A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity

A mutation is described that alters the promoter specificity of sigma 70, the primary sigma factor of Escherichia coli RNA polymerase. In strains carrying both the mutant and wild-type sigma gene (rpoD), the mutant sigma causes a large increase in the activity of mutant P22 ant promoters with A.T or C.G instead of the wild-type, consensus G.C base-pair at position -33, the third position of the consensus -35 hexamer 5'-TTGACA-3'. There is little or no effect on the activities of the wild-type and 23 other mutant ant promoters, including one with T.A at -33. The mutant sigma also activates E. coli lac promoters with A.T or C.G, but not T.A, at the corresponding position. The rpoD mutation (rpoD-RH588) changes a CGT codon to CAT. The corresponding change in sigma 70 is Arg588----His. This residue is in a region that is conserved among most sigma factors, a region that is also homologous with the helix-turn-helix motif of DNA-binding proteins. These results suggest that this region of sigma 70 is directly involved in recognition of the -35 hexamer.     

Characterization of the low pH solution structure and dynamics of the region 4 of Escherichia coli RNA polymerase sigma70 subunit

Solution structure of the region 4 of sigma(70) subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied. Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain (13)C resonances for a recombinant His(6)-tagged protein containing the whole region 4 and a part of region 3.2 of sigma(70) in aqueous solution at pH 2.8 (Poznański, J., Zhukov, I., Bolewska, K., and Wierzchowski, K. L. (2001) J. Biomol. NMR 20, 181-2). The protein proved to be sufficiently soluble and did not aggregate only in the protonated state. In this paper, the structure and dynamics of this state at pH 2.8 have been extensively examined using CD and NMR spectroscopy. Both analysis of CD spectra and NMR observables (secondary chemical shifts of the (13)Calpha, (13)CO, and (1)Halpha nuclei and of vicinal (3)J(HNH)(alpha) coupling constants) indicated that a significant amount of helical structure remained in the protonated protein. The amount of this structure increased upon deprotonation of carboxylic amino acids, as shown by pH titration CD experiments. 2,2,2-Trifluoroethanol induced an even more extensive build up of this structure. Distribution along the protein sequence of the secondary shifts and (3)J(HNH)(alpha) couplings demonstrated partition of the helical secondary structure into three helices located similarly as in the crystal structures of the homologous region 4 of the sigma(A) subunit of Thermus aquaticus RNA polymerase (Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, A., Weinman, O., Trester-Zedlitz, M. L., and Darst, S. A. (2002) Mol. Cell 9, 527-39) and sigma(70) of the Thermus thermophilus RNA polymerase (Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S., and Yokoyama, S. (2002) Nature 417, 712-9.). Spectral density analysis of NMR relaxation parameters, R(1) and R(2), and [(1)H]-(15)N heteronuclear NOEs indicated that backbone fluctuations in the whole region embracing the three helices and intervening nonhelical sequences are severely restricted on the nanosecond time scale as compared with the N- and C-terminal protein segments. Inspection of the side-chain contacts stabilizing the crystal structures well explains the observed folding and solution properties of sigma(70)(4) protein in its protonated state.     

Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of sigma 70 and sigma 38.

The intracellular levels of two principal sigma subunits, sigma 70 (sigma D, the rpoD gene product) and sigma 38 (sigma s, the rpoS gene product), in Escherichia coli MC4100 were determined by a quantitative Western immunoblot analysis. Results indicate that the level of sigma 70 is maintained at 50 to 80 fmol per micrograms of total proteins throughout the transition from the exponential growth phase to the stationary phase, while the level of sigma 38 protein is below the detection level at the exponential growth phase but increases to 30% of the level of sigma 70 when cell growth stops to enter into the stationary phase. Beside the stationary phase, the increase in sigma 38 level was observed in two cases: exposure to heat shock at the exponential phase and osmotic shock at the stationary phase.     

Control by bacteriophage T4 of two sequential phosphorylations of the alpha subunit of Escherichia coli RNA polymerase

The effect of 2′-O-methylation upon the base-stacking properties of dinucleoside monophosphates has been studied by circular dichroism measurements over the temperature range from ?20 °C to +80 °C at high and at low salt concentration of 13 2′-O-methyl derivatives in neutral aqueous solution. It is found that 2′-O methylation generally enhances the stacking propensity of dinucleoside monophosphates except for the dimers with adenine in the 3′-linked nucleoside, where the converse trend is observed. The influence of 2′-O-methylation upon the base-stacking property of a dimer correlates in part with the effect of a reduction in salt concentration, suggesting that the 2′-O-methyl group effects the stacking by displacing ions from the immediate environment of the dimer as well as by intramolecular steric effects. The dimers which exhibit an enhanced stacking due to the 2′-O-methylation are found in a larger than statistical abundance in yeast transfer RNA, whereas those showing a reduced stacking occur in minor abundance. These observations are discussed in relation to some current views on the role of modified nucleosides in the conformation of ribonucleic acids.     

Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase

We have constructed a plasmid that overexpresses 100-fold the sigma subunit of Escherichia coli RNA polymerase. The plasmid was constructed by placing the pLoL promoter-operator of bacteriophage lambda upstream from rpoD, the gene encoding the sigma subunit. A simple procedure for purification of the overexpressed protein has been developed based on guanidine hydrochloride denaturation/renaturation, DEAE cellulose chromatography, and Sephacryl S-200 chromatography. The purified product has been characterized and found to be indistinguishable from normally expressed sigma protein purified by previous protocols as judged by enzymatic activity, heat inactivation, and partial proteolysis.