The opening of specific segments of DNA is required for most types of genetic readout, including σ70‐dependent transcription. To learn how this occurs, a series of single point mutations were introduced into σ70 region 2. These were assayed for duplex DNA binding, DNA opening and DNA double strand–single strand fork junction binding. Band shift assays for closed complex formation implicated a series of arginine and aromatic residues within a minimal 26 amino acid region. Permanganate assays implicated two additional aromatic residues in DNA opening, known to form a parallel stack of the type that can accept a flipped‐out base. Substitution for either of these aromatics had no effect on duplex probe recognition. However, when a single unpaired −11 nucleotide is added to the probe, the mutants fail to bind appropriately to give heparin resistance. A model for DNA opening is presented in which duplex recognition by regions 2.3, 2.4 and 2.5 of sigma positions the pair of aromatic amino acids, which then create the fork junction required for stable opening.

Adaptation to high-salt environments is critical for the survival of a wide range of cells, especially for pathogenic bacteria that colonize the animal gut and urinary tract. The adaptation strategy involves production of the salt potassium glutamate, which induces a specific gene expression program that produces electro-neutral osmolytes while inhibiting general sigma(70) transcription. These data show that in Escherichia coli potassium glutamate stimulates transcription by disengaging inhibitory polymerase interactions at a sigma(38) promoter. These occur in an upstream region that is marked by an osmotic shock promoter DNA consensus sequence. The disruption activates a poised RNA polymerase to transcribe. This transcription program leads to the production of osmolytes that are shown to have only minor effects on transcription and therefore help to restore normal cell function. An osmotic shock gene expression cycle is discussed.

Band shift assays using DNA probes that mimic closed and open complexes were used to explore the determinants of promoter recognition by sigma38 (rpoS) RNA polymerase. Duplex recognition was found to be much weaker than that observed in sigma70 promoter usage. However, binding to fork junction probes, which attempt to mimic melted DNA, was very strong. This binding occurs via the non-template strand with the identity of the two conserved junction nucleotides (؊12T and ؊11A) being of paramount importance. A modified promoter consensus sequence identified these two nucleotides as among only four (underlined) that are highly conserved, and all four were in the ؊10 region (CTAcacT from ؊13 to ؊7). The remaining two nucleotides were shown to have different roles, ؊13C in preventing recognition by the heterologous sigma70 polymerase and ؊7T in directing enzyme isomerization. These ؊10 region nucleotides appear to have their primary function prior to full melting because probes that had a melted start site were relatively insensitive to substitution at these positions. These results suggest the sigma38 mechanism differs from the sigma70 mechanism, and this difference likely contributes to selective use of sigma38 under conditions that exist during stationery phase.The alternative sigma factor, sigma38 (rpoS), is the principle regulator of stress responses in Escherichia coli. The sigma38 regulon is very large and controls from 50 to 100 genes (1). Subsets of these genes are induced during starvation for various nutrients and in response to various stresses such as the accumulation of reactive oxygen species and changes in pH and osmolarity. The highest activity of sigma38 occurs during stationary phase when these and others stresses are actively assaulting the cell. Many factors contribute to this activity, especially the increased stability of sigma38 (2).Subsets of genes in this regulon are often needed at lower levels under conditions when sigma38 is not very active. Such genes can contain multiple promoters that are recognized by sigma38 and sigma70 (3-5) or have a single promoter that can be transcribed by both holoenzymes, although not necessarily to the same level (6, 7). The two holoenzymes can also be effected by common activator proteins, although again not necessarily to the same extent (8, 9). Thus the promoters of genes transcribed by sigma38 appear to have varying degrees of ability to be recognized by sigma70. In principle, the extent to which a promoter will be used by each sigma would be set by its core promoter sequence and by the upstream activator sites.It is not known how promoter sequences specify the extent of transcription by each type of holoenzyme. Sigma38 and sigma70 have very similar amino acid sequences. The regions of sigma70 that recognize promoter DNA, conserved region 4.2 (recognizes the Ϫ35 element) and conserved regions 2.3-2.5 (recognize the Ϫ10 element) are greater than 70% similar to those of sigma38 (10). The two sigmas recognize similar but not identical DNA sequences near Ϫ10, b...

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38 is a non-essential but highly homologous member of the 70 family of transcription factors. In vitro mutagenesis and in vivo screening were used to identify 22 critical amino acids in the promoter interaction domain of Escherichia coli 38 . Electrophoretic mobility shift assay studies showed that residues involved in duplex DNA binding largely segregated into distinct regions that coincided with those of 70 . However, the majority of these amino acids were in non-conserved positions. Analysis indicates that this region of the two s probably has a common overall organization but differs in how its amino acids are used to form functional open complexes. Placement of the mutations on the known 70 holoenzyme structure shows two clusters; one appears to be used for duplex DNA recognition and the other for the subsequent isomerization events. Permanganate assays for DNA melting support this view.The alternative factor, 38 (rpoS), is the principle regulator of the general stress response in Escherichia coli. The 38 regulon controls 50 to 100 genes (1). Subsets of these genes are induced during starvation for various nutrients and in response to various stresses such as the accumulation of reactive oxygen species, changes in pH, and osmolarity. The highest activity of 38 occurs during stationary phase when these and other stresses are present. Many factors contribute to this activity, especially the increased stability of the 38 protein (2). 38 is highly homologous to 70 , the vegetative factor responsible for the transcription of most of the housekeeping genes. The regions of 70 that recognize promoter DNA, conserved region 4.2 of the protein (recognizes the Ϫ35 element), and conserved regions 2.3-2.5 (Ϫ10 element) are over 70% similar (60% identical) to those of 38 (3). However, 38 promoters generally contain only a single DNA recognition element, a Ϫ10 sequence centered between nucleotides Ϫ14 and Ϫ7 (4, 5). The four most conserved of these nucleotides, Ϫ13C, Ϫ12T, Ϫ11A, and Ϫ7T, are involved in directing promoter selectivity and play a dominant role in setting promoter strength (4 -6). Three of these positions, Ϫ12T, Ϫ11A, and Ϫ7T, are also critical for 70 function, although they are utilized at different steps during 70 transcription initiation (7). 38 and 70 do not respond to regulators and the physiological state of the cell in the same manner. It is clear that such regulators as Lrp, CRP, H-NS, and many others can differentially effect transcription by 38 and 70 (8, 9). At certain promoters, a low supercoiled state of the DNA, which is present in stationary phase (10), seems to favor 38 transcription (11). Increased concentrations of trehalose (12), as well as potassium glutamate (13), also preferentially stimulate 38 -dependent transcription at certain promoters.The basis for these diverse properties between the two highly homologous holoenzymes have remained largely unknown. Some differences in 38 function have been attributed to differential recognition of nucleotides Ϫ14 and Ϫ13 (5) and a Cterminal "tail"...