There are large differences between the cellular environment and the conditions widely used to study RNA structure and function in vitro, but until recently no technologies allowed nucleotide-resolution analyses of RNA structure inside cells. We have now examined the structure, dynamics, and ligand-binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution using SHAPE. We compared the in-cell RNA structure with that obtained in aqueous buffer containing 1 mM Mg2+, approximately the concentration that we directly measured inside cells. The fully folded ligand-bound aptamer formed essentially the same structure in cells as in buffer. In contrast, the unbound RNA aptamer in cells was much more highly structured than the ligand-free state in vitro. Even high in vitro concentrations of Mg2+ did not yield the degree of structural organization observed for the free aptamer in cells. The crowded cellular environment thus stabilizes, or pre-organizes, otherwise dynamic RNA conformations significantly more than does Mg2+ alone, demonstrating a profound influence of the cellular environment on RNA structure. To deduce how the structural effects observed in cells may be induced in vitro and how the aptamer ligand binding affinity is affected by crowding, studies in the presence of total cellular RNA, and the synthetic polymer, polyethylene glycol (PEG), were employed. These studies showed that RNA-RNA interactions and PEG had varying effects on aptamer structure. The structure of the aptamer in the presence of both cellular RNA and large PEGs agreed better with the structure observed in cells. However, specific characteristics observed for the aptamer in cells were not observed in vitro under either condition. Crowding the aptamer with PEG had little effect on ligand binding affinity, even when large structural effects were observed. These results suggest that the complex cellular environment is difficult to mimic in vitro, and to do so, may require a combination of crowding agents having different chemical properties. Finally, the observed minimal effects on ligand binding affinity for the aptamer in the presence of PEG, suggests that while RNA structure is affected by crowding in cells, RNA function may not be significantly affected. Advisors/Committee Members: Tyrrell, Jillian, Pielak, Gary J..