Structural and biochemical investigation of functional RNAs: Studies of the group I intron, the GlmS ribozyme and the GEMM riboswitch.

摘要

Functional RNAs play a role in a multitude of cellular processes via RNA-RNA, RNA-protein and RNA-ligand interactions. Structural and biochemical data provide insight into the interactions between three functional RNAs, the group I intron, the GlmS ribozyme and the GEMM riboswitch, and their trans-acting substrates or ligands. During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. The crystal structure of the intron in complex with spliced exons reveals a relaxed active site, in which direct metal coordination by the exons is lost following ligation, while other tertiary interactions are retained between the exon and the intron. Kinetic and thermodynamic measurements also show a change in metal-ligand contacts before and after ligation. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.;Riboswitches act as sensors to small molecules in order to regulate gene expression in many bacteria. The GlmS ribozyme is found upstream of the gene for glucosamine-6-phosphate (GlcN6P) synthase and is sensitive to the protein product GlcN6P. pH studies of GlmS ribozyme activation by GlcN6P suggest the phosphate contributes to the pH profile for ligand binding, with weaker binding at low pH and tighter binding at high pH. The amine, however, contributes to catalysis with a pKa for the reaction that matches the pK a for the amine. These data in combination with structural observations suggest the amine acts as a general acid in the ribozyme reaction.;The GEMM riboswitch recognizes the second messenger molecule c-diGMP. Thermodynamic studies probe the interactions between c-diGMP and the GEMM riboswitch to confirm recognition elements in the ligand binding pocket. Binding of mutant GEMM sequences to alternate ligands confirm an asymmetric recognition of both bases and phosphates in the ligand. A double mutation successfully changes the riboswitch specificity from c-diGMP to c-diAMP, further validating our understanding of the recognition elements seen structurally. Together, these data provide insight into the interactions between functional RNAs and their substrates or ligands as well as a broader understanding of the role of RNA in regulation of proper gene expression within cells.