Gostick Dominic O, Green Jeffrey, Irvine Alistair S, Gasson Michael J, Guest John R
The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank,Sheffield S10 2TN,UK.
Institute of Food Research, Norwich Laboratory, Colney Research Park,Colney, Norwich NR4 7UA,UK.
Microbiology (Reading). 1998 Mar;144 ( Pt 3):705-717. doi: 10.1099/00221287-144-3-705.
FNR (regulator for fumarate and nitrate reduction) and CRP (cAMP receptor protein) are global regulators which regulate the transcription of overlapping modulons of target genes in response to anaerobiosis and carbon source in Escherichia coli. An ORF, designated flp because it encodes an FNR-like protein of the FNR-CRP family, has been found in Lactobacillus casei. The product of the flp coding region (FLP) was overproduced in E. coli, purified and crystallized. FLP is a homodimeric protein in which each subunit can form an intramolecular disulphide bond. The isolated protein also contains non-stoichiometric amounts of Cu and Zn. Although the DNA recognition helix of FLP resembles that of FNR, the flp gene failed to complement the anaerobic respiratory deficiency of an fnr mutant when expressed in E. coli and it neither activated nor interfered with transcription from FNR- or CRP-dependent promoters in E. coli. Site-specific DNA binding by oxidized FLP (the form containing intrasubunit disulphide bonds) was abolished by reduction. The interconversion between disulphide and dithiol forms thus provides the basis for a novel redox-mediated transcriptional switch. Two non-identical FLP-binding sites, distinct from FNR- and CRP-binding sites, were identified in the meIR region of E. coli by gel-retardation analysis. A further eight FLP-binding sites were selected from a random library. A synthetic oligonucleotide conforming to a putative FLP site consensus, CA/CTGA-N4-TCAG/TG (the most significant bases are underlined), was retarded by FLP. Functional tests showed that FLP represses the aerobic transcription of a semi-synthetic promoter in E. coli. A C5S variant of FLP lacking the ability to form intramolecular disulphide bonds was unable to bind to FLP sites and failed to repress transcription in vivo.
延胡索酸和硝酸盐还原调节蛋白（FNR）及环腺苷酸受体蛋白（CRP）是全局调节因子，它们在大肠杆菌中响应厌氧状态和碳源，调节重叠的靶基因调节子的转录。在干酪乳杆菌中发现了一个开放阅读框（ORF），因其编码FNR-CRP家族中一种类似FNR的蛋白而被命名为flp。flp编码区的产物（FLP）在大肠杆菌中过量表达、纯化并结晶。FLP是一种同二聚体蛋白，其中每个亚基都能形成分子内二硫键。分离得到的蛋白还含有非化学计量的铜和锌。尽管FLP的DNA识别螺旋与FNR的相似，但flp基因在大肠杆菌中表达时不能弥补fnr突变体的厌氧呼吸缺陷，它既不激活也不干扰大肠杆菌中FNR或CRP依赖性启动子的转录。还原作用可消除氧化型FLP（含有亚基内二硫键的形式）的位点特异性DNA结合。因此，二硫键和二硫醇形式之间的相互转化为一种新型的氧化还原介导的转录开关提供了基础。通过凝胶阻滞分析，在大肠杆菌的meIR区域鉴定出两个与FNR和CRP结合位点不同的、不相同的FLP结合位点。从一个随机文库中又筛选出另外八个FLP结合位点。一个符合假定的FLP位点共有序列CA/CTGA-N4-TCAG/TG（最重要的碱基加下划线）的合成寡核苷酸被FLP阻滞。功能测试表明，FLP抑制大肠杆菌中一个半合成启动子的有氧转录。缺乏形成分子内二硫键能力的FLP的C5S变体无法与FLP位点结合，也不能在体内抑制转录。
