Study of riboswitch regulatory mechanisms in Escherichia coli

核糖开关是一种结构化的RNA基序，它与细胞代谢物结合，并经历变构变化来控制基因表达。这些RNA开关在所有三个生命王国中都存在，这表明它们可能是一种早期基因控制的代表。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，因此由自主的RNA细胞传感器组成，介导细胞调节。在大肠杆菌中发现的大多数核糖开关通过调节核糖体对核糖体结合位点(RBS)和/或AUG起始密码子的访问来控制基因的表达。然而，最近观察到，除了控制翻译起始外，一些大肠杆菌核糖开关还利用额外的机制来调节mRNA水平。例如，我们的实验室证明了大肠杆菌lysC核糖开关调节RNase E依赖的mRNA降解。我们还获得了一些证据表明，几种大肠杆菌核糖开关--如lysC核糖开关--通过使用Rho蛋白效应器来调节mRNA水平。因此，lysC核糖开关是第一个至少涉及三个调控过程的例子。这是普遍规律还是罕见情况，目前尚不得而知。重要的是，尽管我们和其他人在过去几年中已经描述了大肠杆菌核糖开关的调控机制，但关于核糖开关如何协调调控机制以确保细胞内稳态，仍然存在一个不完整的图景。在这个研究方案中，(1)我们将破译Rho如何参与lysC核糖开关的转录调控。我们将使用LacZ报告基因分析来确定Rho转录终止所需的lysC mRNA的最小区域。接下来，(2)我们将结合使用生物化学方法，如核酸酶S1和3‘-O-甲基核苷酸序列作图，在体外研究Rho结合和终止位点的位置。最后，(3)我们将探索其他大肠杆菌核糖开关是否同时依赖RNaseE和Rho来控制代谢物感应的mRNA水平。这一目标非常重要，因为它将提供关于核糖开关如何与细胞机制相互作用以调节基因表达的更广泛的图景。显然，了解代谢产物与核糖开关结合是如何控制基因表达的，是获得关于细菌内稳态的关键知识。从这项研究中获得的数据可能会导致基于核糖开关的新型遗传工具被用作生化传感器或作为对细胞变化做出反应的人造基因诱导/抑制因子。

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.