Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
Chembiochem. 2010 May 3;11(7):972-7. doi: 10.1002/cbic.201000041.
A light-activatable bacteriophage T7 RNA polymerase (T7RNAP) has been generated through the site-specific introduction of a photocaged tyrosine residue at the crucial position Tyr639 within the active site of the enzyme. The photocaged tyrosine disrupts polymerase activity by blocking the incoming nucleotide from reaching the active site of the enzyme. However, a brief irradiation with nonphototoxic UV light of 365 nm removes the ortho-nitrobenzyl caging group from Tyr639 and restores the RNA polymerase activity of T7RNAP. The complete orthogonality of T7RNAP to all endogenous RNA polymerases in pro- and eukaryotic systems allowed for the photochemical activation of gene expression in bacterial and mammalian cells. Specifically, E. coli cells were engineered to produce photocaged T7RNAP in the presence of a GFP reporter gene under the control of a T7 promoter. UV irradiation of these cells led to the spatiotemporal activation of GFP expression. In an analogous fashion, caged T7RNAP was transfected into human embryonic kidney (HEK293T) cells. Irradiation with UV light led to the activation of T7RNAP, thereby inducing RNA polymerization and expression of a luciferase reporter gene in tissue culture. The ability to achieve spatiotemporal regulation of orthogonal RNA synthesis enables the precise dissection and manipulation of a wide range of cellular events, including gene function.
一种光激活的噬菌体 T7 RNA 聚合酶（T7RNAP）通过在酶的活性位点内关键位置 Tyr639 处特异性引入光笼酪氨酸残基而产生。光笼酪氨酸通过阻止进入的核苷酸到达酶的活性位点来破坏聚合酶活性。然而，用非光毒性的 365nm 紫外光短暂照射会从 Tyr639 上去除邻硝基苄基笼状基团，并恢复 T7RNAP 的 RNA 聚合酶活性。T7RNAP 与原核和真核生物系统中所有内源性 RNA 聚合酶的完全正交性允许在细菌和哺乳动物细胞中进行光化学基因表达的激活。具体来说，通过 GFP 报告基因在 T7 启动子的控制下，工程菌细胞被设计为在存在 GFP 报告基因的情况下产生光笼 T7RNAP。对这些细胞进行 UV 照射会导致 GFP 表达的时空激活。以类似的方式，将光笼 T7RNAP 转染到人胚肾（HEK293T）细胞中。用紫外光照射会导致 T7RNAP 的激活，从而诱导 RNA 聚合酶和组织培养中荧光素酶报告基因的表达。实现正交 RNA 合成时空调节的能力使我们能够精确剖析和操纵广泛的细胞事件，包括基因功能。
