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.
Spiro
S
S
Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Gaston
K L
KL
Bell
A I
AI
Roberts
R E
RE
Busby
S J
SJ
Guest
J R
JR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002872:Chromosome Deletion; D002373:Cyclic AMP Receptor Protein; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007506:Iron-Sulfur Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D011953:Receptors, Cyclic AMP / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D011953:Receptors, Cyclic AMP