NONSPECIFIC DNA BINDING OF GENOME-REGULATING PROTEINS AS A BIOLOGICAL-CONTROL MECHANISM - MEASUREMENT OF DNA-BOUND ESCHERICHIA-COLI LAC REPRESSOR INVIVO
NONSPECIFIC DNA BINDING OF GENOME-REGULATING PROTEINS AS A BIOLOGICAL-CONTROL MECHANISM - MEASUREMENT OF DNA-BOUND ESCHERICHIA-COLI LAC REPRESSOR INVIVO
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DOI：
10.1073/pnas.74.10.4228
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发表时间：
1977-01-01
影响因子：
11.1
通讯作者：
VONHIPPEL, PH
中科院分区：
文献类型：
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作者：
KAOHUANG, Y;REVZIN, A;VONHIPPEL, PH
基因组调控蛋白与非特异性DNA位点的结合可能在控制这些蛋白与其特异性靶DNA序列相互作用的热力学和动力学中起重要作用。估计了E. coli lac阻遏物分子在体内与E.大肠杆菌染色体是通过测量阻遏物在产生小细胞的E.大肠杆菌菌株（P678-54）和由其衍生的无DNA小细胞。假设小细胞的细胞质是亲本E.在大肠杆菌细胞中，平均细胞中不到10%的阻遏物四聚体在溶液中是游离的;其余的被认为与细菌染色体结合。从这些结果计算的阻遏物到散装非操纵基因DNA（KRD）的结合常数的最小体内值约为103 M-1，并且对小细胞实验中的误差来源的分析表明，KRD的实际体内值可能更大。KRD的值，结合体外实验数据对离子强度的依赖性，可以用来估计有效的细胞内阳离子活性的E。大肠杆菌中的Na+浓度不大于约0.24 M（可能不小于0.17 M）。小细胞分布实验还证实，诱导物-阻遏物复合物与大量非操纵基因DNA（KRID）结合的缔合常数为约。体内KRD。这些结果用于计算KRO和KRIO（阻遏物和诱导物-阻遏物复合物与操纵子结合的结合常数）的最小体内值，分别为约1012 M-1和约109 M-1。这些结果符合操纵子调控的定量模型，其中非特异性DNA-阻遏物复合物在确定基因表达的基础和组成水平中发挥关键作用。
Binding of genome regulatory proteins to nonspecific DNA sites may play an important role in controlling the thermodynamics and kinetics of the interactions of these proteins with their specific target DNA sequences. An estimate of the fraction of E. coli lac repressor molecules bound in vivo to the operator region and to nonoperator sites on the E. coli chromosome is derived by measurement of the distribution of repressor between a minicell-producing E. coli strain (P678-54) and the DNA-free minicells derived therefrom. Assuming the minicell cytoplasm to be representative of that of the parent E. coli cells, less than 10% of the repressor tetramers of the average cell are free in solution; the remainder are presumed to be bound to the bacterial chromosome. The minimum in vivo value of the association constant for repressor to bulk nonoperator DNA (KRD) calculated from these results is about 103 M-1, and analysis of the sources of error in the minicell experiment suggests that the actual in vivo value of KRD could be substantially greater. The value of KRD, coupled with in vitro data on the ionic strength dependence of this parameter, can be used to estimate that the effective intracellular cation activity of E. coli is no greater than about 0.24 M (and probably no less than 0.17 M) in terms of Na+ equivalents. The minicell distribution experiments also confirm that the association constant for the binding of inducer-repressor complex to bulk nonoperator DNA (KRID) is .simeq. KRD in vivo. These results are used to calculate minimum in vivo values of KRO and KRIO (association constants for repressor and for inducer-repressor complex binding to operator) of about 1012 M-1 and about 109 M-1, respectively. The results fit a quantitative model for operon regulation in which nonspecific DNA-repressor complexes play a key role in determining basal and constitutive levels of gene expression.