其实转录因子结合DNA吗?DNA足迹和凝胶转变化验

研究人员之一,这是常识转录因素带来的变化基因表情,至少部分通过直接绑定到特殊网站内细胞的DNA。但是科学家决定是否和如何发生这种绑定在哪里?几个技术可用于研究转录因子结合,包括DNA足迹和电泳迁移率改变分析(emsa),也被称为凝胶转变化验。这两种技术是分析的基础基因调控。

4 (lane 5), CoSO₄ (lane 6), or NiSO₄ (lane 7).", "true", "All rights reserved.", '230', '629', 'http://www.cell.com');">

4 (lane 5), CoSO₄ (lane 6), or NiSO₄ (lane 7).", "true", "All rights reserved.", '230', '629', 'http://www.cell.com');">图1

4 (lane 5), CoSO₄ (lane 6), or NiSO₄ (lane 7).", "true", "All rights reserved.", '230', '629', 'http://www.cell.com');"> 4 (lane 5), CoSO₄ (lane 6), or NiSO₄ (lane 7).", '230','http://www.cell.com', "Each lane shows a series of bands of continually decreasing size from top to bottom. Lane 1 shows DNA fragments after digestion in the absence of the transcription factor Sp1. Lane 2 shows DNA fragments after digestion in the presence of Sp1. In this lane, a range of bands is missing, indicating that there is a region of DNA that is protected from digestion by the binding of Sp1. Lane 3 shows the digestion results of Sp1 plus EDTA, a compound that chelates metal ions. The banding pattern is similar to digestion of DNA without Sp1. The next four lanes show digestion results of aklpoSp1 with a variety of metal sulfates. ApoSp1 was incubated with water as a negative control in lane 4, with zinc sulfate (ZnSO₄) in lane 5, with cobalt (II) sulfate (CoSO₄) in lane 6, and with nickel (II) sulfate (NiSO₄) in lane 7. When incubated with zinc sulfate, apoSp1 is able to bind to DNA and protect the DNA from digestion. In contrast, when incubated with water, cobalt (II) sulfate, or nickel (II) sulfate, apoSp1 is unable to bind to DNA and the DNA is digested in a similar pattern as DNA without Sp1.")" class="inlineLinks">细节图

DNA碳足迹是一个在体外技术用于研究蛋白质绑定到特定区域的DNA。当这种技术巧妙地利用了事实转录因子绑定到DNA与一定的亲和力,DNA免受核酸酶降解。感兴趣的转录因子从而叶子对DNA的“足迹”。

典型的足迹实验涉及放射性标记的DNA区域interest-usually一块更大的DNA可能包含一个或多个转录因子结合位点。这个片段是放射性标记的一端,然后孵化在体外都有或没有感兴趣的转录因子。接下来,DNA被核酸酶,如DNAse我消化只保护DNA。最后,产生的DNA产品消化分离聚丙烯酰胺凝胶,这是干,对x射线胶片实现可视化。由此产生的图像被称为一个放射能照像。

图1显示了一个示例的放射能照像造成典型的DNA足迹分析。看到足迹,比较两个左边的车道。梯子的乐队不断减少大小的结果DNAse消化的DNA。然而,在转录因子的存在Sp1,一系列从巷带尺寸缺失,指示一个保护区域的DNA被绑定到Sp1。重要的是要注意,DNAse消化通常不允许继续完成;这些部分消化导致一系列片段长度。的长度包含Sp1结合位点的DNA片段可以由包括标准车道(即车道包含DNA片段的大小)。

如图1所示的结果还要考虑锌的作用(锌)Sp1-DNA绑定。Sp1是一个蛋白质有多个锌指图案,或地区被绑定到锌;然而,EDTA的包容,一个代理,金属离子螯合物,抑制锌绑定。因此,注意在EDTA的车道,Sp1和DNA之间的绑定。此外,请注意,当调查人员添加离子锌(5道),钴(巷6),和镍(巷7),他们看到,只有锌离子的加入导致Sp1绑定。

