Book of Abstracts: Albany 2005
Guanine Riboswitch Crystal Structure as a Basis for Ligand Specificity by a Natural RNA Aptamer
Riboswitches are a level of transcriptional or translational control that is independent of protein cofactors. Molecular recognition of a metabolite is carried out by a regulatory element in the 5' UTR of the mRNA molecule leading to the formation or disruption of a terminator. Riboswitches have been discovered for a number of molecules across several species of bacteria and are known to regulate > 2% of all genes in B. subtilis. In the case of the guanine riboswitch, a loosely associated binding pocket is created to detect the presence of guanine or hypoxanthine to control purine metabolism and synthesis genes. Upon binding of the metabolite the riboswitch encourages the formation of a downstream terminator that halts transcription.
We have solved 1.9 Å resolution crystal structures of the guanine riboswitch bound to the metabolites hypoxanthine and xanthine individually. Both structures revealed the strikingly complex features that contributed to the recognition and specificity of binding. A stearically restricting binding pocket completely engulfed the nucleobases and organized itself identically in both structures, indicative of a rigidity in metabolite binding not commonly seen in RNA binding interactions. In addition, an intricate tertiary loop-loop interaction, distant from the binding pocket, was shown to be required for metabolite binding. Complimentary studies of binding done by isothermal titration calorimetry have measured the thermodynamic contribution of each contact to the purine analogues. Concurrently, we are investigating the binding tolerance of nucleotides with alternative functional groups, probing the kinetics of riboswitch function, and focusing on obtaining structures of other nucleotides bound to the riboswitch in order to refine our knowledge of the specificity and complexity involved in metabolite recognition and transcription termination.
Sunny D. Gilbert
Department of Chemistry and Biochemistry