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Hung DC Downey JS Ayala EA Kreth J Mair R Senadheera DB Qi F Cvitkovitch DG Shi W Goodman SD 《Journal of bacteriology》2011,193(14):3642-3652
In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT. 相似文献
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Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. 总被引:708,自引:149,他引:559
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We describe the use of gel electrophoresis in studies of equilibrium binding, site distribution, and kinetics of protein-DNA interactions. The method, which we call protein distribution analysis, is simple, sensitive and yields thermodynamically rigorous results. It is particularly well suited to studies of simultaneous binding of several proteins to a single nucleic acid. In studies of the lac repressor-operator interaction, we found that binding to the so-called third operator site (03) is 15-18 fold weaker than operator binding, and that the binding reactions with the first and third operators are uncoupled, implying that there is no communication between the sites. Pseudo-first order dissociation kinetics of the repressor-203 bp operator complex were found to be temperature sensitive, with delta E of 80 kcal mol-1 above 29 degrees C and 26 kcal mol-1 below. The half life of the complex (5 min at 21 degrees C) is shorter than that reported for very high molecular weight operator-containing DNAs, but longer than values reported for much shorter fragments. The binding of lac repressor core to DNA could not be detected by this technique: the maximum binding constant consistent with this finding is 10(5) M-1. 相似文献
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CS3纤毛抗原表达调控机理的研究 总被引:2,自引:0,他引:2
CS3是某些肠毒素大肠杆菌菌体表面上的多聚物,它能使病原菌粘附于宿主的小肠上皮细胞上,是致病的重要因素.为了探索CS3菌毛抗原基因的表达调控机制,根据CS3亚基结构基因的核苷酸序列分析表明,在其翻译起始位点的上游存在着rbs位点及原核启动子的-10区和-35区DNA序列.采用基因重组技术将CS3结构基因上游120bp的DNA片段亚克隆进缺乏启动子而只含报告基因lacZ的质粒pCB267中.凝胶滞留和启动报告基因表达的实验证明了CS3亚基结构基因具有自身的启动子(Ps).将该启动子上游区域不同长度的核苷酸片段克隆进pCB267中,报告基因表达结果表明CS3结构基因的表达受其上游区域的抑制.核苷酸序列分析发现,在Ps-35区上游550bp和840bp处各存在一个富A-T簇.结合原核启动子的一般作用规律推知,CS3的表达可能受DNA结合蛋白型的正向调节因子的作用.用CFA/1菌毛抗原基因的正向调节基因cfaD对CS3基因进行的互补表达试验表明cfaD基因不仅可消除上游区对Ps的抑制,而且可大幅度地提高Ps的启动能力.在分析表达调控的基础上获得CS3重组高效表达.同时提出了其表达调控模型. 相似文献
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Footprinting studies of specific complexes formed by RepA, a replication initiator of plasmid pCU1, and its binding site
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The basic replicon of plasmid pCU1 contains three different replication origins. Replication initiated from the oriB origin requires pCU1-encoded protein RepA. Previously, information analysis of 19 natural RepA binding sequences predicted a 20-bp sequence as a RepA binding site. Guanines contacting RepA in the major groove of DNA have also been determined. In this study, we used the missing-nucleoside method to determine all of the bases relevant to RepA binding. The importance of some thymine bases was also confirmed by a missing-thymine site interference assay. Participation of the 5-methyl groups of two thymines (at positions -6 and 7) in RepA binding was pointed out by a missing-thymine methyl site interference assay. Phosphate groups of the DNA backbone which strongly interfered with RepA binding upon ethylation were also identified. The pattern of contacting positions mapped by hydroxyl radical protection footprinting indicates that RepA binds to one face of B-form DNA. The length of the binding site was found to be 20 bp by dissociation rate measurement of complexes formed between RepA and a variety of binding sequences. The symmetry of the binding site and that of the contacting bases, particularly the reacting 5-methyl groups of two thymines, suggest that pCU1-encoded RepA may contact its site as a homodimer. 相似文献
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Interaction of the noncovalent molecular adapter, beta-cyclodextrin, with the staphylococcal alpha-hemolysin pore
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Cyclodextrins act as noncovalent molecular adapters when lodged in the lumen of the alpha-hemolysin (alphaHL) pore. The adapters act as binding sites for channel blockers, thereby offering a basis for the detection of a variety of organic molecules with alphaHL as a biosensor element. To further such studies, it is important to find conditions under which the dwell time of cyclodextrins in the lumen of the pore is extended. Here, we use single-channel recording to explore the pH- and voltage-dependence of the interaction of beta-cyclodextrin (betaCD) with alphaHL. betaCD can access its binding site only from the trans entrance of pores inserted from the cis side of a bilayer. Analysis of the binding kinetics shows that there is a single binding site for betaCD, with an apparent equilibrium dissociation constant that varies by >100-fold under the conditions explored. The dissociation rate constant for the neutral betaCD molecule varies with pH and voltage, a result that is incompatible with two states of the alphaHL pore, one of high and the other of low affinity. Rather, the data suggest that the actual equilibrium dissociation constant for the alphaHL. betaCD complex varies continuously with the transmembrane potential. 相似文献