Jacobs Ruth Q, Bellis Nathan F, Lucius Aaron L, Schneider David A
Deparment of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
STAR Protoc. 2023 Mar 24;4(2):102191. doi: 10.1016/j.xpro.2023.102191.
Here we present an optimized protocol for monitoring and analyzing single nucleotide incorporation by RNA polymerases. This protocol describes the assembly of Saccharomyces cerevisiae RNA polymerase I elongation complexes in a promoter-independent system in vitro. We describe how to collect a time course using a quench-flow, a rapid mixing instrument, and subsequently resolve reactions on a polyacrylamide gel. Finally, we detail how to quantify the gel images. For complete details on the use and execution of this protocol, please refer to Appling et al. (2015)..
在此，我们展示了一种用于监测和分析RNA聚合酶单核苷酸掺入的优化方案。该方案描述了在体外启动子非依赖系统中酿酒酵母RNA聚合酶I延伸复合物的组装。我们描述了如何使用淬灭流动仪（一种快速混合仪器）收集时间进程，随后在聚丙烯酰胺凝胶上解析反应。最后，我们详细说明了如何对凝胶图像进行定量分析。有关该方案使用和执行的完整详细信息，请参考阿普林等人（2015年）的文献。
