Conformation and DNA binding properties of a single-stranded DNA binding region of sigma 70 subunit from Escherichia coli RNA polymerase are modulated by an interaction with the core enzyme

We have explored how IL-15 influences Th1 or Th2 type immune response in vivo. Intraperitoneal application of an IL-15-IgG2b fusion protein (FP) to mice did neither significantly affect the footpad swelling nor the production of hemagglutinizing antibodies in a delayed type hypersensitivity reaction to sheep red blood cells. In contrast, in an established murine Th2 model of sensitization to ovalbumin (OVA), IL-15-IgG2b FP plus OVA sensitization resulted in massively accelerated and enhanced allergen-specific IgE and IgG1 antibody production. In vitro, stimulation of spleen cells from OVA-sensitized mice with OVA+IL-15 or OVA+IL-15-IgG2b resulted in a significantly enhanced IgE production. IL-4 secretion was significantly induced by IL-15 but not by IL-15-IgG2b. An IL-2-IgG2b FP with the same Fc tail as the IL-15-IgG2b FP was used as control in both models. In striking contrast to the IL-15-IgG2b FP, IL-2-IgG2b significantly inhibited the Th2 type antibody production in vivo. The current study suggests that IL-15-IgG2b may be employed as a potent accelerator and enhancer of Th2 type immune responses in vivo, while IL-2-IgG2b can suppress the latter.     

The C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase is required for efficient rho-dependent transcription termination

Type I Helicobacter pylori strains frequently recognize the Lewisb (Leb) blood group antigen. This binding property and expression of the Leb oligosaccharide were required for adherence to fixed normal or pathologic gastric tissue. In contrast, both type I and type II strains adhered to cultured cells in the absence of the Leb epitope. For the gastric cell line AGS, adherence was significantly higher when viable type I strains were allowed to interact with viable AGS cells compared with fixed cells. The observation that chloramphenicol and cycloheximide, inhibitors of bacterial and eukaryotic protein synthesis, respectively, significantly reduced adherence of type I but not type II isolates suggests that in type I strains, adherence depends on the up-regulation of one or more host cell receptors triggered by the bacterium.     

Sigma s-dependent promoters in Escherichia coli are located in DNA regions with intrinsic curvature

Expression of a number of genes during stationary phase in Escherichia coli is controlled by the alternative sigma factor sigma s (KatF). Promoters recognized by sigma s do not present a well-defined consensus sequence in their -10 and -35 regions. By polyacrylamide gel electrophoresis of DNA fragments performed at different temperatures, and by computer prediction analyses, we have found that sigma s-regulated promoters are located in regions where DNA shows intrinsic curvatures. This feature does not appear in a stationary-phase-induced promoter which is not controlled by sigma s. We propose that DNA bending may help in recognition and/or binding of sigma s to stationary-phase-induced promoters.     

Scanning force microscopy of Escherichia coli RNA polymerase.sigma54 holoenzyme complexes with DNA in buffer and in air

Scanning force microscopy (SFM) was used to visualize complexes of Escherichia coli RNA polymerase.sigma54 (RNAP.sigma54) and a 1036 base-pair linear DNA fragment containing the glnA promoter. In order to preserve the native hydration state of the protein-DNA complexes, the samples were injected directly into the SFM fluid cell and imaged in buffer. With this protocol, an apparent bending angle of 26(+/-34) degrees was determined for the specific complexes at the promoter. The bending angle of the unspecifically bound RNAP.sigma54 showed a somewhat broader distribution of 49(+/-48) degrees, indicating the existence of conformational differences as compared to the closed complex. In about two-thirds of the closed complexes, the RNA polymerase holoenzyme was located in a lateral position with respect to the DNA and the bend of the DNA was pointing away from the protein. This conformation was consistent with the finding that for the complexes at the promoter, the apparent contour length was reduced by only about 6 nm in buffer as compared to the free DNA. From these results we conclude that in the closed complex of RNAP. sigma54, the DNA was not wrapped around the polymerase, and we present a model for the trajectory of the DNA with respect to the RNA polymerase. The images acquired in buffer were compared to samples that were washed with water and then dried before imaging. Two artefacts of the washing and drying process were detected. First, extensive washing of the sample reduced the number of the specific complexes bound at the promoter (closed complex of RNAP.sigma54) from about 70% to 30%. This is likely to be a result of sliding of the RNAP.sigma54 holoenzyme along the DNA induced by the washing process. Second, the apparent DNA shortening of the contour length of RNAP.sigma54-DNA complexes at the promoter as compared to the contour length of the free DNA was 22 nm for the dried samples as opposed to only 6 nm for the undried samples imaged in buffer. This suggests an artefact of the drying process.     

