Gracia Brant, Russell Rick
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.
Methods Mol Biol. 2014;1086:225-37. doi: 10.1007/978-1-62703-667-2_13.
For structured RNAs that possess catalytic activity, this activity provides a powerful probe for measuring the progress of folding and the effects of RNA chaperone proteins on the folding rate. The crux of this approach is that only the natively folded RNA is able to perform the catalytic reaction. This method can provide a quantitative measure of the fraction of native RNA over time, and it can readily distinguish the native state from all misfolded conformations. Here we describe an activity-based method measuring native folding of ribozymes derived from self-splicing group I introns, and we show how the assay can be used to monitor acceleration of native folding by DEAD-box RNA helicase proteins that function as general RNA chaperones. By measuring the amount of substrate that is converted to product in a rapid first turnover, we describe how to determine the fraction of the ribozyme population that is present in the native state. Further, we describe how to perform a two-stage or discontinuous assay in which folding proceeds in stage one and then solution conditions are changed in stage two to permit catalytic activity and block further folding. This protocol allows folding to be followed under a broad range of solution conditions, including those that do not support catalytic activity, and facilitates studies of chaperone proteins.
对于具有催化活性的结构化RNA而言，这种活性为测量折叠进程以及RNA伴侣蛋白对折叠速率的影响提供了一种强大的探针。该方法的关键在于只有天然折叠的RNA能够进行催化反应。此方法能够提供天然RNA随时间变化的比例的定量测量，并且能够轻易地将天然状态与所有错误折叠的构象区分开来。在这里，我们描述一种基于活性的方法来测量源自自我剪接I组内含子的核酶的天然折叠，并且我们展示了该检测方法如何用于监测作为通用RNA伴侣发挥作用的DEAD-box RNA解旋酶蛋白对天然折叠的加速作用。通过测量在快速的首次周转中转化为产物的底物量，我们描述了如何确定处于天然状态的核酶群体的比例。此外，我们描述了如何进行两阶段或不连续检测，其中折叠在第一阶段进行，然后在第二阶段改变溶液条件以允许催化活性并阻止进一步折叠。该方案允许在广泛的溶液条件下跟踪折叠过程，包括那些不支持催化活性的条件，并有助于对伴侣蛋白的研究。
