Jones Brittnee N, Quang-Dang Duc-Uy, Oku Yuko, Gross John D
Program in Chemistry and Chemical Biology, University of California, San Francisco, California, USA.
Methods Enzymol. 2008;448:23-40. doi: 10.1016/S0076-6879(08)02602-5.
The stability of all RNA polymerase II transcripts depends on the 5'-terminal cap structure. Removal of the cap is a prerequisite for 5' to 3'-decay and is catalyzed by distinct cellular and viral decapping activities. Over the past decade, several decapping enzymes have been characterized through functional and structural studies. An emerging theme is that function is regulated by protein interactions; however, in vitro assays to dissect the effects on enzyme activity are unavailable. Here we present a kinetic assay to monitor decapping by the heterodimeric yeast Dcp1/Dcp2 complex. Kinetic constants related to RNA binding and the rate of the catalytic step can be determined with recombinant enzyme and cap-radiolabeled RNA substrate, allowing substrate specificity and the role of activating factors to be firmly established.
所有RNA聚合酶II转录本的稳定性都依赖于5'-末端帽结构。帽的去除是5'至3'衰变的前提条件，并且由不同的细胞和病毒去帽活性催化。在过去十年中，通过功能和结构研究对几种去帽酶进行了表征。一个新出现的主题是功能受蛋白质相互作用调节；然而，尚无用于剖析对酶活性影响的体外测定方法。在这里，我们提出了一种动力学测定方法，以监测异二聚体酵母Dcp1/Dcp2复合物的去帽过程。与RNA结合以及催化步骤速率相关的动力学常数可以用重组酶和帽放射性标记的RNA底物来确定，从而能够牢固地确定底物特异性和激活因子的作用。