凝胶转变化验,或者emsa,另一种方法被用来研究转录因子结合。在这里,基本概念是一段DNA迁移通过凝胶更慢如果是绑定到一个蛋白质,如转录因子。差异,或“转变”,的速度迁移转录因子的存在和没有因此作为绑定的证据。

科学家可以用分子生物学技术来创建不同的蛋白质片段,以确定哪些特定的蛋白质结合一段DNA的一部分。例如,在一个测定凝胶转变,可以诱导表达质粒编码多肽截断蛋白质。唯一的蛋白质碎片,一定会在这个实验中使用的DNA是那些包含在一个特定的最小大小。当然,时间越长氨基酸链,越大产生的蛋白质,这意味着蛋白质需要更长的时间通过凝胶。因此,通过检查结果和注意绑定时不发生氨基酸的蛋白质含有n #但却发生在蛋白质n + 100 #的氨基酸,科学家们就能够得出这样的结论:氨基酸n和n + 100之间protein-DNA所必需的交互在考虑。

凝胶转变的一个重要要求化验是胶体电泳条件不扰乱protein-DNA交互。这些被称为nondenaturing条件,它们不同于用于常规DNA可视化条件。

32P-labelled double-stranded Foot A was incubated with nuclear extracts from HepG2 cells in the presence or absence of antibodies as indicated. Arrows indicate the supershifted band generated with antibodies to HNF-4α and HNF-4γ. F, free DNA probes; N.E., nuclear extracts from HepG2 cells.", "true", "All rights reserved.", '700', '453', 'http://www.biochemj.org/bj');">

32P-labelled double-stranded Foot A was incubated with nuclear extracts from HepG2 cells in the presence or absence of antibodies as indicated. Arrows indicate the supershifted band generated with antibodies to HNF-4α and HNF-4γ. F, free DNA probes; N.E., nuclear extracts from HepG2 cells.", "true", "All rights reserved.", '700', '453', 'http://www.biochemj.org/bj');">图2

32P-labelled double-stranded Foot A was incubated with nuclear extracts from HepG2 cells in the presence or absence of antibodies as indicated. Arrows indicate the supershifted band generated with antibodies to HNF-4α and HNF-4γ. F, free DNA probes; N.E., nuclear extracts from HepG2 cells.", "true", "All rights reserved.", '700', '453', 'http://www.biochemj.org/bj');"> 32P-labelled double-stranded Foot A was incubated with nuclear extracts from HepG2 cells in the presence or absence of antibodies as indicated. Arrows indicate the supershifted band generated with antibodies to HNF-4α and HNF-4γ. F, free DNA probes; N.E., nuclear extracts from HepG2 cells.", '700','http://www.biochemj.org/bj', "A) The results of the gel shift and competition experiments indicate that the protein hepatocyte nuclear factor-4 (HNF-4) is involved in gene regulation. Lane 1 shows the results for a sample containing only the radioactive DNA probe (Foot A) for the DD4 gene promoter. Addition of nuclear extract to Foot A produces a shift in the location of a band, indicating that the probe bound to a protein in the nuclear extract (lane 2). When an excess of unlabeled Foot A was added, the shifted band was not visible (lane 3). However, when an unlabeled mutated Foot A sequence (Foot Am) was added, the shifted band was still present (lane 4). Addition of an unlabeled sequence containing a known HNF-4 binding site, α1-AT-A, interfered with formation of the shifted complex (lane 5). Interference did not occur upon addition of a sequence with a known HNF-1 binding site (α1-AT-B; lane 6). When radiolabeled Foot Am was added in the absence (lane 7) or presence (lane 8) of nuclear extract, no DNA-protein complex was observed. B) To identify specific protein components of the DNA-protein complex, the researchers performed a supershift assay and added antibodies that only recognized one specific protein. Lane 1 shows the results for a sample containing only the radioactive DNA probe (Foot A) for the DD4 gene promoter. Addition of nuclear extract to Foot A produced a shift in the location of a band. Lanes 3 to 6 show the results following addition of antibodies to the labeled probe and nuclear extract. Lane 3 shows a supershift following addition of the HNF-4α antibody. In lane 4, a supershift is also observed upon addition of the HNF-4γ antibody. In lanes 5 and 6, no supershift was observed when HNF-1α or HNF-1β antibodies were added, respectively.")" class="inlineLinks">细节图

