Mutational Analysis of an Extracytoplasmic-Function Sigma Factor To Investigate Its Interactions with RNA Polymerase and DNA

Abstract

The extracytoplasmic-function (ECF) family of sigma factors comprises a large group of proteins required for synthesis of a wide variety of extracytoplasmic products by bacteria. Residues important for core RNA polymerase (RNAP) binding, DNA melting, and promoter recognition have been identified in conserved regions 2 and 4.2 of primary sigma factors. Seventeen residues in region 2 and eight residues in region 4.2 of an ECF sigma factor, PvdS from Pseudomonas aeruginosa, were selected for alanine-scanning mutagenesis on the basis of sequence alignments with other sigma factors. Fourteen of the mutations in region 2 had a significant effect on protein function in an in vivo assay. Four proteins with alterations in regions 2.1 and 2.2 were purified as His-tagged fusions, and all showed a reduced affinity for core RNAP in vitro, consistent with a role in core binding. Region 2.3 and 2.4 mutant proteins retained the ability to bind core RNAP, but four mutants had reduced or no ability to cause core RNA polymerase to bind promoter DNA in a band-shift assay, identifying residues important for DNA binding. All mutations in region 4.2 reduced the activity of PvdS in vivo. Two of the region 4.2 mutant proteins were purified, and each showed a reduced ability to cause core RNA polymerase to bind to promoter DNA. The results show that some residues in PvdS have functions equivalent to those of corresponding residues in primary sigma factors; however, they also show that several residues not shared with primary sigma factors contribute to protein function.Bacterial core RNA polymerase (RNAP; ␣ 2 ␤␤Ј) requires a fifth subunit, the sigma factor, in order to initiate specific transcription from double-stranded templates. Core RNAPsigma factor complexes bind to gene promoters in a sequencespecific manner (reviewed in references 18, 25, and 46). Sigma factor proteins of the 70 family bind at specific DNA sequences that are usually centered at about the Ϫ35 and Ϫ10 region of the promoter. Multiple sigma factor proteins can be present in a single bacterium, and each has an important role in controlling gene expression, with different sigma factors recognizing different promoter sequences (25,66). Primary sigma factors, such as 70 in Escherichia coli, are essential proteins that are responsible for the majority of RNA synthesis in exponentially growing cells. Alternative sigma factors are nonessential proteins required under certain circumstances and, except for 54 and related proteins, have various degrees of sequence similarity to 70 . The extracytoplasmic-function (ECF) family is a large group of sigma proteins that regulate the production of various extracytoplasmic products in a variety of bacteria (reviewed in references 20 and 40). Although they have conserved sequence features present in 70 -type proteins (34), ECF sigma factor proteins are much smaller than most other sigma factors (typically 20 to 25 kDa) and therefore may have significant differences in their interactions with DNA, core RNAP, or other proteins.Sigma fac...

A two-subunit bacterial σ-factor activates transcription in Bacillus subtilis

“…S5C), it copurified with RNAP to a much lesser extent than WT YvrHa. This supports the hypothesis that this region 2.2 motif in YvrHa is involved in core binding, consistent with results for conventional -factors (13)(14)(15).…”

“…3, Bottom), implying functional importance. Previously, mutations in -factor that negatively influence holoenzyme formation (presumably by disrupting interaction with the ␤Ј-subunit coiled coil) were mapped to this subsequence (13)(14)(15). To determine if this region is also critical for YvrHa function, we engineered mutations in this motif.…”

“…Amino acids in regions 2.1 and 2.2 form a pair of ␣-helices that mediate core binding by virtue of interaction with a coiled-coil element in the amino terminus of the ␤Ј-subunit of core RNAP (8). This interaction is critical for holoenzyme formation, because a number of mutations in these interacting elements negatively influence -binding to core (9)(10)(11)(12)(13)(14)(15). This interaction is also important for -region 2.3-2.4 recognition of the single-and double-stranded -10 element within promoter DNA (16,17).…”

Role of Cell Surface Signaling in Proteolysis of an Alternative Sigma Factor inPseudomonas aeruginosa

“…Recombinant PvdS fused to a hexahistidine tag (hPvdS) was purified from E. coli BL21 (pPROEx::pvdS) as described previously (41,42). Purity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining (5); no contaminating proteins were detected.…”

Residues Involved in FecR Binding Are Localized on One Side of the FecA Signaling Domain in Escherichia coli

“…The results obtained with FecA in E. coli most likely also apply for transcription initiation in Shigella flexneri, various Bordetella strains, Serratia marcescens, Pseudomonas aeruginosa (10,13), Pseudomonas putida, and Ralstonia solanacearum. In these species, a FecIRA type of transcription initiation has been demonstrated experimentally, and many predicted FecIRA-type regulatory devices have been predicted from sequenced genomes (listed in reference 1).…”