Direct evidence for the preferential binding of Escherichia coli RNA polymerase holoenzyme to the ends of deoxyribonucleic acid restriction fragments.
Direct evidence for the preferential binding of Escherichia coli RNA polymerase holoenzyme to the ends of deoxyribonucleic acid restriction fragments.
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大肠杆菌 RNA 聚合酶全酶优先结合脱氧核糖核酸限制性片段末端的直接证据。
DOI：
10.1021/bi00291a017
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发表时间：
1983
期刊：
影响因子：
2.9
通讯作者：
RecordJr,MT
中科院分区：
文献类型：
--
作者：
Melançon,P;Burgess,RR;RecordJr,MT
Paul Melanfon，** Richard R. Burgess和M. Jesus，Jr.*摘要：在用硝酸纤维素滤膜测定法进行的实验中，已经观察到大肠杆菌RNA聚合酶全酶与DNA限制性片段形成多种非启动子复合物[Melanfon，P.，伯吉斯河R.，& Record，M. T.，Jr.（1982）Biochemistry 21，4318-4331]，在此，我们报告了使用该测定来研究RNA聚合酶核心和全酶与1600个碱基对（bp）的片段的弱（肝素敏感的）相互作用的方面。T7 DNA不含启动子或TB（紧密结合;肝素抗性）位点。在研究的离子条件下[50 mM NaCl/10 mM MgCl 2/10 mM N-（2-羟乙基）哌嗪-A-乙磺酸钠（pH 7.7）]，核心和全酶均与线性DNA片段结合，并导致相当水平的过滤器保留。当DNA片段自连接成环状分子（非超螺旋）时，全酶的结合程度（但不是核心的结合程度）显著降低。这直接证明了我们先前的假设，即全酶识别并优先结合DNA片段的末端，这种结合模式是非启动子片段的大部分肝素敏感性过滤器保留的原因。大肠杆菌RNA聚合酶（RNAP）是大肠杆菌中负责RNA合成的多亚基酶。大肠杆菌中存在两种主要形式：核心（亚基结构ce 2 PP '）和全酶（a2 PP' a）。额外的α亚基的存在允许全酶识别DNA上的特定区域（启动子），从那里RNA合成正确有效地启动。这两种酶都对DNA表现出一般的亲和力。全酶的一些非启动子相互作用可能在启动子搜索机制中起作用[参见von Hippel et al.（1982）]。此外，在用于研究与启动子结合的大多数体外条件下，它们显著降低全酶的溶液浓度。当使用硝酸纤维素滤膜结合试验时，除了游离酶浓度的降低外，还必须考虑结合在非启动子位点的RNAP对DNA的保留潜力。广泛的技术已被用于研究这种非特异性相互作用。Shaner等人提供了对结果的调查。（1983年）。最近，我们通过使用硝酸纤维素过滤结合试验（Melanfon et al.
Paul Melanfon,** Richard R. Burgess, and M. ThomasRecord, Jr.* abstract: Escherichia coli RNA polymerase holoenzyme has been observed to form a variety of nonpromoter complexes with DNA restriction fragments in experiments performed with the nitrocellulose filter assay [Melanfon, P., Burgess, R. R., & Record, M. T., Jr.(1982) Biochemistry 21, 4318-4331], Here we report the use of this assay to investigate aspects of the weak (heparin-sensitive) interactions of RNA polymerase core and holoenzyme with a 1600 base pair (bp) fragment of. T7 DNA whichcontains no promoters or TB (tight binding; heparin-resistant) sites. Under the ionic conditions investigated [50 mM NaCl/10 mM MgCl2/10 mM sodium N-(2-hydroxyethyl) piperazine-A-ethanesulfonic acid (pH 7.7)], both core and holoenzyme bind to the linear DNA fragment and cause comparable levels of filter retention. When the DNA fragment is self-ligated into a circular molecule (non-supercoiled), the extent of binding of holoenzyme (but not that of core) is dramatically reduced. This directly proves our previous hypotheses that holoenzyme recognizes and prefer-entially binds to the ends of DNA fragments and that this mode of binding is responsible for most of the heparin-sensitive filter retention of nonpromoter fragments. The residual modeEscherichia coli RNA polymerase (RNAP), 1 the multi-subunit enzyme responsible for the synthesis of RNA in E. coli, exists in two major forms: core (subunit structure ce2PP') and holoenzyme (a2PP'a). Theadditional presence of the a subunit allows the holoenzyme to recognize specific (promoter) regions on the DNA, from which RNA synthesis is correctly and efficiently initiated. Bothforms of the enzyme exhibit general affinities for DNA. Some nonpromoter interactions of holoenzyme may play a role in the promoter search mechanism [see von Hippel et al.(1982)]. In addition, they reduce significantly the solution concentration of holoenzyme under most in vitro conditions used to investigate binding to promoters. When the nitrocellulose filter binding assay is used, the potential for retention of DNA by RNAP bound at non-promoter sites must be considered in addition to the reduction of free enzyme concentration. A wide range of techniques has been used to study such nonspecific interactions. A survey of the results is provided by Shaner et al.(1983). We have recently studied the interactions between RNAP holoenzyme and an unfractionated HaeIII digest of T7 DNA by using the nitrocellulose filter binding assay (Melanfon et