High-Resolution Ribosome Profiling for Determining Ribosome Functional States During Translation Elongation.
High-Resolution Ribosome Profiling for Determining Ribosome Functional States During Translation Elongation.
复制标题
用于确定翻译延伸过程中核糖体功能状态的高分辨率核糖体分析。
DOI：
10.1007/978-1-0716-1975-9_11
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Wu,ColinChih-Chien
中科院分区：
文献类型：
--
作者：
Shafieinouri,Mohammad;Membreno,BritnieSantiago;Wu,ColinChih-Chien
翻译延伸是一个高度精心设计的过程，涉及核糖体的大量构象变化，以适应氨酰-tRNA并沿着沿着mRNA模板遍历。为了捕获核糖体的不同功能状态，已经开发了一种基于高分辨率核糖体分析的方法。通过对不同大小的核糖体保护的mRNA片段进行深度测序，该方法不仅捕获核糖体位置，而且还捕获它们在酵母转录组中的体内功能状态，具有密码子解析。本章提出了一个浓缩的和一步一步的协议，用于保存核糖体在其功能状态，使用抗生素的鸡尾酒，陷阱延长核糖体的不同步骤，并构建一个cDNA文库来自核糖体保护的mRNA片段的深度测序。
Translation elongation is a highly choreographed process that involves substantial conformational changes of the ribosome to accommodate aminoacyl-tRNAs and traverse along the mRNA template. To capture distinct functional states of the ribosome, a high-resolution ribosome profiling-based approach has been developed. By deep-sequencing differently sized ribosome-protected mRNA fragments, this approach captures not only ribosome positions but also their functional states in vivo across theSaccharomyces cerevisiaetranscriptome with codon resolution. This chapter presents a condensed and step-by-step protocol for preserving ribosomes in their functional states using a cocktail of antibiotics that traps distinct steps of elongating ribosomes and for constructing a cDNA library derived from the ribosome-protected mRNA fragments for deep sequencing.