We wish to understand the relationships between the three-dimensional (3D)architectures of biological RNAs and their mechanisms of action. We use a combination of X-ray crystallography and biochemistry to study both, RNAs that fold and function autonomously, and cellular complexes of RNAs and proteins. Our model systems are riboswitches and pseudouridine syntheses. Riboswitches are RNAs that control gene expression in archaea, bacteria and eukaryotes. Riboswitches bind small-molecule metabolites with high affinity and specificity and switch conformation upon binding. Despite being 'naked' RNAs, riboswitches display sophisticated biochemical behaviors. Some of them bind their effectors through multiple sites that exhibit cooperativity. Others are catalytic RNAs that are allosterically activated by their ligands. Pseudouridine synthases are protein enzymes responsible for the most abundant type of post-transcriptional modification of cellular RNAs. We are particularly interested in an RNA-protein complex formed by the pseudouridine synthase Dyskerin, 3 accessory proteins, and the box H/ACA RNAs. This ribonucleoprotein (RNP) complex is conserved between archaea and human, and is essential for processing and maturation of ribosomal RNA. In vertebrates, a box H/ACA RNP is also part of the telomerase RNP. Correct association of the H/ACA domain of telomerase RNA with Dyskerin is essential both for telomerase assembly and stability. We will use X-ray crystallography to visualize riboswitches and box H/ACA RNPs in multiple functional states. We will combine structural information with biochemical probing and solution enzymology to deduce how these molecular machines work. Lay Description: Proteins and RNAs, the molecular machines that make life possible, have complicated architectures. In order to understand how cellular RNA machines work, we will trap them at different times while they carry out their function, and use a technique called X-ray crystallography to find out where their constituent atoms are at different points in time. These 'molecular movies' will provide the basic understanding that will help manipulate the functions of RNAs in human health and disease, and in the life cycle of pathogenic organisms. ? ? ?
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