Study of riboswitch regulatory mechanisms in Escherichia coli

核糖开关是结构化的RNA基序，其结合细胞代谢物并经历变构变化以控制基因表达。这些RNA开关存在于所有三个生命王国中，这表明它们可能是早期遗传控制形式的代表。与其他遗传控制系统不同，核糖开关不需要代谢物结合蛋白，因此由介导细胞调节的自主RNA细胞传感器组成。在大肠杆菌中鉴定的大多数核糖开关已经显示通过调节核糖体接近核糖体结合位点（RBS）和/或AUG起始密码子来控制基因表达。然而，最近观察到，除了控制翻译起始外，一些E.大肠杆菌核糖开关利用额外的机制来调节mRNA水平。例如，我们的实验室证明，E. colilysC核糖开关调节RNaseE依赖的mRNA降解。我们还获得了证据表明，几个E。大肠杆菌核糖开关-如lysC核糖开关-通过使用Rho蛋白效应子调节mRNA水平。因此，lysC核糖开关是第一个例子，其中至少有三个参与的调控过程。目前尚不清楚这是普遍规律还是罕见现象。重要的是，尽管我们和其他人已经描述了E。大肠杆菌核糖开关调控机制的研究近年来，关于核糖开关如何协调调控机制以确保细胞内稳态的研究仍不完整。在本研究计划中，（1）我们将破译Rho如何参与lysC核糖开关的转录调控。我们将采用lacZ报告基因测定来确定Rho转录终止所需的lysC mRNA的最小区域。接下来，（2）我们将通过使用生物化学方法如核酸酶S1和3 '-O-甲基核苷酸序列作图的组合在体外定位Rho结合位点和终止位点的基因座。最后，（3）我们将探讨其他E.大肠杆菌核糖开关依赖于RNase E和Rho来控制代谢物传感后的mRNA水平。这一目标非常重要，因为它将为核糖开关如何与细胞机制相互作用以调节基因表达提供更广泛的图景。显然，了解代谢物如何结合核糖开关控制基因表达是获得有关细菌体内平衡的关键知识。从这项研究中获得的数据可能会导致新的核糖开关为基础的遗传工具，可用作生化传感器或作为人工基因诱导剂/抑制剂响应细胞的变化。

Riboswitches are structured RNA motifs that bind to cellular metabolites and undergo allosteric changes to control gene expression. These RNA switches are present in all three kingdoms of life suggesting that they might be representatives of an early form of genetic control. Unlike other genetic control systems, riboswitches do not require metabolite-binding proteins and thus consists of autonomous RNA cellular sensors mediating cellular regulation. The majority of riboswitches identified in Escherichia coli have been shown to control gene expression by modulating ribosome access to the ribosome binding site (RBS) and/or AUG start codon. However, it has recently been observed that in addition to control translation initiation, some E. coli riboswitches make use of additional mechanisms to regulate mRNA levels. For example, our laboratory demonstrated that the E. coli lysC riboswitch modulates RNase E-dependent mRNA degradation. We also gained evidence that several E. coli riboswitches-such as the lysC riboswitch-regulate mRNA levels by using the Rho protein effector. Thus, the lysC riboswitch is the first example where there is at least three involved regulatory processes. Whether this is a general rule or a rare occurrence is not currently known. Importantly, although we and others have characterized E. coli riboswitch regulatory mechanisms in the last years, there is still an incomplete picture about how riboswitches orchestrate regulation mechanisms to ensure cellular homeostasis. In this research proposal, (1) we will decipher how Rho is involved in the transcriptional regulation of the lysC riboswitch. We will employ lacZ reporter gene assays to determine the minimal region of lysC mRNA that is required for Rho transcription termination. Next, (2) we will address in vitro the locus of the Rho binding and termination sites by using a combination of biochemical approaches such as nuclease S1 and 3'-O-methyl nucleotide sequence mapping. Finally, (3) we will explore whether other E. coli riboswitches rely on both RNase E and Rho to control mRNA levels upon metabolite sensing. This goal is very important as it will provide a broader picture about how riboswitches interact with the cellular machinery to regulate gene expression. Clearly, understanding how metabolite binding to riboswitches controls genetic expression is key to gain crucial knowledge about bacterial homeostasis. The data obtained from this research could lead to novel riboswitch-based genetic tools to be used as biochemical sensors or as artificial gene inducers/repressors responding to cellular changes.