Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Penn State University, University Park, PA 16802, United States.
Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Penn State University, University Park, PA 16802, United States.
Methods. 2019 Apr 15;159-160:70-81. doi: 10.1016/j.ymeth.2019.01.011. Epub 2019 Jan 24.
Transcription of DNA into RNA is critical for all life, and RNA polymerases are enzymes tasked with this activity. In eukaryotes, RNA Polymerase II (RNAPII) is responsible for transcription of all protein coding genes and many non-coding RNAs. RNAPII carries out the remarkable feat of unwinding the stable double-stranded DNA template, synthesizing the transcript and re-forming the double helix behind it with great precision and speed. In vitro, RNAPII is capable of carrying out templated RNA chain elongation in the absence of any accessory proteins. However, in cells, the transcription of genes is influenced by several factors, including DNA structure, chromatin, co-transcriptional processes, and DNA binding proteins, which impede the smooth progression of RNAPII down the template. Many transcription elongation proteins have evolved to mitigate the complications and barriers encountered by polymerase during transcription. Many of these elongation factors physically interact with components of the RNAPII elongation complex, including the growing RNA transcript and the DNA template entering and exiting RNAPII. To better understand how transcription elongation factors (EFs) regulate RNAPII, elegant methods are required to probe the structure of the elongation complex. Here, we describe a collection of biochemical assays to interrogate the structure of the RNAPII elongation complex of Saccharomyces cerevisiae that are capable of providing insights into the function of EFs and the elongation process.
DNA 转录为 RNA 对于所有生命都是至关重要的，而 RNA 聚合酶就是负责这项活动的酶。在真核生物中，RNA 聚合酶 II（RNAPII）负责转录所有蛋白质编码基因和许多非编码 RNA。RNAPII 完成了一项非凡的壮举，它能够解开稳定的双链 DNA 模板，以极高的精度和速度合成转录本，并在其后重新形成双链螺旋。在体外，RNAPII 能够在没有任何辅助蛋白的情况下进行模板指导的 RNA 链延伸。然而，在细胞中，基因的转录受到多种因素的影响，包括 DNA 结构、染色质、共转录过程和 DNA 结合蛋白，这些因素阻碍了 RNAPII 在模板上的顺利延伸。许多转录延伸蛋白已经进化出了减轻聚合酶在转录过程中遇到的复杂性和障碍的方法。许多这些延伸因子与 RNAPII 延伸复合物的组成部分物理相互作用，包括正在生长的 RNA 转录本和进入和离开 RNAPII 的 DNA 模板。为了更好地理解转录延伸因子 (EF) 如何调节 RNAPII，需要采用巧妙的方法来探测延伸复合物的结构。在这里，我们描述了一系列用于研究酿酒酵母 RNAPII 延伸复合物结构的生化分析方法，这些方法能够深入了解 EF 的功能和延伸过程。
