Imaging metabolite dynamics in living cells using a Spinach-based riboswitch
Imaging metabolite dynamics in living cells using a Spinach-based riboswitch
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DOI：
10.1073/pnas.1504354112
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发表时间：
2015-05-26
影响因子：
11.1
通讯作者：
Jaffrey, Samie R.
中科院分区：
文献类型：
--
作者：
You, Mingxu;Litke, Jacob L.;Jaffrey, Samie R.
核糖开关是天然的配体感应 RNA，通常存在于 mRNA 的 5' UTR 中。已经发现了许多类别的核糖开关，使 mRNA 能够受到多种和生理上重要的细胞代谢物和小分子的调节。在这里，我们描述了菠菜核糖开关，这是一种源自天然核糖开关的新型基因编码代谢物传感器。利用天然核糖开关的结构转换机制，我们证明菠菜可以交换为各种核糖开关的表达平台，从而允许代谢物结合直接诱导菠菜荧光。在编码羟乙基噻唑激酶的大肠杆菌 thiM 基因中的硫胺素 5'-焦磷酸 (TPP) 核糖开关中，我们发现菠菜的插入会导致 RNA 传感器在结合 TPP 时表现出荧光。这种 TPP 菠菜核糖开关以类似于内源性核糖开关的亲和力和选择性结合 TPP，并能够使用简单的荧光读数发现 TPP 核糖开关的激动剂和拮抗剂。此外，TPP 菠菜核糖开关在大肠杆菌中的表达能够对单个细胞中细胞内 TPP 浓度的动态变化进行实时成像。此外，我们还表明，使用与TPP核糖开关类似的结构机制的其他核糖开关，包括来自编码黄嘌呤磷酸核糖基转移酶的枯草芽孢杆菌xpt基因的鸟嘌呤和腺嘌呤核糖开关，以及来自编码蛋氨酸合酶的枯草芽孢杆菌yitJ基因的S-腺苷-甲硫氨酸-I核糖开关，可以转化为菠菜核糖开关。因此，菠菜核糖开关构成了一类新型的基于 RNA 的荧光代谢物传感器，它利用了天然存在的配体结合核糖开关的多样性。
Riboswitches are natural ligand-sensing RNAs typically that are found in the 5' UTRs of mRNA. Numerous classes of riboswitches have been discovered, enabling mRNA to be regulated by diverse and physiologically important cellular metabolites and small molecules. Here we describe Spinach riboswitches, a new class of genetically encoded metabolite sensor derived from naturally occurring riboswitches. Drawing upon the structural switching mechanism of natural riboswitches, we show that Spinach can be swapped for the expression platform of various riboswitches, allowing metabolite binding to induce Spinach fluorescence directly. In the case of the thiamine 5'-pyrophosphate (TPP) riboswitch from the Escherichia coli thiM gene encoding hydroxyethylthiazole kinase, we show that insertion of Spinach results in an RNA sensor that exhibits fluorescence upon binding TPP. This TPP Spinach riboswitch binds TPP with affinity and selectivity similar to that of the endogenous riboswitch and enables the discovery of agonists and antagonists of the TPP riboswitch using simple fluorescence readouts. Furthermore, expression of the TPP Spinach riboswitch in Escherichia coli enables live imaging of dynamic changes in intracellular TPP concentrations in individual cells. Additionally, we show that other riboswitches that use a structural mechanism similar to that of the TPP riboswitch, including the guanine and adenine riboswitches from the Bacillus subtilis xpt gene encoding xanthine phosphoribosyltransferase, and the S-adenosyl-methionine-I riboswitch from the B. subtilis yitJ gene encoding methionine synthase, can be converted into Spinach riboswitches. Thus, Spinach riboswitches constitute a novel class of RNA-based fluorescent metabolite sensors that exploit the diversity of naturally occurring ligand-binding riboswitches.