几个修改可以被纳入基本测定凝胶转变过程加强质量的结果。例如,提供进一步证明protein-DNA绑定交互是独一无二的,或者针对一个特定的序列,研究人员经常执行所谓的竞争化验。这样的分析不仅涉及放射性标记(“热”)DNA,但也添加过多的标记(“冷”)的DNA序列相同。在这里,冷DNA序列应该取代热DNA转录因子结合的伴侣,从而消除检测放射能照像乐队转变,只有热DNA检测。逆转冷凝胶转变的已知序列的DNA序列提供了证据的重要性,在转录因子结合。

虽然竞争分析提供了DNA序列的确认参与绑定,确认身份的蛋白质引起的转变是由supershift化验。supershift控制尤为重要,如果源核转录因子的提取,因为多个转录因子将出现在这样一个提取;然而,如果一个纯化转录因子用于凝胶实验转变,supershift控制不太重要。supershift分析利用事实抗体特别感兴趣的结合蛋白可以被孤立。在这里,添加一个抗体具体到一个已知的转录因子的DNA DNA蛋白质复杂的怀疑是应该引起更大nondenaturing凝胶迁移率的变化,因此引发了“supershift。”

为了更好地理解这项技术是如何工作的,考虑的一项研究,调查人员检查摘要基因的调控DDR。检查启动子地区的DDR肝细胞的核因子显示共识序列(HNF)蛋白质(图2;大关et al .,2001)。具体地说,研究人员怀疑这种蛋白质家族的一个成员,HNF-4,参与基因调控,而另一个家庭成员,HNF-1,不是。该研究小组因此放射性探针启动子区域他们学习和称之为脚;他们的证据表明,蛋白质在细胞中核绑定到这个调查。之后,结合标记探针与核提取物(“N.E.”表示为图),研究人员指出一个明确的转变探针的位置(巷2)。

接下来,研究者进行了竞争实验使用过多的脚,无标号;在这种情况下,将不再是可见的。事实上,混合多余的标记探针(巷3脚)导致一个完整的转移产品的损失。(需要注意的是,核中的蛋白质提取仍是绑定到探测器,但这个特定protein-DNA复杂无法看到,因为大多数绑定标记探针,这个实验不能检测到。)调查人员还可以干扰或竞争,蛋白质的核提取时添加了一个序列中含有一个已知HNF-4结合位点(α₁在一个),但是这个干扰没有发生时,添加了一个序列与已知HNF-1结合位点(α₁在b)。

虽然这第一个实验证实,有核提取物中绑定到探测器,特定的相互作用分子没有确定。为了明确表明,这是一个HNF-4蛋白质绑定子,调查人员执行supershift化验。通过混合标记探针和核提取物,研究小组改革复合物。然后,为了识别dna蛋白质的特定蛋白质成分复杂,他们加入了抗体只识别一个特定的蛋白质。结果supershifts(如图2 b)透露,HNF-4α和HNF-4γ,但不是HNF-1α或HNF-1β,绑定到脚探针。在这些实验中,antibody-protein-DNA复合物迁移更慢于protein-DNA复合物(由箭头的位置指出),从而允许调查人员跟踪特定的绑定supershifted乐队。

在一起,DNA足迹和凝胶转变分析提供基本信息物理转录因子与DNA之间的相互作用。因此,这些技术都留下了不朽的足迹在基因调控的研究。