Spiro S, Gaston K L, Bell A I, Roberts R E, Busby S J, Guest J R
Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Mol Microbiol. 1990 Nov;4(11):1831-8. doi: 10.1111/j.1365-2958.1990.tb02031.x.
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.
在大肠杆菌中，FNR和CRP是同源转录调节因子，它们通过含有类似螺旋-转角-螺旋基序的DNA结合结构域识别相似的核苷酸序列。通过在相应的DNA识别螺旋中进行定向氨基酸替换，研究了它们识别和区分靶位点的分子基础。在FNR中，Glu-209和Ser-212是识别FNR位点的必需残基。V208R替换赋予了CRP位点特异性，同时不丧失FNR特异性，但这对厌氧生长有不利影响。相比之下，FNR中两个（V208R和E209D）或三个（V208R、S212G和G216K）位置的变化赋予了单一的CRP位点结合特异性。在反向实验中，需要两个替换（R180V和G184S）才能将CRP的结合特异性转化为FNR的结合特异性。与CRP中可比位点的替换不同，改变FNR中的Asp-199未能产生阳性对照表型。讨论了FNR和CRP序列区分机制的相关问题。
