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

Abstract

The alternative sigma factor S , mainly active in stationary phase of growth, recognizes in vitro a ؊10 promoter sequence almost identical to the one for the main sigma factor, 70 , thus raising the problem of how specific promoter recognition by S -RNA polymerase (E S ) is achieved in vivo. We investigated the promoter features involved in selective recognition by E S at the strictly S -dependent aidB promoter. We show that the presence of a C nucleotide as first residue of the aidB ؊10 sequence (؊12C), instead of the T nucleotide canonical for 70 -dependent promoters, is the major determinant for selective recognition by E S . The presence of the ؊12C does not allow formation of an open complex fully proficient in transcription initiation by E 70 . The role of ؊12C as specific determinant for promoter recognition by E S was confirmed by sequence analysis of known E S -dependent promoters as well as site-directed mutagenesis at the promoters of the csgB and sprE genes. We propose that E S , unlike E 70 , can recognize both C and T as the first nucleotide in the ؊10 sequence. Additional promoter features such as the presence of a C nucleotide at position ؊13, contributing to open complex formation by E S , and a TG motif found at the unusual ؊16/؊15 location, possibly contributing to initial binding to the promoter, also represent important factors for S -dependent transcription. We propose a new sequence, TG(N) 0 -2 CCATA(c/a)T, as consensus ؊10 sequence for promoters exclusively recognized by E S .Bacterial cells adapt to changing environmental and physiological conditions by modulating gene expression. Sigma ( ) factors of RNA polymerase, as the subunits responsible for promoter recognition, play a major role in programming gene expression. At least seven different subunits have been identified in Escherichia coli; 70 is the main subunit and can carry out transcription from the majority of E. coli promoters. The alternative subunits can direct transcription toward specific sets of genes (i.e. heat-shock, extracellular proteins, etc.) whose transcription is directed by -specific promoter sequences. 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 (1-3). 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). The alignment of E S -dependent promoters and the search for an optimal promoter for E S in vitro using the systematic evolution of ligands by exponential enrichment (SELEX) procedure have pointed to a Ϫ10 consensus sequence for E S , CTATA(c/a)T that is very similar to the canonical TATAAT sequence for 70 (6 -10). These results suggest that promoter selectivity between 70 and S might be determined by factors other than promote...

σ S -Dependent Gene Expression at the Onset of Stationary Phase in Escherichia coli : Function of σ S -Dependent Genes and Identification of Their Promoter Sequences

“…However, we expect that most promoters induced at the onset of stationary phase should belong to the class of promoters recognized with high affinity by the S subunit of RNA polymerase and thus should display promoter features favorable for E S . Indeed, the sequences of the Ϫ10 regions of the promoters tested are in good agreement with previous compilations of S -dependent promoters (23,47,54) and reflect the recently proposed motif TGN 0-2 CYATAMT (47). The Ϫ10 sequence of around 70% (12 of 18) of the newly characterized S -dependent promoters is immediately preceded by a C at the position conventionally indicated as Ϫ13; Ϫ13C is a widely conserved feature for promoter recognition by E S (9,15).…”

“…The Ϫ10 sequence of around 70% (12 of 18) of the newly characterized S -dependent promoters is immediately preceded by a C at the position conventionally indicated as Ϫ13; Ϫ13C is a widely conserved feature for promoter recognition by E S (9,15). Six (30%) promoters (ansPp 1 , ansPp 2 , fbaB, talAp 2 , pfkBp 1 , and yggEp 2 ) display a C as the first nucleotide of the Ϫ10 promoter element (conventionally Ϫ12C), a feature that might prevent their transcription by E 70 (47). A TG motif is present upstream of the Ϫ10 element in 10 (50%) of the newly determined promoters, at positions from Ϫ17 to Ϫ14.…”

“…Thus, at least for a subclass of promoters, promoter selectivity between E 70 and E S in vivo might be determined by factors other than DNA sequence, such as increased intracellular salt concentrations, degree of DNA supercoiling, modulation by additional regulators (37,41; reviewed in reference 34), or signal molecules such as the ppGpp alarmone (43,46). However, specific deviations from the common consensus sequence might favor either form of RNAP; for instance, the presence of a C immediately upstream of the Ϫ10 element (Ϫ13C [9]), flexibility in the location of the TG motif, and the presence of a C as the first nucleotide of the Ϫ10 sequence might favor promoter recognition by E S (47). In this work, we have studied the expression of rpoS-dependent genes at the onset of stationary phase in cell grown in rich medium.…”

“…Among these, the rpoS-encoded alternative sigma factor S plays a major role as a regulator of genes involved in stress responses (32). The S protein accumulates in stationary phase as well as in response to stress conditions and directs transcription from about 100 genes (41,47,85). The high conservation (61) and the tight control of RpoS accumulation in response to a wide range of environmental stimuli are consistent with its crucial role in bacterial physiology (36).…”

Borrelia burgdorferi bba74Is Expressed Exclusively during Tick Feeding and Is Regulated by Both Arthropod- and Mammalian Host-Specific Signals

“…Alignment of the bba74, ospA, ospE, and ospC upstream sequences from strains B31-MI and 297. The 70 consensus Ϫ35 and Ϫ10 sites for ospA, bba74, and ospE are based on those of E. coli (50,99) while the RpoS-dependent extended Ϫ10 for ospC is from Eggers et al (26). The ospA direct repeats (DR1 and DR2) and ospC operator region (IR1 and IR2) are based on Sohaskey et al (84) and Xu et al (101), respectively.…”