Escherichia coli integration host factor inhibits the NusA stimulation of RNA polymerase sigma subunit synthesis in vitro

As reported previously, Integration Host Factor (IHF) stimulates cII expression but the stimulatory effect is prevented by the NusA protein (Peacock and Weissbach, 1985, Biochem. Biophys. Res. Commun. 127, 1026-1031). The interaction between IHF and the NusA protein has been investigated further in studies on the in vitro expression of the genes for the beta (rpoB) and sigma (rpoD) subunits of RNA polymerase, both known to be stimulated by NusA. The NusA stimulation of rpoD expression can be prevented by IHF, but IHF has no effect by itself on rpoD expression. IHF does not influence rpoB expression either in the presence or absence of NusA.     

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.     

Principal sigma subunit of the Caulobacter crescentus RNA polymerase.

We have identified the gene encoding the Caulobacter crescentus principal sigma subunit, RpoD. The rpoD gene codes for a polypeptide of 653 amino acids with a predicted molecular mass of 72,623 Da (sigma 73). The C. crescentus sigma subunit has extensive amino acid sequence homology with the principal sigma factors of a number of divergent procaryotes. In particular, the segments designated region 2 that are involved in core polymerase binding and promoter recognition were identical among these bacteria despite the fact that the -10 region recognized by the C. crescentus sigma 73 differs significantly from that of the other bacteria. Thus, it appears that additional sigma factor regions must be involved in -10 region recognition. This conclusion was strengthened by a heterologous complementation assay in which C. crescentus sigma 73 was capable of complementing the Escherichia coli rpoD285 temperature-sensitive mutant. Furthermore, C. crescentus sigma 73 conferred new specificity on the E. coli RNA polymerase, allowing the expression of C. crescentus promoters in E. coli. Thus, the C. crescentus sigma 73 appears to have a broader specificity than does the sigma 70 of the enteric bacteria.     

细菌RNA聚合酶亚单位研究进展

细菌的转录过程是一个由多种分子共同调控的复杂过程,其中RNA聚合酶(RNA polymerase,RNAP)是催化转录合成RNA的重要酶.作为RNAP中一个独立的亚单位,σ因子(sigma factor)在转录起始过程中起着至关重要的作用.最近的研究表明σ因子参与了转录起始的各个过程,包括启动子的定位、启动子的解链、起始RNA合成、脱离启动子等过程.由于其在细菌转录过程中的重要作用,σ因子正在成为抗菌药物研究的新靶点.本文对σ因子的结构、分类、功能以及以它为中心的调控网络的研究进行综述.     

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.     

Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of four species of sigma subunit under various growth conditions.

By a quantitative Western immunoblot analysis, 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), and of two minor sigma subunits, sigma 54 (sigma N, the rpoN gene product) and sigma 28 (sigma F, the rpoF gene product), were determined in two Escherichia coli strains, W3110 and MC4100. The results indicated that the levels of sigma 54 and sigma 28 are maintained at 10 and 50%, respectively, of the level of sigma 70 in both strains growing at both exponential and stationary phases, but in agreement with the previous measurement for strain MC4100 (M. Jishage and A. Ishihama, J. Bacteriol. 177:6832-6835, 1995), the level of sigma 38 was undetectable at the exponential growth phase but increased at 30% of the level of sigma 70 at the stationary phase. Stress-coupled change in the intracellular level was observed for two sigma subunits: (i) the increase in sigma 38 level and the decrease in sigma 28 level upon exposure to heat shock at the exponential phase and (ii) the increase in sigma 38 level under high-osmolality conditions at both the exponential and stationary phases.     

Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase.

An improved purification procedure is described for the sigma subunit of escherichia coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70, and finally Ultragel AcA44. The sigma factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical--physical properties of sigma are presented. A molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of sigma have been determined. The isoelectric focusing properties of sigma are presented. Denaturation--renaturation studies indicate that sigma is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000-dalton fragment is generated from sigma by mild trypsin treatment.     

Chemical modifications of the sigma subunit of the E. coli RNA polymerase.

The function of arginine, cysteine and carboxylic amino acid (glutamic and aspartic) residues of sigma was studied using chemical modification by group specific reagents. Following modification of 3 arginine residues with phenylglyoxal or 3 cysteine residues with N-ethylmaleimide (NEM) sigma activity was lost. Analysis of the kinetic data for inactivation indicated that one arginine or cysteine residue is essential for sigma activity. At low NEM concentration alkylation was limited to a non-critical cysteine which was identified as cysteine-132. Modification of arginine or cysteine residues had no observable effect on the binding of the inactivated sigma to the core polymerase. Modification of aspartic and/or glutamic acid residues with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) or 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMC) resulted in loss of sigma activity. The inactivation data indicated that one carboxylic amino acid residue is essential for sigma activity. Sigma modified with EDC, CMC or EDC in the presence of glycine was inactive in supporting promoter binding and initiation by core polymerase. Reaction with EDC plus (3H)glycine resulted in the incorporation of glycine into sigma. The (3H)glycine-sigma was unable to form a stable holoenzyme complex.     

Purification and characterization of the DNA-dependent RNA polymerase and its subunit sigma from Micrococcus luteus

DNA-dependent RNA polymerase from Micrococcus luteus can be isolated from cell extracts after removal of an excess of nucleic acids by fractionation with ammonium sulfate, followed by two consecutive gel filtrations through agarose and chromatography on cellulose phospate. Either homogeneous holoenzyme or a mixture of core and holoenzyme is obtained in this way, as is indicated by electrophoresis in polyacrylamide gels in the absence of detergent, where core enzyme migrates ahead of holoenzyme. Homogeneous core enzyme can be isolated from holoenzyme by chromatography on DEAE-cellulose. Core enzyme contains the subunits alpha, beta and beta' previously described [U.I. Lill et al., (1975) Eur. J. Biochem. 52, 411-420] in a molar ratio of 2:1:1. Holoenzyme contains an additional subunit sigma of 80 000 molecular weight (molar subunit composition alpha2 betabeta' sigma) and two relatively small polypeptides (molecular weight 14 000 and 25 000, respectively). Subunit sigma may be isolated from holoenzyme by chromatography on DEAE-cellulose at pH 6.9 in the presence of low concentrations of glycerol. The behaviour of holoenzyme during sedimentation in a glycerol gradient at low ionic strength indicates its occurrence as a dimer of the alpha2betabeta'sigma-protomer, whereas the monomeric form is preferred by core enzyme. Holoenzyme is much more active than core enzyme in RNA synthesis on bacteriophage T4DNA as template. The activity of the latter is stimulated by isolated sigma. M. luteus sigma as well as holoenzyme enhances also the activity of core enzyme fro- Escherichia coli. The formation of a hybrid between micrococcal sigma and E. coli core polymerase is also suggested by the influence of sigma on the oligomerisation of the enzyme from E. coli.