Discovery and Characterization of Novel RNA Switch Chemistry and Biology via RNA Structure-seq

Abstract

The ability of an organism to respond to changing environmental conditions is critical for its survival. Bacteria can be beneficial, for example by helping with digestion, or pathogenic, causing illness and even death. It is therefore important to understand how bacteria regulate gene expression, which can ultimately lead to new medicines against bacterial disease, such as new antibiotics. Genes, which are encoded in DNA, are transcribed by RNA polymerase into an intermediary called mRNA. The code in the mRNA is then translated by ribosomes into proteins, which function as essential structural elements within cells and as enzymes that catalyze the large number of chemical reactions necessary to sustain life. Bacteria have evolved elaborate mechanisms that regulate this gene expression process in response to environmental cues. Transcription and translation are regulated by RNA structure and RNAbinding proteins in all organisms, with mechanisms that regulate the cessation of mRNA synthesis (transcription termination) and the initiation of protein synthesis (translation initiation) being particularly common in bacteria. However, the extent of RNA-based regulatory mechanisms remains vastly underexplored. We propose to extend and apply novel genome-wide RNA structure determination methodologies that we invented (RNA Structure-seq) to uncover new paradigms of RNA-based gene regulation in bacteria. Although various methods have been developed to analyze the effects of RNA structure, RNA-binding proteins, and metabolites on RNA function, these methods are largely limited to assessment of single RNAs, and are typically applied under highly artificial test-tube (in vitro) conditions. Here we propose transformative advances in RNA chemistry, molecular biology, and microbiology to advance novel methodologies for the determination of the “RNA structurome”: the structures of all RNAs (i.e. thousands of RNAs in one experiment), in living cells (in vivo). We will identify, in unprecedented molecular detail, global effects of metabolites, stressors and RNA-binding proteins on the RNA structurome of Escherichia coli (Gram-negative bacteria) and Bacillus subtilis (Gram-positive bacteria); these species represent the two major groups of bacteria and reside in the mammalian gut and the soil, respectively. These model organisms, which are more diverged from each other than humans are from yeast, contain many examples of genes that are regulated by RNA structural switches. We will identify genome-wide RNA structural changes that occur in response to some of the most common and important environmental cues a bacterium faces–metabolites, salinity, heavy metals, and temperature–and then elucidate the mechanisms of these RNA switches. Because we are approaching this problem with an entirely new genome-wide method, we will discover new principles of gene regulation such as new categories of RNAs that respond to environmental signals, as well as general sequence/structure rules that govern protein-RNA interaction. Our studies will have a substantial impact on the broad field of life science, as our methodologies will be readily adaptable to any organism or cell type. This research has the potential for long-term applications to benefit humankind such as the development of new therapeutics to cure diseases.

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