ISOLATION OF LAC REPRESSOR
ISOLATION OF LAC REPRESSOR
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DOI：
10.1073/pnas.56.6.1891
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发表时间：
1966-01-01
影响因子：
11.1
通讯作者：
MULLERHI.B
中科院分区：
文献类型：
--
作者：
GILBERT, W;MULLERHI.B
蛋白质的合成通常受阻遏物的控制，这一认识提出了分子生物学中的一个中心问题：控制物质的性质是什么？雅各布和莫诺提出的负控制方案设想，某些基因，即调节基因，产生的产物可以通过细胞质发挥作用，阻止其他基因发挥功能。这些其他基因被组织成具有顺式显性操纵子的操纵子，这些操纵子充当阻遏物的受体。适当的小分子可以作为诱导物，通过阻止阻遏，或者作为辅阻遏物，导致活性阻遏物的存在。诱导系统最简单的明确假设是，控制基因的直接产物本身就是阻遏物，并且该阻遏物与DNA分子上的操纵子位点结合以阻止操纵子的转录。诱导物将与阻遏物联合收割机结合，产生一个不再与操纵子结合的分子，操纵子产生的酶的合成将开始。然而，其他模型也将拟合数据。阻遏物几乎可以有任何靶点来阻断蛋白质合成所需的任何起始过程。对控制过程的分子理解一直在等待一个或多个阻遏物的分离。我们已经开发了一种测定乳糖阻遏物的方法，乳糖阻遏物是乳糖操纵子的控制基因（i基因）的产物。该测定法通过测量其与诱导剂的结合来检测和定量该阻遏物，如在这种情况下通过针对放射性IPTG（异丙基硫代半乳糖苷）的平衡透析所示。为了寻找乳糖阻遏物，我们需要一些不依赖于实际阻遏机制的特定模型的方法。该试验所依据的最低限度的假设是，阻遏物和诱导物之间应该存在某种相互作用。然而，加入到细胞中的诱导物质通常在它们能够触发诱导之前被修饰。乳糖的情况就是如此，它必须被半乳糖苷酶裂解才能诱导，但硫代半乳糖苷似乎是真正的无偿诱导剂;尚未检测到与其作为诱导剂的作用相关的化学修饰。IPTG稳定某些温度敏感的i基因突变体的能力4强烈表明i基因产物直接与该诱导剂相互作用。此外，竞争性诱导抑制剂ONPF（邻硝基苯基岩藻糖苷）的存在也支持了这一论点，ONPF也可以稳定某些泄漏i突变体，其行为就像它驱动阻遏物进入关闭操作子的形式。5实验设计为了检测。通过平衡透析将IPTG与阻遏物结合，必须达到与复合物的解离常数相当的阻遏物浓度。阻遏物对诱导物有什么亲和力？在无渗透酶的菌株中，在2-10-4 M IPTG下发生半最大诱导。单凭这一事实并不能直接得出平衡常数的估计值，因为酶的水平在1891年的变化是1,000倍
The realization that the synthesis of proteins is often under the control of repressors', 2 has posed a central question in molecular biology: What is the nature of the controlling substances? The scheme of negative control proposed by Jacob and Monod envisages that certain genes, regulatory genes, make products that can act through the cytoplasm to prevent the functioning of other genes. These other genes are organized into operons with cis-dominant operators, such operators behaving as acceptors for the repressor. Appropriate small molecules act either as inducers, by preventing the repression, or as corepressors, leading to the presence of active repressor. The simplest explicit hypothesis for inducible systems is that the direct product of the control gene is itself the repressor and that this repressor binds to the operator site on a DNA molecule to prevent the transcription of the operon. The inducer would combine with the repressor to produce a molecule which can no longer bind to the operator, and the synthesis of the enzymes made by the operon would begin. However, other models will also fit the data. Repressors could have almostany target that would serve as a block to any of the initiation processes required to make a protein. A molecular understanding of the control process has waited on the isolation of one or more repressors. We have developed an assay for the lactose repressor, the product of the control gene (i gene) of the lactose operon. The assay detects and quantitates this repressor by measuring its binding to an inducer, as seen in this case by equilibrium dialysis against radioactive IPTG (isopropyl-thio-galactoside). In order to seek the lactose repressor, we desired some means that would not depend on the specific models that might be imagined for the actual mechanism of repression. The minimal assumptionon which the assay is based is that there should besome interaction between the repressor and the inducer.,. However, in-ducing substances added to the cell are often modified before they can trigger induc-tion. Such is the case with lactose which must be split by fl-galactosidase in order to induceI but the thiogalactosides appear to be true gratuitousinducers; no chem-ical modification has yet been detected associated with their action as inducers. The ability of IPTG to stabilize certain temperature-sensitive i-gene mutants4 argues strongly that the i-gene product interacts directly with this inducer. Furthermore, the existence of a competitive inhibitor of induction, ONPF (o-nitrophenylfucoside), which can also stabilize certain leaky i mutants and which behaves as though it drives therepressor into the form that shuts off the operator, also supports this thesis. 5Design of the Experiment.-In order to detect. the binding of IPTG to the re-pressor by equilibrium dialysis, one must achieve concentrations of repressor that are comparable to the dissociation constant of the complex. What affinity does the repressor have for the inducer? Half-maximal induction occurs at 2-10-4 M IPTG in a permeaseless strain. This fact alone does not lead directly to an estimate of the equilibrium constant because the enzyme level changes by a factor of 1,000 on 1891