Functional map of the alpha subunit of Escherichia coli RNA polymerase: amino acid substitution within the amino-terminal assembly domain

The alpha subunit of Escherichia coli RNA polymerase plays a key role in assembly of the core enzyme. In previous studies the amino-terminal domain consisting of 215 amino acid residues between positions 21 and 235 was identified to be involved in this assembly, and the sites for beta and beta' association were suggested to be located within or near the two conserved regions in this amino-terminal assembly domain of alpha. For detailed functional mapping, Ala was substituted for 26 highly conserved amino acids around residues 40, 80 and 170 to 210. The alpha-point mutants were analyzed in vitro for their abilities to form dimers and to assemble beta beta' subunits. New types of assembly-deficient mutants were identified: alpha-R45A (having substituted Ala for Arg at residue 45) dimerized but did not assemble beta (and beta') subunits; and alpha-L48A showed a decreased level of alpha 2 beta subassembly formation, indicating that this region (residues 45 to 48) is responsible for beta-binding. Isolation of two mutants, alpha-K86A and alpha-V173A, both forming alpha 2 beta but not alpha 2 beta beta' complex, confirmed our previous conclusion that two separated regions participate in beta'-binding.     

Architecture of a complex between the sigma70 subunit of Escherichia coli RNA polymerase and the nontemplate strand oligonucleotide. Luminescence resonance energy transfer study

We used luminescence energy transfer measurements to determine the localization of 5'- and 3'-ends of a 12-nucleotide nontemplate strand oligonucleotide bound to sigma70 holoenzyme. Five single reactive cysteine mutants of sigma70 (cysteine residues at positions 1, 59, 366, 442, and 596) were labeled with a europium chelate fluorochrome (donor). The oligonucleotide was modified at the 5'- or at the 3'-end with Cy5 fluorochrome (acceptor). The energy transfer was observed upon complex formation between the donor-labeled sigma70 holoenzyme and the acceptor-labeled nontemplate strand oligonucleotide, whereas no interaction was observed with the template strand oligonucleotide. The oligonucleotide was bound in one preferred orientation. This observation together with the sequence specificity of single-stranded oligonucleotide interaction suggests that two mechanisms of discrimination between the template and nontemplate strand are used by sigma70: sequence specificity and strand polarity specificity. The bound oligonucleotide was found to be close to residue 442, confirming that the single-stranded DNA binding site of sigma70 is located in an alpha-helix containing residue 442. The 5'-end of the oligonucleotide was oriented toward the COOH terminus of the helix.     

Iron regulates transcription of the Escherichia coli ferric citrate transport genes directly and through the transcription initiation proteins

Water compartments were studied in 72 black and 128 white women, aged 20 to 70 years. Total body water (TBW) was measured by tritiated water dilution, and extracellular water (ECW) was measured by using delayed gamma neutron activation for the determination of total body chloride. Intracellular water (ICW) was assessed as the difference between TBW and ECW. Fat-free mass (FFM) was estimated by the measurement of total body potassium (TBK) and total body fat (TBF) by measurement of total body carbon (TBC). Total body protein was calculated from total body nitrogen (TBN). TBW was found to decline with age and to also be significantly influenced by body size. The extracellular water space was 41.5% of TBW. The decline of TBW with age is due primarily to a decline in ICW. The hydration of the FFM increased with age for the overall population because of a decline in TBK and TBN and an increase in ECW. Body composition models that have assumed constancy of hydration should be adjusted not only for gender but for age and body size.