INVIVO THERMODYNAMIC ANALYSIS OF REPRESSION WITH AND WITHOUT LOOPING IN LAC CONSTRUCTS - ESTIMATES OF FREE AND LOCAL LAC REPRESSOR CONCENTRATIONS AND OF PHYSICAL-PROPERTIES OF A REGION OF SUPERCOILED PLASMID DNA INVIVO
INVIVO THERMODYNAMIC ANALYSIS OF REPRESSION WITH AND WITHOUT LOOPING IN LAC CONSTRUCTS - ESTIMATES OF FREE AND LOCAL LAC REPRESSOR CONCENTRATIONS AND OF PHYSICAL-PROPERTIES OF A REGION OF SUPERCOILED PLASMID DNA INVIVO
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DOI：
10.1006/jmbi.1993.1133
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发表时间：
1993-03-05
影响因子：
5.6
通讯作者：
RECORD, MT
中科院分区：
文献类型：
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作者：
LAW, SM;BELLOMY, GR;RECORD, MT
强结合初级 (O1)lac 操纵子位于 alac 启动子控制区上游 100 至 200 个碱基对 (bp) 处，可将大肠杆菌中多拷贝质粒上较弱结合 (Oc)lac 操纵子控制的 alac 启动子的表达降低 3 至 20 倍。大肠杆菌。我们将这种效应归因于环的形成，其中双齿抑制子四聚体与 O1 和 Ocoperators 之间形成热力学稳定的复合物。开发了抑制的热力学模型，以根据 Ocsite 处的游离抑制蛋白浓度和局部抑制蛋白浓度（来自循环）的复合效应来解释这些数据。发现局部阻遏蛋白浓度随着 O1 和 Ocooperators 之间碱基对的距离而周期性变化，范围比 F'Iq 过量产生菌株中阻遏蛋白的游离浓度（即总体热力学活性）大 2 至 20 倍（估计为 ≲0·5 μM）。表达和局部浓度的周期性变化的幅度似乎随着检查范围内操作者距离的增加而减小。循环数据的定量分析提供了体内插入 DNA 区域物理特性的估计。对于127至197 bp范围内的距离，调制周期一致为11·28(±0·04) bp，我们将其解释为体内超螺旋质粒DNA该区域的螺旋重复。这种改变的螺旋重复的可能起源包括超螺旋DNA的全局连接缺陷和由来自包围互操作子区域的启动子的不同转录诱导的任何局部连接缺陷。 DNA环化分析得出该互操作区域的表观体内持久长度为64(±26) Å（约为体内结果的15%），体内扭转刚性常数为1·1(±0·1) × 10-19erg cm，处于体外发现值范围的下限。
A strong-binding primary (O1)lacoperator located 100 to 200 base-pairs (bp) upstream from alacpromoter control region reduces expression from alacpromoter controlled by a weaker-binding (Oc)lacoperator between 3 and 20-fold on a multicopy plasmid inE. coli. We attribute this effect to loop formation in which a thermodynamically stable complex is formed between bidentatelacrepressor tetramers and the O1and Ocoperators. A thermodynamic model for repression is developed to interpret these data in terms of the composite effects of freelacrepressor concentration and of local repressor concentration (from looping) at the Ocsite. The local repressor concentration is found to vary periodically with the distance in base-pairs between the O1and the Ocoperators, ranging from 2 to 20-fold larger than the free concentration (i.e. the bulk thermodynamic activity) of repressor in this F′Iqoverproducing strain (estimated to be ≲0·5 μM). The amplitude of the periodic variation in expression and in local concentration appears to decrease with increasing interoperator distance in the range examined. Quantitative analysis of the looping data provides estimates of the physical properties of the intervening DNA regionin vivo. For distances in the range 127 to 197 bp, the periodicity of modulation is uniformly 11·28(±0·04) bp, which we interpret as the helical repeat of this region of supercoiled plasmid DNAin vivo. Possible origins of this altered helical repeat include the global linking deficit of the supercoiled DNA and any local linking deficit induced by divergent transcription from promoters bracketing the interoperator region. DNA cyclization analysis yields an apparentin vivopersistence length of this interoperator region of 64(±26) Å (which is ∼15% of thein vivoresult) and anin vivotorsional rigidity constant of 1·1(±0·1) × 10-19erg cm, which is at the lower end of the range of values foundin vitro.