Han Lu, Marcus Erin, Phizicky Eric M
Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, NY, United States; Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, United States; Department of Genetics, The Blavatnik Institute, Harvard Medical School, Boston, MA, United States.
Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, NY, United States.
Methods Enzymol. 2021;658:359-377. doi: 10.1016/bs.mie.2021.06.010. Epub 2021 Jul 14.
A tRNA interacts with numerous proteins throughout its biogenesis and during translation, and a significant portion of these interacting proteins are involved in post-transcriptional modifications. While some of the modifying enzymes use relatively simple recognition elements for substrate recognition, many enzymes selectively modify a specific subset of tRNA species without obvious recognition rules. In this chapter we describe a semi-quantitative pull-down assay to study tRNA substrate specificity of modification enzymes, by using the yeast Saccharomyces cerevisiae mC methyltransferase Trm140 as an example. We also discuss some overall considerations for a successful pull-down experiment, with a focus on practical applications of the dissociation constant K between the protein and the tRNA and the off-rate.
转运RNA（tRNA）在其生物合成过程以及翻译过程中会与众多蛋白质相互作用，并且这些相互作用的蛋白质中有很大一部分参与转录后修饰。虽然一些修饰酶使用相对简单的识别元件来识别底物，但许多酶会选择性地修饰特定的tRNA种类子集，却没有明显的识别规则。在本章中，我们以酿酒酵母mC甲基转移酶Trm140为例，描述一种用于研究修饰酶的tRNA底物特异性的半定量下拉实验。我们还讨论了成功进行下拉实验的一些总体注意事项，重点关注蛋白质与tRNA之间的解离常数K和解离速率的实际应用。
