Chemical synthesis and biological studies on mutated gene-control regions.
Chemical synthesis and biological studies on mutated gene-control regions.
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突变基因控制区域的化学合成和生物学研究。
DOI：
10.1101/sqb.1983.047.01.048
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发表时间：
1983
期刊：
影响因子：
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通讯作者：
Stabinsky,Y
中科院分区：
文献类型：
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作者：
Caruthers,MH;Beaucage,SL;Efcavitch,JW;Fisher,EF;Goldman,RA;deHaseth,PL;Mandecki,W;Matteucci,MD;Rosendahl,MS;Stabinsky,Y
许多重要的生物化学过程依赖于各种蛋白质与确定的DNA序列的相互作用。一些实例是复制、转录、某些形式的重组、基因表达的阳性和阴性控制以及宿主限制性修饰酶。然而，这些蛋白质识别和结合独特的多核苷酸序列的机制知之甚少。我们研究的一个主要重点是了解这些识别过程。我们的方法是通过化学合成来操纵和修饰特定的基因控制区域，然后研究这些序列改变的DNA如何与适当的蛋白质相互作用。在过去，我们的研究主要集中在lac阻遏物-lac操纵子（Camthers 1980）和cI阻遏物-k操纵子（Kawashima et al.1977）系统上。目前，我们也在研究SV 40 T抗原、大肠杆菌RNA聚合酶、cAMP受体蛋白（CRP）和cro阻遏物与适当基因控制区的相互作用。在本文中，我们概述了我们最近的结果，旨在了解如何E。coli RNA聚合酶和CRP识别E. coli启动子并激活转录过程。我们在几个重要的生物化学问题上的进展，传统上一直受到限制，我们不能快速合成脱氧寡核苷酸的定义序列。例如，我们涉及/ac操纵子-/ac阻遏子系统的研究需要准备大约50个操纵子。综合这些算子是一项艰巨的任务。因此，我们启动了一项计划，设计一种快速、有效、非化学家也能使用的合成方法。通过这种方式，重要的生物化学问题的研究将是我们研究中的速率决定步骤，而不是合成脱氧寡核苷酸所需的时间。与该方法相关的化学反应先前已有报道（Beaucage和卡鲁瑟斯1981; Matteucci和卡鲁瑟斯1981）。在本文中，我们概述了我们目前的合成周期，并总结了我们的整体合成方法。
Many biochemicaUy important processes are dependent on the interaction of various proteins with defined sequences of DNA. Some examples are replication, transcription, certain forms of recombination, positive and negative control of gene expression, and host restriction-modification enzymes. However, the mechanisms whereby these proteins recognize and bind to unique polynucleotide sequences are poorly understood. A major emphasis of our research is to understand these recognition processes. Our approach is to manipulate and modify specifically a gene-control region by chemical synthesis and then to study how these sequence-altered DNAs interact with the appropriate proteins. In the past, our research has focused on the lac repressor-lac operator (Camthers 1980) and the cI repressor-k operator (Kawashima et al. 1977) systems. Currently, we are also examining the interaction of SV40 T antigen, Escherichia coli RNA polymerase, cAMP receptor protein (CRP), and cro repressor with appropriate gene-control regions. In this paper we outline our recent results directed toward understanding how E. coli RNA polymerase and CRP recognize E. coli promoters and activate the transcription process. Our progress on several important biochemical problems has traditionally been constrained by our inability to synthesize rapidly deoxyoligonucleotides of defined sequence. For example, our research involving the/ac operator-/ac repressor system required the preparation of approximately 50 operators. The synthesis of these operators was an enormous task. We therefore initiated a program to design a synthetic methodology that was rapid, efficient, and accessible to nonchemists. In this way the investigation of important biochemical problems would be the rate-determining step in our research, rather than the time required to synthesize deoxyoligonucleotides. The chemistry associated with this methodology has been reported previously (Beaucage and Caruthers 1981; Matteucci and Caruthers 1981). In this paper we outline our current synthesis cycle and summarize our overall synthetic approach.