Study of riboswitch regulatory mechanisms in Escherichia coli

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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.