依赖新霉素的锤头状核酶可直接控制酿酒酵母中的基因表达。

Neomycin-dependent hammerhead ribozymes for the direct control of gene expression in Saccharomyces cerevisiae.

机构信息

Department of Chemistry, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany; Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany.

Department of Biology, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany.

出版信息

Methods. 2019 May 15;161:35-40. doi: 10.1016/j.ymeth.2019.01.004. Epub 2019 Jan 9.

DOI:10.1016/j.ymeth.2019.01.004

PMID:30639182

Abstract

Hammerhead ribozyme-based RNA switches have been proven to be powerful tools for conditional gene regulation in various organisms. We present neomycin-dependent hammerhead ribozymes (HHR) that influence gene expression in a ligand- and dose-dependent manner in S. cerevisiae. We utilized a novel design of fusing the aptamer domain to the HHR enabling for the first time the identification of genetic ON- and OFF-switches within the same library. For this purpose a neomycin aptamer was fused to stem 1 of a type 3 hammerhead ribozyme via an addressable three-way junction that shows high flexibility at the connection site. An in vivo screening approach identified sequences that allow to induce or repress gene expression 2- to 3-fold in response to neomycin addition. The ribozyme switches operate at neomycin concentrations that show no toxic effect on cell growth and pose powerful genetic tools to study and modulate cellular function in yeast.

摘要

锤头核酶 RNA 开关已被证明是在各种生物体中进行条件基因调控的有力工具。我们在酿酒酵母中展示了依赖新霉素的锤头核酶（HHR），这些核酶以配体和剂量依赖的方式影响基因表达。我们利用了一种将适体结构域融合到 HHR 的新设计，首次在同一个文库中鉴定出遗传的 ON 和 OFF 开关。为此，通过可寻址的三链连接体将新霉素适体融合到 3 型锤头核酶的茎 1 上，该连接体在连接位点具有很高的灵活性。体内筛选方法鉴定出了能够在新霉素添加时响应诱导或抑制基因表达 2-3 倍的序列。这些核酶开关在新霉素浓度下工作，该浓度对细胞生长没有毒性作用，是研究和调节酵母细胞功能的强大遗传工具。