The downstream regulatory element of the proU operon of Salmonella typhimurium inhibits open complex formation by RNA polymerase at a distance

The intracellular concentration of K(+)-glutamate, chromatin-associated proteins, and a downstream regulatory element (DRE) overlapping with the coding sequence, have been implicated in the regulation of the proU operon of Salmonella typhimurium. The basal expression of the proU operon is low, but it is rapidly induced when the bacteria are grown in media of high osmolarity (e.g. 0.3 M NaCl). It has previously been suggested that increased intracellular concentrations of K(+)-glutamate activate the proU promoter in response to increased extracellular osmolarity. We show here that the activation of the proU promoter by K(+)-glutamate in vitro is nonspecific, and the in vivo regulation cannot simply be mimicked in vitro. In vivo specificity requires both the chromatin-associated protein H-NS and the DRE; they are both needed to maintain repression of proU expression at low osmolarity. How H-NS and the DRE repress the proU promoter in vivo has so far been unclear. We show that, in vivo, the DRE acts at a distance to inhibit open complex formation at the proU promoter.     

Replacement of potassium chloride by potassium glutamate dramatically enhances protein-DNA interactions in vitro

Although protein-nucleic acid interactions exhibit dramatic dependences on both ion concentration and type in vitro, large variations in intracellular ion concentrations can occur in Escherichia coli and other organisms without apparent effects on gene expression in vivo. E. coli accumulates K+ and glutamate as cytoplasmic osmolytes. The cytoplasmic K+ concentration in E. coli varies from less than 0.2 to greater than 0.9 m as a function of external osmolarity; corresponding cytoplasmic glutamate concentrations range from less than 0.03 to greater than 0.25 m. Only low levels of chloride occur in the cytoplasm of E. coli at all osmotic conditions. Since most in vitro studies have been performed in chloride salts, whereas glutamate is the more relevant physiological anion, we have measured the effects of the substitution of potassium glutamate (KGlu) for KCl on the kinetics and equilibria of a variety of site-specific protein-DNA interactions in vitro. Both the interaction of E. coli RNA polymerase with two phage lambda promoters and the interactions of various restriction enzymes with their DNA cleavage sites are enhanced by this substitution. Using the abortive initiation assay, we find a greater than 30-fold increase in the second-order rate constant for open complex formation at the lambda PR promoter and a 10-fold increase at the lambda PR' promoter, when KGlu is substituted for KCl. Replacement of KCl by KGlu does not affect the strong salt dependences of these interactions; increasing either KCl or KGlu concentrations decreases both reaction rates and extents. Substitution of glutamate for chloride does, however, shift the range of salt concentrations over which these interactions are observable to higher K+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of Escherichia coli RNA polymerase holoenzyme containing sigma 32 with heat shock promoters. DNase I footprinting and methylation protection

The DNase I protection pattern of E sigma 32 was assayed on three heat shock promoters, the E sigma 32 promoter for the groESL operon, P2 of the dnaKJ operon, and rpoD PHS, the E sigma 32 promoter upstream from rpoD. E sigma 32 protected each of these promoters from DNase I digestion from around -60 to around +20. Protection from dimethyl sulfate methylation was assayed at the groE promoter. E sigma 32 binding altered the sensitivity to methylation of bases in the vicinity of both the -10 and -35 regions. The DNase I footprints for the E sigma 32 promoters were very similar to the DNase I footprint of E sigma 70 on the lacUV5 promoter. After analyzing the DNase I footprints by taking into account the contacts predicted to be made by DNase I, it appeared that E sigma 32, like E sigma 70, contacts the DNA primarily on one face of the helix in the -35 region and on both faces in the -10 region.