CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex.
CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex.
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DOI：
10.7554/elife.08505
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发表时间：
2015-09-08
期刊：
影响因子：
7.7
通讯作者：
Campbell EA
中科院分区：
文献类型：
--
作者：
Bae B;Chen J;Davis E;Leon K;Darst SA;Campbell EA
调控基因表达的关键点是转录起始，激活因子起主要作用。CardD是结核分枝杆菌中的一种必需激活剂，存在于许多细菌中，包括栖热菌属，但在大肠杆菌中不存在。为了阐明CardD的分子机制，我们测定了含CardD的栖热菌转录起始复合物的晶体结构。结构表明，CardD与转录泡上游双链/单链DNA连接处呈现的独特DNA拓扑结构相互作用。我们确认，我们的结构对应于功能激活复合物，并扩展我们的理解保守的卡D色氨酸残基的作用，作为一个小沟楔，防止崩溃的转录泡稳定的转录起始复合物。与E. coli RNAP中，许多细菌RNAP形成不稳定的启动子复合物，解释了对CardD的需要。在细胞内，双链DNA分子编码制造蛋白质所需的指令。为了制造蛋白质，组成基因的两条DNA链被分离，其中一条链作为模板来制造信使核糖核酸（或简称mRNA）分子。这个过程称为转录。然后将mRNA用作组装蛋白质的模板。一种称为RNA聚合酶的酶进行转录，并在从细菌到人类和其他动物的所有细胞中发现。细菌具有最简单的RNA聚合酶形式，并提供了一个很好的系统来研究它如何控制转录。它由几种蛋白质组成，这些蛋白质共同作用，以DNA为模板制造RNA。然而，它需要另一种称为sigma因子的蛋白质的帮助，将其引导到称为启动子的DNA区域，该区域位于基因开始之前。当RNA聚合酶和sigma因子相互作用时，产生的蛋白质组被称为RNA聚合酶"全酶"。转录分几个阶段进行。首先，RNA聚合酶全酶定位并结合启动子DNA。接下来，它将两条DNA链分开，并暴露出模板链的一部分。在这一点上，DNA和全酶被认为是在一个“开放的启动子复合物”和部分的启动子DNA是在它被称为“转录泡”。另一种名为CardD的蛋白质有助于加速转录，但尚不清楚这一过程的工作原理。Bae等人现在已经使用X射线晶体学来揭示与RNA聚合酶holoenzme和DNA启动子结合的CardD的结构。结构表明，CardD的一部分在转录泡的开始与DNA相互作用，另一部分与RNA聚合酶结合。CardD位于启动子中的两条DNA链之间，就像一个楔子，使两条DNA链保持分离。因此，CardD稳定了开放启动子复合物并防止转录泡破裂。这些发现揭示了一种以前从未见过的参与激活转录的机制，并将指导进一步的实验探索CardD在活细胞中的作用。Bae，Feklistov等人的另一项研究-这项研究的一些研究人员也参与了这项研究-揭示了sigma因子也在转录泡的开始与DNA结合。这些研究概述的一般原则可能有助于识别其他调节转录的蛋白质。DOI：www.example.com网站
A key point to regulate gene expression is at transcription initiation, and activators play a major role. CarD, an essential activator in Mycobacterium tuberculosis, is found in many bacteria, including Thermus species, but absent in Escherichia coli. To delineate the molecular mechanism of CarD, we determined crystal structures of Thermus transcription initiation complexes containing CarD. The structures show CarD interacts with the unique DNA topology presented by the upstream double-stranded/single-stranded DNA junction of the transcription bubble. We confirm that our structures correspond to functional activation complexes, and extend our understanding of the role of a conserved CarD Trp residue that serves as a minor groove wedge, preventing collapse of the transcription bubble to stabilize the transcription initiation complex. Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter complexes, explaining the need for CarD. DOI: http://dx.doi.org/10.7554/eLife.08505.001 Inside cells, molecules of double-stranded DNA encode the instructions needed to make proteins. To make a protein, the two strands of DNA that make up a gene are separated and one strand acts as a template to make molecules of messenger ribonucleic acid (or mRNA for short). This process is called transcription. The mRNA is then used as a template to assemble the protein. An enzyme called RNA polymerase carries out transcription and is found in all cells ranging from bacteria to humans and other animals. Bacteria have the simplest form of RNA polymerase and provide an excellent system to study how it controls transcription. It is made up of several proteins that work together to make RNA using DNA as a template. However, it requires the help of another protein called sigma factor to direct it to regions of DNA called promoters, which are just before the start of the gene. When RNA polymerase and the sigma factor interact the resulting group of proteins is known as the RNA polymerase ‘holoenzyme’. Transcription takes place in several stages. To start with, the RNA polymerase holoenzyme locates and binds to promoter DNA. Next, it separates the two strands of DNA and exposes a portion of the template strand. At this point, the DNA and the holoenzyme are said to be in an ‘open promoter complex’ and the section of promoter DNA that is within it is known as a ‘transcription bubble’. Another protein called CarD helps to speed up transcription but it is not clear how this stage of the process works. Bae et al. have now used X-ray crystallography to reveal the structure of CarD bound to the RNA polymerase holoenyzme and a DNA promoter. The structures show that one part of CarD interacts with the DNA at the start of the transcription bubble, and another part binds to the RNA polymerase. CarD fits between the two strands of DNA in the promoter, like a wedge, to keep the strands apart. Therefore, CarD stabilizes the open promoter complex and prevents the transcription bubble from collapsing. These findings reveal a previously unseen mechanism involved in activating transcription and will guide further experiments probing the role of CarD in living cells. Another study by Bae, Feklistov et al.—which involves some of the same researchers as this study—reveals that the sigma factor also binds to DNA at the start of the transcription bubble. The general principles outlined by these studies may help to identify other proteins that regulate transcription. DOI: http://dx.doi.org/10.7554/eLife.08505.002