Mapping of the Rsd contact site on the sigma 70 subunit of Escherichia coli RNA polymerase.

Rsd (regulator of sigma D) is an anti-sigma factor for the Escherichia coli RNA polymerase sigma(70) subunit. The contact site of Rsd on sigma(70) was analyzed after mapping of the contact-dependent cleavage sites by Rsd-tethered iron-p-bromoacetamidobenzyl EDTA and by analysis of the complex formation between Ala-substituted sigma(70) and Rsd. Results indicate that the Rsd contact site is located downstream of the promoter -35 recognition helix-turn-helix motif within region 4, overlapping with the regions involved in interaction with both core enzyme and sigma(70) contact transcription factors.

Two extracytoplasmic function sigma subunits, sigma(E) and sigma(FecI), of Escherichia coli: promoter selectivity and intracellular levels.

The promoter selectivity of two extracytoplasmic function (ECF) subfamily sigma subunits, sigma(E) (sigma(24)) and sigma(FecI) (sigma(18)), of Escherichia coli RNA polymerase was analyzed by using an in vitro transcription system and various promoters. The Esigma(E) holoenzyme recognized only the known cognate promoters, rpoEP2, rpoHP3, and degP, and the Esigma(FecI) recognized only one known cognate promoter, fecA. The strict promoter recognition properties of sigma(E) and sigma(FecI) are similar to those of other minor sigma subunits. Transcription by Esigma(E) and Esigma(FecI) was enhanced by high concentrations of glutamate, as in the case of other minor sigma subunits. The optimum temperature for transcription by Esigma(FecI) was low, around 25 degrees C, apparently in agreement with the high rate of iron sequestration by E. coli at low temperatures. By quantitative Western blot analysis, the intracellular levels of sigma(E) and sigma(FecI) in the uninduced steady-state culture of E. coli W3110 (type A) were determined to be 0.7 to 2.0 and 0.1 to 0.2 fmol per microg of total proteins (or 3 to 9 and 0.4 to 0.9 molecules per cell), respectively, and less than 1% of the level of the major sigma(70) subunit.

Transcriptional organization and in vivo role of the Escherichia coli rsd gene, encoding the regulator of RNA polymerase sigma D.

The regulator of sigma D (Rsd) was identified as an RNA polymerase sigma70-associated protein in stationary-phase Escherichia coli with the inhibitory activity of sigma70-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953-4958, 1998). Primer extension analysis of rsd mRNA indicated the presence of two promoters, sigmaS-dependent P1 and sigma70-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expression of a reporter gene fused to either the sigma70- or sigmaS-dependent promoter was analyzed in the absence of Rsd or the presence of overexpressed Rsd. In the rsd null mutant, the sigma70- and sigmaS-dependent gene expression was increased or decreased, respectively. On the other hand, the sigma70- or sigmaS-dependent transcription was reduced or enhanced, respectively, after overexpression of Rsd. The repression of the sigmaS-dependent transcription in the rsd mutant is overcome by increased production of the sigmaS subunit. Together these observations support the prediction that Rsd is involved in replacement of the RNA polymerase sigma subunit from sigma70 to sigmaS during the transition from exponential growth to the stationary phase.

A stationary phase protein in Escherichia coli with binding activity to the major sigma subunit of RNA polymerase.

Switching of the transcription pattern in Escherichia coli during the growth transition from exponential to stationary phase is accompanied by the replacement of the RNA polymerase-associated sigma70 subunit (sigmaD) with sigma38 (sigmaS). A fraction of the sigma70 subunit in stationary phase cell extracts was found to exist as a complex with a novel protein, designated Rsd (Regulator of sigma D). The intracellular level of Rsd starts to increase during the transition from growing to stationary phase. The rsd gene was identified at 90 min on the E. coli chromosome. Overexpressed and purified Rsd protein formed complexes in vitro with sigma70 but not with other sigma subunits, sigmaN, sigmaS, sigmaH, sigmaF, and sigmaE. Analysis of proteolytic fragments of sigma70 indicated that Rsd binds at or downstream of region 4, the promoter -35 recognition domain. The isolated Rsd inhibited transcription in vitro to various extents depending on the promoters used. We propose that Rsd is a stationary phase E. coli protein with regulatory activity of the sigma70 function.

Variation in RNA polymerase sigma subunit composition within different stocks of Escherichia coli W3110.

The composition of RNA polymerase sigma subunits was analyzed for stock strains of Escherichia coli K-12 W3110 in Japan. Heterogeneity was discovered with respect to two sigma subunits, sigma28 (sigmaF, the rpoF gene product) and sigma38 (sigmaS, the rpoS gene product). Five different types of W3110 were identified: A-type lineages have both sigma subunits in intact forms; B-type lineages carry a truncated sigma38 subunit and an intact sigma28 subunit; C-type lineages carry an intact sigma28 subunit but lack a sigma38 subunit; D-type lineages have only a sigma38 subunit without a sigma28 subunit; and E-type stocks lack both sigma subunits. All the lineages examined, however, contain the intact forms of sigma70 (sigmaD, the rpoD gene product) and sigma54 (sigmaN, the rpoN gene product). As expected from the lack of a sigma28 subunit, cells of D- and E-type lineages are nonmotile. The truncated form of the sigma38 subunit in B-type stocks carries two mutations near its N terminus and lacks C-terminal proximal region 4 due to an amber mutation. The failure of C- and E-type W3110 cells to express sigma38 and that of D- and E-type cells to express sigma28 were found to be due to defects in transcription even though the respective sigma subunit genes remain intact. These findings emphasize the importance of paying attention to possible variations in the genetic background between laboratory stocks originating from the same strain.

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.

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.