Interconversion of the DNA-binding specificities of two related transcription regulators, CRP and FNR.

In Escherichia coli, FNR and CRP are homologous transcriptional regulators which recognize similar nucleotide sequences via DNA-binding domains containing analogous helix-turn-helix motifs. The molecular basis for recognition and discrimination of their target sites has been investigated by directed amino acid substitutions in the corresponding DNA-recognition helices. In FNR, Glu-209 and Ser-212 are essential residues for the recognition of FNR sites. A V208R substitution confers CRP-site specificity without loss of FNR specificity, but this has adverse effects on anaerobic growth. In contrast, changes at two (V208R and E209D) or three (V208R, S212G and G216K) positions in FNR endow a single CRP-site binding specificity. In reciprocal experiments, two substitutions (R180V and G184S) were required to convert the binding specificity of CRP to that of FNR. Altering Asp-199 in FNR failed to produce a positive control phenotype, unlike substitutions at the comparable site in CRP. Implications for the mechanism of sequence discrimination by FNR and CRP are discussed.

Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP.

Expression from the E.coli melR promoter (pmelR) is normally totally dependent on the transcription activator protein, CRP. We describe experiments with a genetically engineered DNA fragment carrying pmelR in which the wild type CRP binding site was replaced with synthetic oligonucleotides containing either FNR or CRP binding sequences. When the synthetic oligonucleotide contains the 22 bp consensus for FNR binding sites, expression from pmelR is dependent on FNR but not CRP. Single changes at either of two symmetrically-related positions create sites that are recognised by both FNR and CRP. Changes at both positions result in a site that is not recognised by FNR but which binds CRP tightly.

The nirB promoter of Escherichia coli: location of nucleotide sequences essential for regulation by oxygen, the FNR protein and nitrite.

Using recombinant DNA techniques, nested deletions have been made upstream of the Escherichia coli nirB transcription start site and their effects on the regulation of nirB promoter activity have been measured. Nucleotide sequences downstream of -73 are sufficient for FNR-dependent induction of activity by anaerobic growth conditions. However, nucleotide sequences between -87 and -149 are essential for further induction by nitrite in the growth medium. The nucleotide sequence at the galP1 CRP binding site located from -31 to -52 displays some similarities with the same region at the nirB promoter. When the galP1 sequence from -30 to -54 was replaced by the corresponding nirB sequence, expression from galP1 became inducible by FNR under anaerobic growth conditions.