SUMMARY RNA structures can influence many aspects of gene expression including transcription, translation, splicing, and polyadenylation. As RNA folding occurs immediately during transcription, this raises a fundamental question as to how nascent RNA structures influence RNA processing and gene expression. Here we will address aspects of this question through detailed structure-function studies of riboswitches, broadly distributed regulatory RNAs that in response to binding specific ligands can control transcription, translation and splicing. Riboswitches consist of two domains – a highly structured aptamer that can bind a specific ligand, and a downstream expression platform that changes structure and regulates expression due to aptamer-ligand interactions. Riboswitches sense an array of metabolites, metals, ions and other small molecules to regulate essential and virulence genes in medically important pathogens such as Listeria monocytogenes, Staphylococcus aureus and Vibrio cholerae, making them of great interest as targets for novel antimicrobial therapies. They are also being developed as novel biosensors for biomedical applications. Riboswitches are powerful model systems for understanding diverse areas of RNA biology including RNA-ligand interactions, mechanisms of gene regulation, cellular RNA structures, and structure-based drug discovery. In addition, a critical feature of many riboswitches is that regulation only occurs during transcription, making them ideal model systems to study the impacts of nascent RNA structure on gene expression. Towards our long-term goal of developing a molecular understanding of how cotranscriptional RNA folding regulates and coordinates gene expression and RNA processing, we are using diverse riboswitches that regulate transcription as model systems. This proposal details a set of complementary specific aims that address fundamental questions: (1) what are the sequence determinants and transcription dynamics that promote efficient expression platform folding through cotranscriptional strand displacement, and (2) what are the mechanisms by which aptamer-ligand interactions block cotranscriptional strand displacement to enact the regulatory decision. One of the main approaches we will use to address these questions involve cellular gene expression assays to detect changes in gene expression caused by riboswitches in response to varying ligand conditions. Mutating riboswitch sequences allows us to directly test hypotheses about how different sequences and structures affect the riboswitch mechanism. Quantitative cellular gene expression assays are conducted by measuring the amounts of fluorescent reporter genes through plate reader assays. This supplementary proposal therefore requests funds to support the purchase of a new plate reader to be used for these studies.