体外RNA聚合酶I转录延伸的定量分析。
Quantitative analysis of transcription elongation by RNA polymerase I in vitro.
作者信息
Schneider David Alan
机构信息
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA.
出版信息
Methods Mol Biol. 2012;809:579-91. doi: 10.1007/978-1-61779-376-9_37.
Abstract
The elongation step in transcription has gained attention for its roles in regulation of eukaryotic gene expression and for its influence on RNA processing. Sophisticated genetic analyses have identified factors and/or conditions that may affect transcription elongation rate or processivity; however, differentiation of direct and indirect effects on transcription is difficult using in vivo strategies. Therefore, effective, reproducible in vitro assays have been developed to test whether a given factor or condition can have a direct effect on the kinetics of transcription elongation. We have adapted a fully reconstituted transcription system for RNA polymerase I (Pol I) for kinetic analysis of transcription elongation rate in vitro. The assay described here has proven to be effective in the characterization of defects or enhancement of wild-type transcription elongation by RNA Pol I. Since transcription elongation by RNA Pol I has only recently gained significant attention, this assay will be a valuable resource for years to come.
摘要
转录过程中的延伸步骤因其在真核基因表达调控中的作用以及对RNA加工的影响而受到关注。复杂的遗传分析已经确定了可能影响转录延伸速率或持续合成能力的因素和/或条件；然而，使用体内策略很难区分对转录的直接和间接影响。因此，已经开发出有效、可重复的体外测定方法，以测试给定的因素或条件是否会对转录延伸动力学产生直接影响。我们采用了一种完全重组的RNA聚合酶I（Pol I）转录系统，用于体外转录延伸速率的动力学分析。这里描述的测定方法已被证明在表征RNA Pol I对野生型转录延伸的缺陷或增强方面是有效的。由于RNA Pol I介导的转录延伸直到最近才受到广泛关注，该测定方法在未来几年将是一项宝贵的资源。
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