Bacteria use cis-regulatory RNA structures called riboswitches to control the expression of many genes. Riboswitches are found in the 5’-untranslated regions of mRNAs where they regulate transcription or translation of downstream genes by forming a stabilized conformation upon specific ligand binding. These regulatory elements are important for bacterial survival because they quickly alter gene expression in response to changes in the intracellular environment. Studying riboswitches in atomic detail using X-ray crystallography has allowed me to reveal how the structure of these RNAs connects specific recognition of a ligand to subsequent changes in gene expression. Riboswitch-like regulatory elements called T-boxes sense amino acid abundance by binding tRNA. Conserved structural domains in the T-box allow it to decode tRNA and determine whether it is aminoacylated. To gain a mechanistic understanding of these processes and how they affect gene regulation, I solved a high-resolution crystal structure of a full-length T-box-tRNA complex. The structure reveals the organization of T-box domains and their contributions to decoding and aminoacylation sensing. This was the first glimpse of aminoacylation sensing by the T-box, which is facilitated by a three-domain module that makes specific contacts to the uncharged tRNA terminus. I solved another structure of the T-box bound to a charged tRNA mimic to understand how the aminoacylation status of tRNA determines T-box regulatory output. Together, these structures reveal how charged and uncharged tRNA influence T-box conformation selection to affect gene expression. Although atomic level analysis is necessary for understanding the relationship between structure and function in riboswitches, understanding the biological contexts in which riboswitches are involved is an equally critical endeavor. After solving the structure of a guanidine riboswitch, I became interested in studying the biological role of its ligand. Guanidine was not appreciated as a biologically relevant metabolite, but the wide distribution of guanidine riboswitches in bacteria suggests otherwise. To understand the toxicity and intracellular source of guanidine, I used genetic approaches in the model bacterium Bacillus subtilis. My results have uncovered novel guanidine efflux pump genes and a potential non-enzymatic route of guanidine production. Expanding beyond the study of riboswitch function to understanding the processes they regulate is a promising new avenue to undiscovered biology.