RNA polymerase spoiled for choice as transcription begins.
RNA polymerase spoiled for choice as transcription begins.
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当转录开始时，RNA 聚合酶的选择就变多了。
DOI：
10.1073/pnas.2110640118
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发表时间：
2021
影响因子：
11.1
通讯作者：
Busby SJW
中科院分区：
文献类型：
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作者：
Busby SJW
关于转录及其在启动子中的调控的论文在科学文献中比比皆是，但关于起始的报道很少，起始定义为新转录物的第一个3′− 5′磷酸二酯键形成的时刻。在PNAS中，Skalenko et al. (1)与细菌多亚基DNA依赖性RNA聚合酶（RNAP）合作，将联合收割机高通量DNA碱基测序方法与结构分析相结合，以深入了解RNAP如何管理创建第一个键所需的两个伴侣分子，因为每个新的RNA转录本都是如此。教科书描绘了一幅看似简单的转录起始画面：在大多数细菌启动子中，RNAP σ（sigma）亚基协调启动子识别，然后驱动双链DNA的一个多圈打开以形成“转录泡”，这对于读取模板链至关重要（2）。这几乎是由σ亚基的结构域2单独完成的，其与启动子DNA的非模板链产生特异性碱基依赖性相互作用，这使得单链模板链接近RNAP催化位点（3-5）。教科书告诉我们，然后，通过与模板链碱基配对选择两个核苷三磷酸（NTP），表示为“TSS”（转录起始位点）和“TSS+ 1”（下游一个碱基），并且，一旦在产物和添加位点，RNAP催化活性促进磷酸二酯键形成。然而，现实往往并不那么简单，在过去的十年里，罗格斯大学的布莱斯·尼克尔斯（Bryce Nickels）、理查德·埃布赖特（Richard Ebright）和他们的同事们发表了一系列出色的论文，逐步揭开了启蒙的奥秘。第一个复杂性来自RNAP如何管理转录泡中的两条DNA链的不平等（图1）。在起始之前，非模板链中所谓的-10六聚体元件（共有5′-TATAAT-3′）被牢固而精确地固定，A-2和T-6被插入RNAP σ亚基结构域2的识别口袋中（3，5）。相反，单链模板链的定位具有灵活性。因此，TSS和TSS+ 1碱基相对于-10元件的位置因启动子而异，气泡与双链模板DNA的下游连接也是如此。来自Rutgers的先前出版物描述了TSS和TSS+ 1的选择如何由RNAP和模板链之间的多重相互作用驱动（5）。这可以涉及延伸或减少被拉入酶活性位点的单链模板链的长度（6），并且Vvedenskaya等人（7，8）先前设计了一种聪明的高通量方法来找到这方面的规则，从而避免了一次研究一个启动子的需要。为此，通过扰乱-10六聚体元件下游末端后的10个碱基对的序列，产生了一个包含100多万（410）个已充分表征的大肠杆菌乳糖（lac）操纵子启动子变体的质粒载体文库。每个变体在转录物起始点周围携带独特的碱基序列，并且由乱序序列的第二下游区块（其在设定启动子活性中不起作用）条形码化。Vvedenskaya等人使用更下游的引物和连接到从细菌培养物中提取的RNA的5′端的寡核苷酸接头，然后能够使用高通量碱基测序来定位每个启动子变体的转录起始位点。因此，他们可以推导出驱动每个启动子的起始选择的编码链决定簇。这些作者的独创性...
Papers concerning transcription, and its regulation at promoters, abound in the scientific literature, but reports about initiation, defined as the moment that the first 3′− 5′ phosphodiester bond of a new transcript is forged, are scarce. In PNAS, Skalenko et al.(1), working with the bacterial multisubunit DNA-dependent RNA polymerase (RNAP), combine high-throughput DNA base-sequencing methods with structural analysis, to provide insights into how RNAP manages the two partner molecules needed to create that first bond, as each new RNA transcript is born. Textbooks paint a deceptively simple picture of transcript initiation: At the majority of bacterial promoters, the RNAP σ (sigma) subunit orchestrates promoter recognition, and then drives the opening of just over one turn of the duplex DNA to form a “transcription bubble,” which is essential for the template strand to be read (2). This is done almost singlehandedly by domain 2 of the σ subunit, which makes specific base-dependent interactions with the nontemplate strand of the promoter DNA, and this allows the single-stranded template strand access to the RNAP catalytic site (3–5). The textbook tells us that, then, two nucleoside triphosphates (NTPs) are selected by base pairing with the template strand bases, denoted “TSS”(for transcription start site) and “TSS+ 1”(one base downstream), and, once in the product and addition sites, the RNAP catalytic activity facilitates phosphodiester bond formation. However, as so often, the reality is not quite so simple, and, over the past decade, Bryce Nickels, Richard Ebright, and their colleagues at Rutgers University have produced a stellar series of papers that, step by step, have unveiled the mysteries of initiation. The first complication arises from inequalities in how RNAP manages the two DNA strands in the transcription bubble (Fig. 1). Prior to initiation, the socalled− 10 hexamer element (consensus 5′-TATAAT-3′) in the nontemplate strand is held firmly and precisely, with A-2 and T-6 being inserted into recognition pockets in domain 2 of the RNAP σ subunit (3, 5). In contrast, there is flexibility in positioning of the single-stranded template strand. Hence, the location of the bases TSS and TSS+ 1, with respect to the− 10 element, varies from promoter to promoter, as does the downstream junction of the bubble with double-stranded template DNA. Previous publications from Rutgers describe how the choice of TSS and TSS+ 1 is driven by multiple interactions between RNAP and the template strand (5). This can involve extending or reducing the length of the single-stranded template strand drawn into the enzyme active site (6), and Vvedenskaya et al.(7, 8) previously devised a clever high-throughput method to find the rules for this, thereby avoiding the need to study one promoter at a time. To do this, a plasmidborne library of over a million (410) variants of the wellcharacterized Escherichia coli lactose (lac) operon promoter was generated by scrambling the sequence of 10 base pairs following the downstream end of the− 10 hexamer element. Each variant carries a unique base sequence around the transcript start point, and is barcoded by a second downstream block of scrambled sequence (that plays no part in setting promoter activity). Using a farther-downstream primer, and an oligonucleotide adapter, which is ligated to the 5′-end of RNA extracted from bacterial cultures, Vvedenskaya et al. were then able to use high-throughput base sequencing to map the transcript start site at each one of the promoter variants. Thus, they could deduce the coding strand determinants that drive selection of the start at each promoter. The ingenuity of these authors …