Scott S, Busby S, Beacham I
Faculty of Science and Technology, Griffith University, Nathan, Brisbane, Queensland, Australia.
Mol Microbiol. 1995 Nov;18(3):521-31. doi: 10.1111/j.1365-2958.1995.mmi_18030521.x.
Previous work with semi-synthetic promoters containing a single CRP binding site centred at 41.5 bp from the transcription start site has demonstrated enhanced transcription (synergism) when a second binding site, for CRP or FNR, is placed upstream at around -91 bp. The ansB promoter in Escherichia coli is co-activated in a co-dependent manner by one dimer each of CRP and FNR protein whose binding sites are at around -91 and -41 bp, respectively, from the transcription start site. Similarly, the homologous ansB promoter in Salmonella is co-activated by two dimers of CRP which function synergistically. The binding sites at the E. coli promoter have been changed by mutation to provide a number of active promoter derivatives carrying other combinations of FNR and CRP binding sites. The co-dependent versus synergistic interaction of these activators and their requirement for known activating regions have been examined. The results demonstrate that FNR can co-activate when located upstream at around -91 bp in combination with either FNR or CRP downstream. When FNR occupies the downstream site the promoter is co-dependent on an upstream activator, but not when CRP occupies this site. Activating region 1 in CRP (defined by substitutions at residue H159) and its putative equivalent in FNR (defined by substitutions at S73) are mainly required in the upstream activator; the putative equivalent in FNR of activating region 3 of CRP (defined by substitutions at G85 and K52, respectively) is mainly required in the dimer which binds downstream. Activating region 1 of FNR is required only in the downstream subunit of the upstream activator in a promoter which is co-dependent on two FNR dimers. These data suggest that both bound upstream and downstream activators interact with RNA polymerase to promote transcription, and that co-dependence is determined by the nature of the activator plus the promoter context.
先前对含有单个CRP结合位点且该位点位于距转录起始位点41.5 bp处的半合成启动子的研究表明，当第二个用于CRP或FNR的结合位点位于上游约-91 bp处时，转录会增强（协同作用）。大肠杆菌中的ansB启动子由CRP和FNR蛋白的各一个二聚体以共依赖的方式共同激活，其结合位点分别距转录起始位点约-91和-41 bp。同样，沙门氏菌中的同源ansB启动子由两个具有协同作用的CRP二聚体共同激活。通过突变改变了大肠杆菌启动子上的结合位点，以提供许多携带FNR和CRP结合位点其他组合的活性启动子衍生物。已经研究了这些激活剂的共依赖与协同相互作用以及它们对已知激活区域的需求。结果表明，当FNR位于上游约-91 bp处且与下游的FNR或CRP结合时，它可以共同激活。当FNR占据下游位点时，启动子共依赖于上游激活剂，但当CRP占据该位点时则不然。上游激活剂主要需要CRP中的激活区域1（由H159残基处的取代定义）及其在FNR中的假定等效区域（由S73处的取代定义）；CRP激活区域3在FNR中的假定等效区域（分别由G85和K52处的取代定义）主要在结合下游的二聚体中需要。在共依赖于两个FNR二聚体的启动子中，FNR的激活区域1仅在上游激活剂的下游亚基中需要。这些数据表明，结合在上游和下游的激活剂都与RNA聚合酶相互作用以促进转录，并且共依赖性由激活剂的性质加上启动子背景决定。
