Measuring riboswitch activity in vitro and in artificial cells with purified transcription-translation machinery.
Measuring riboswitch activity in vitro and in artificial cells with purified transcription-translation machinery.
复制标题
使用纯化的转录翻译机器测量体外和人工细胞中的核糖开关活性。
作者：
L. Martini;S. Mansy
我们提出了一种简单的方法来测量体外和人工细胞内纯化组分的核糖开关的实时活性。通常，核糖开关活性是通过利用β-半乳糖苷酶编码结构和假定的非翻译区域的核糖开关序列在体内测量的。额外的体外鉴定通常使用在线探测来探索配体与mRNA结合引起的构象变化或分析存在和不存在配体的转录物长度。然而，核糖开关最终控制蛋白质水平，并且经常需要辅助因子。因此，一个能够监测蛋白质生产的体外系统，具有完全定义的成分，可以补充辅助因子，将极大地有助于核开关的研究。在此，我们提出了一个适用于这种分析的系统。此外，由于所描述的系统可以很容易地在隔室内重建以构建具有传感能力的人工细胞模拟物，因此提供了在油包水乳剂隔室和脂质囊泡内构建传感响应系统的方案。仅使用标准实验室设备和市售材料进行所描述的分析，包括DNA，纯化的转录-翻译机器，即PURE系统和荧光光谱仪。
We present a simple method to measure the real-time activity of riboswitches with purified components in vitro and inside of artificial cells. Typically, riboswitch activity is measured in vivo by exploiting β-galactosidase encoding constructs with a putative riboswitch sequence in the untranslated region. Additional in vitro characterization often makes use of in-line probing to explore conformational changes induced by ligand binding to the mRNA or analyses of transcript lengths in the presence and absence of ligand. However, riboswitches ultimately control protein levels and often times require accessory factors. Therefore, an in vitro system capable of monitoring protein production with fully defined components that can be supplemented with accessory factors would greatly aid riboswitch studies. Herein we present a system that is amenable to such analyses. Further, since the described system can be easily reconstituted within compartments to build artificial, cellular mimics with sensing capability, protocols are provided for building sense-response systems within water-in-oil emulsion compartments and lipid vesicles. Only standard laboratory equipment and commercially available material are exploited for the described assays, including DNA, purified transcription-translation machinery, i.e., the PURE system, and a spectrofluorometer.
影响因子：
16
作者：
Wickiser, JK;Winkler, WC;Crothers, DM
通讯作者：
Crothers, DM
影响因子：
--
作者：
Nahvi, A;Sudarsan, N;Breaker, RR
通讯作者：
Breaker, RR
影响因子：
4.8
作者：
Artsimovitch, Irina;Henkin, Tina M.
通讯作者：
Henkin, Tina M.