidase from cells with patA-lacZ transcriptional and translational fusions indicate dual control of patA transcription and putrescine-stimulated patA translation. Similar assays for PatD indicate that patD transcription required σ(S) -RNA polymerase, and Nac, an Ntr regulator, enhanced the σ(S) -dependent transcription. Since Nac activation via σ(S) -RNA polymerase is without precedent, transcription with purified components was examined and the results confirmed this conclusion. This result indicates that the Ntr regulon can intrude into the σ(S) regulon. Strains lacking both polyamine catabolic pathways have defective responses to oxidative stress, high temperature and a sublethal concentration of an antibiotic. These defects and the σ(S) -dependent expression indicate that polyamine catabolism is a core metabolic response to stress.

SRBC/cavin-3 is a caveolin adapter protein that regulates caveolae function.

Caveolae are a major membrane domain common to most cells. One of the defining features of this domain is the protein caveolin. The exact function of caveolin, however, is not clear. One possible function is to attract adapter molecules to caveolae in a manner similar to how clathrin attracts molecules to coated pits. Here, we characterize a candidate adapter molecule called SRBC. SRBC binds PKCdelta and is a member of the STICK (substrates that interact with C-kinase) superfamily of PKC-binding proteins. We also show it co-immunoprecipitates with caveolin-1. A leucine zipper in SRBC is essential for both co-precipitation with caveolin and localization to caveolae. SRBC remains associated with caveolin when caveolae bud to form vesicles (cavicles) that travel on microtubules to different regions of the cell. In the absence of SRBC, intracellular cavicle traffic is markedly impaired. We conclude that SRBC (sdr-related gene product that binds to c-kinase) and two other family members [PTRF (Pol I and transcription release factor) and SDPR] function as caveolin adapter molecules that regulate caveolae function.

Conversion of active promoter-RNA polymerase complexes into inactive promoter bound complexes in E. coli by the transcription effector, ppGpp.

Guanosine tetraphosphate (ppGpp) is a signal of nutritional stress that regulates transcription. An RNA polymerase rudder mutant rpoC (Delta 312-315) is found to suppress ppGpp deficiency phenotypes by restoring both negative and positive activities of promoter fusions in vivo, as if ppGpp were present. Measurements of defects in transcription of the PargT tRNA promoter with mutant RNA polymerase reveal that the mutant enzyme quantitatively mimics the presence of added ppGpp. DNaseI footprints and mobility shifts under RNA polymerization conditions reveal that the promoter-specific transcription defect of the mutant enzyme can be ascribed to the presence of inactive dead-end promoter complexes with features similar to those of a stable closed complex. We propose that formation of such inactive complexes represents an alternative explanation of "stringent RNA polymerase" mutant behavior to those currently published, and it represents a newly discovered mode of action of ppGpp.

Direct stimulation of the lambdapaQ promoter by the transcription effector guanosine-3',5'-(bis)pyrophosphate in a defined in vitro system.

The bacterial response to nutritional deprivation, called the stringent response, results in the introduction of the specific nucleotide guanosine-3',5'-(bis) pyrophosphate (ppGpp). This nucleotide interacts with RNA polymerase and alters its action so that transcription from certain promoters is inhibited, whereas transcription from others seems to be activated. The exact mechanism of transcriptional stimulation by ppGpp in vivo remains unknown. A passive control model has been proposed according to which transcription inhibition during the stringent response at several very active promoters, like those for rRNA and tRNA genes, makes more free RNA polymerase (RNAP) molecules available for transcription at promoters with weak binding affinities for RNAP, thus leading to their passive activation. Among promoters whose transcription is activated by ppGpp in vivo is the histidine operon promoter (hisGp). However, in vitro it is only possible to demonstrate this effect in a coupled transcription-translation system. Here we demonstrate, using another in vivo ppGpp-stimulated promoter, the phage lambdapaQ promoter, that activation by ppGpp in a defined in vitro system is direct. A systematic study of ppGpp effects on the stimulation of paQ revealed that, as in the case of promoters inhibited by this nucleotide, ppGpp decreases the half-life of paQ open complexes. Our results also indicate that the equilibrium binding affinity of RNA polymerase to paQ seems not to be affected in the presence of ppGpp. Our data indicate that the mechanism underlying ppGpp stimulation of paQ is due to an increased rate of productive open complex formation.

Inducible expression, enzymatic activity, and origin of higher plant homologues of bacterial RelA/SpoT stress proteins in Nicotiana tabacum.

