Active RNA polymerases: mobile or immobile molecular machines?
Active RNA polymerases: mobile or immobile molecular machines?
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DOI：
10.1371/journal.pbio.1000419
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发表时间：
2010-07-13
期刊：
影响因子：
9.8
通讯作者：
Cook PR
中科院分区：
文献类型：
--
作者：
Papantonis A;Larkin JD;Wada Y;Ohta Y;Ihara S;Kodama T;Cook PR
尽管人们普遍认为，活性RNA聚合酶是沿着它的模板运动的，但我们发现是DNA而不是聚合酶运动的，这表明聚合酶是通过旋转模板来工作的。人们普遍认为，活性RNA聚合酶沿着它们的模板来产生转录物。我们使用染色体构象捕获来测试这一点，人类基因被肿瘤坏死因子α (TNFα)快速同步打开；一种是221 kbp的SAMD4A，聚合酶转录需要1个多小时。刺激10分钟后，SAMD4A启动子与其他tnf α响应启动子聚集在一起。随后，这些接触随着新的下游接触的出现而消失；被转录的序列之间总是有接触。超分辨率显微镜证实新生转录本（通过RNA荧光原位杂交检测）在相关时间共定位。结果与另一种转录观点是一致的：固定在工厂中的聚合酶卷绕在各自的模板中，因此模板的不同部分暂时放在一起。我们都学过，RNA聚合酶通过扩散到启动子，开始转录，然后像机车一样沿着DNA模板跟踪而变得活跃。我们使用肿瘤坏死因子α (TNFα)来打开两个人类基因的转录，这两个基因在遗传图谱上相距很远，然后测量它们在三维核空间中的距离。如果我们被教导的是真的，就没有理由认为这两种基因会同时说谎。我们使用两种不同的技术（切割/连接附近的序列和超分辨率显微镜）发现，这两个基因最初是分开的；然后，在特定时刻被转录的基因部分会短暂地接近。我们的研究结果与一个模型是一致的，在这个模型中，基因扩散到一群聚合酶（一个转录工厂）中，转录本作为固定的聚合酶在它们各自的模板中旋转而产生。移动的是DNA，而不是聚合酶。
Although it is widely assumed that active RNA polymerase tracks along its template, we find that DNA, not the polymerase, moves, suggesting that polymerase works by reeling in the template. It is widely assumed that active RNA polymerases track along their templates to produce a transcript. We test this using chromosome conformation capture and human genes switched on rapidly and synchronously by tumour necrosis factor alpha (TNFα); one is 221 kbp SAMD4A, which a polymerase takes more than 1 h to transcribe. Ten minutes after stimulation, the SAMD4A promoter comes together with other TNFα-responsive promoters. Subsequently, these contacts are lost as new downstream ones appear; contacts are invariably between sequences being transcribed. Super-resolution microscopy confirms that nascent transcripts (detected by RNA fluorescence in situ hybridization) co-localize at relevant times. Results are consistent with an alternative view of transcription: polymerases fixed in factories reel in their respective templates, so different parts of the templates transiently lie together. We were all taught that an RNA polymerase becomes active by diffusing to a promoter, initiating transcription, and then tracking like a locomotive down the DNA template. We test this using tumour necrosis factor alpha (TNFα) to switch on transcription of two human genes which lie far apart on the genetic map and then measure how close the two are in 3D nuclear space. If what we were taught were true, there is no reason to expect the two genes to lie together. What we find—using two different techniques (cutting/ligating nearby sequences, and super-resolution microscopy)—is that the two genes are initially apart; then the parts of the genes being transcribed at a particular moment transiently come into close proximity. Our results are consistent with a model in which genes diffuse to a cluster of polymerases—a transcription factory—with transcripts being made as immobile polymerases reel in their respective templates. The DNA moves, not the polymerase.
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