Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli.

Abstract

The rpoS gene of Escherichia coli encodes a putative RNA polymerase a factor that is considered to be the central regulator of gene expression in stationary phase. The gene product (ar3) was overproduced using the cloned rpoS gene and purified to homogeneity. Reconstituted RNA polymerase holoenzyme (Ea38) was found to recognize in vitro a number of typical a70-type promoters, including the lacUV5 and thp promoters. Some, however, were recognized exclusively or preferentially by Ear70, whereas at least one, fic, was favored by E&rm. Thus E. coli promoters can be classified into three groups: the first group is recognized by Ea70 and Ea,38 but the second and third groups are recognized substantially by either Ea70 or Eam alone. In contrast to other minor afactors, am shares a set of amino acid sequences common among the principal a factors of eubacteria and is therefore a member of the RpoD-related protein family. The intracellular level of a38 was demonstrated to increase in vivo upon entry into stationary phase. These results together indicate that am is a second principal a factor in stationary-phase E. coli.The promoter selectivity of RNA polymerase holoenzyme, controlled by replacing cr factors, is the primary mechanism for the global switching of gene expression in eubacteria. The principal Cr factor or70 (the rpoD gene product) in exponentially growing Escherichia coli potentiates the transcription of genes controlled by the consensus promoters consisting of two hexanucleotide sequences, TTGACA and TATAAT (1, 2). Other minor species of alternative cr factors recognize different sets of promoter sequences, each being associated with a limited number of genes that are mostly expressed in response to various stress conditions (1, 2). The rpoS (katF) gene of E. coli was first identified as a positive regulator of the katE gene encoding hydroperoxidase II (HPII) (3). Later, several other genes that are, for the most part, expressed in the stationary growth phase were shown to be regulated by the rpoS allele (4, 5). Thus the rpoS gene was assumed to be the central regulator of gene expression in the stationary phase (4). From the nucleotide sequence ofthe rpoS gene, the gene product has been believed to be an RNA polymerase cr factor (6, 7). However, a, activity has not been detected previously for the rpoS gene product.In this study, we determined the nucleotide sequence ofthe rpoS gene § and found that the gene product is structurally similar to or70 and the principal or factors from divergent eubacterial species (7-12). To assess the functional similarity between the rpoS gene product (or38) and cr70, we reconstituted Eor38 holoenzyme using a highly purified rpoS gene product and examined its promoter selectivity in in vitro transcription assays using various E. coli promoters. MATERIALS AND METHODS Bacterial Strains. E. coli strains DH1 (13), XLlblue (14), MC4100 (4), and BL21(DE3) (15) were used in this study. Plasmid pTZ19R was obtained from Pharmacia. pET3b, pLysS, and pLysE plasmids are as describ...

Inorganic polyphosphate and the induction of rpoS expression

“…Polyethyleneimine-cellulose thin-layer chromatography (PEl-TLC) plastic plates were from Merck. Anti-RpoS antibody was prepared as described (15). Purified histidine-tagged PPX1 enzyme used in poly(P) estimation was prepared as described (17).…”

“…The heat sensitivity of the ppk mutant is suppressed by extra copies of rpoS, the gene encoding the stationary-phase-specific RNA polymerase factor (12). Expression of rpoS is a central element in a regulatory network that governs the expression of many stationary-phase-induced and osmotically regulated genes, including katE (14)(15)(16). These lines of evidence indicate that poly(P) relates to rpoS in its transcription or translation or in stabilization of the RpoS ( 38 ) protein, inasmuch as the cellular content of 38 is regulated at both the transcriptional and post-transcriptional levels (16).…”

Nucleotides from –16 to –12 Determine Specific Promoter Recognition by Bacterial σS-RNA Polymerase

“…Substitutions that either disrupt the TG motif (15G3 C; designated G15C in Figs. [1][2][3][4] or eliminate the Ϫ13C (13C3 G; designated C13G in Figs. 1-4) show a rather small (1.5-to 2-fold) albeit significant and reproducible reduction in promoter activity, suggesting a possible role for these two promoter elements in modulation of affinity by E S for PaidB.…”

“…KMnO 4 Reactivity Experiments-To investigate whether PaidB mutations affect open complex formation by either form of RNA polymerase, we performed permanganate (KMnO 4 ) reactivity experiments. This assay takes advantage of KMnO 4 reactivity with thymine residues in single-stranded DNA, thus allowing us to probe open complex formation by RNA polymerase (42).…”

“…A partial exception to the typical role for alternative subunits is represented by S , the product of the rpoS gene, mainly expressed in the stationary phase of growth (1)(2)(3). Unlike the other subunits, S -RNA polymerase (E S ) 1 can initiate transcription from several promoters also recognized by E 70 , suggesting that they recognize similar promoter sequences (4). The recognition of similar promoter sequences by S and 70 is reflected by their strong similarity in the DNA binding domains (5).…”