All living cells possess adaptive responses to environmental stress that are essential to ensuring cell survival. For motile organisms, this can culminate in avoidance or attractile behavior, but for sessile organisms such as plants, stress adaptation is a process of success or failure within the confines of a given environment. Nearly all bacterial species possess a highly evolved system for stress adaptation, known as the "stringent response." This ancient and ubiquitous regulatory response is mediated by production of a second messenger of general stress, the nucleotide guanosine-3',5'-(bis)pyrophosphate (ppGpp), which mediates reprogramming of the global transcriptional output of the cell. Accumulation of ppGpp is stress-induced through the enzymatic activation of the well known bacterial ppGpp synthetases, RelA and SpoT. We have recently discovered homologues of bacterial relA/spoT genes in the model plant Nicotiana tabacum. We hypothesize that these homologues (designated RSH genes for RelA/SpoT homologues) serve a stress-adaptive function in plants analogous with their function in bacteria. In support of this hypothesis, we find 1) inducibility of tobacco RSH gene expression following treatment with jasmonic acid; 2) bona fide ppGpp synthesis activity of purified recombinant Nt-RSH2 protein, and 3) a wide spread distribution of RSH gene expression in the plant kingdom. Phylogenetic analyses identifies a distinct phylogenetic branch for the plant RSH proteins with two subgroups and supports ancient symbiosis and nuclear gene transfer as a possible origin for these stress response genes in plants. In addition, we find that Nt-RSH2 protein co-purifies with chloroplasts in subcellular fractionation experiments. Taken together, our findings implicate a direct mode of action of these ppGpp synthetases with regard to plant physiology, namely regulation of chloroplast gene expression in response to plant defense signals.

Changes in conserved region 3 of Escherichia coli sigma 70 reduce abortive transcription and enhance promoter escape.

Mutations within the Escherichia coli rpoD gene encoding amino acid substitutions in conserved region 3 of the sigma(70) subunit of E. coli RNA polymerase restore normal stress responsiveness to strains devoid of the stress alarmone, guanosine-3',5'-(bis)pyrophosphate (ppGpp). The presence of a mutant protein, either sigma(70)(P504L) or sigma(70)(S506F), suppresses the physiological defects in strains devoid of ppGpp. In vitro, when reconstituted into RNA polymerase holoenzyme, these sigma mutants confer unique transcriptional properties, namely they reduce the probabilities of forming abortive RNAs. Here we investigated the behavior of these mutant enzymes during transcription of the highly abortive cellular promoter, gal P2. No differences between mutant and wild-type enzymes were observed prior to and including open complex formation. Remarkably, the mutant enzymes produced drastically reduced levels of gal P2 abortive RNAs and increased production of full-length gal P2 RNAs relative to the wild-type enzyme, leading to greatly reduced ratios of abortive to productive RNAs. These results are attributed mainly to a decreased formation of unproductive initial transcribing complexes with the mutant polymerases and increased rates of promoter escape. Altered transcription properties of these mutant polymerases arise from an alternative structure of the sigma(70) region 3.2 segment that permits efficient positioning of the nascent RNA into the RNA exit channel displacing sigma and facilitating sigma release.

Multiple mechanisms of transcription inhibition by ppGpp at the lambdap(R) promoter.

General stress conditions in bacterial cells cause a global cellular response called the stringent response. The first event in this control is production of large amounts of a regulatory nucleotide, guanosine-3',5'-(bis)pyrophospahte (ppGpp). It was proposed recently that ppGpp acts by decreasing stability of open complexes at promoters that make short-lived open complexes, e.g. the rRNA promoters. However, here we report that the bacteriophage lambdap(R) promoter, which forms long-lived open complexes, is inhibited by ppGpp in vitro as observed in vivo. We performed a systematic investigation of the ppGpp-specific inhibition of transcription initiation at lambdap(R) and found that ppGpp does decrease stability of open complexes at lambdap(R), but only slightly. Likewise the equilbrium binding constant and rate of open complex formation by RNA polymerase at lambdap(R) are only slightly affected by ppGpp. The major effect of ppGpp-mediated inhibition is to decrease the rate of promoter escape. We conclude that ppGpp-mediated inhibition of transcription initiation is not restricted to promoters that make short-lived open complexes. Rather we conclude that the initial catalytic step of transcript formation is affected by ppGpp, specifically formation of the first phosphodiester bond is inhibited by ppGpp at lambdap(R).

Low concentrations of free hydrophobic amino acids disrupt the Escherichia coli RNA polymerase core-sigma(70) protein-protein interaction.

Studies of the Escherichia coli RNA polymerase subunit sigma-70 employing limited proteolytic digestion and binding by monoclonal antibodies indicate that conserved region 3 is solvent accessible in the free protein and in the RNA polymerase holoenzyme. Conversely, when sigma-70 binds to core RNA polymerase, proteolytic cleavage of region 3 is dramatically reduced. The former set of results seems to indicate the physical presence of region 3 on or near the surface of the holoenzyme while the latter of these results suggest that region 3 is sequestered in a direct protein-protein contact within the RNA holoenzyme which alters its protease sensitivity. To further investigate these possibilities we inserted an internal histidine-tag within region 3 of sigma(70) (sigma(70)-R3-His6) between amino acids 508 and 509. Confirmation that the internal His-tag insertion does not disrupt normal sigma(70) function was verified by genetic complementation. His-tagged protein was immobilized on nickel-agarose and core RNAP was tethered via the sigma-core interaction. Our results are consistent with the localization of region 3 on or near the surface both of free sigma(70) and of RNA polymerase holoenzyme. Furthermore, we find that the sigma(70)-core interaction is resistant to high ionic conditions but is completely disrupted by the presence of the low-molecular-weight hydrophobic amino acids phenylalanine and leucine free in solution. These results demonstrate the general usefulness of this approach to the disruption of protein-protein interactions and its potential application for protein purification.