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Abstract
We report here an in-depth characterization of the aptamer domain of the transcriptional adenine-sensing riboswitch (pbuE) by NMR and fluorescence spectroscopy. By NMR studies, the structure of two aptamer sequences with different lengths of the helix P1, the central element involved in riboswitch conformational switching, was characterized. Hydrogen-bond interactions could be mapped at nucleotide resolution providing information about secondary and tertiary structure, structure homogeneity and dynamics. Our study reveals that the elongation of helix P1 has pronounced effects not only on the local but on the global structure of the apo aptamer domain. The structural differences induced by stabilizing helix P1 were found to be linked to changes of the ligand binding affinity as revealed from analysis of kinetic and thermodynamic data obtained from stopped-flow fluorescence studies. The results provide new insight into the sequence-dependent fine tuning of the structure and function of purine-sensing riboswitches.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Funding
Work in the groups of H.S. and J.W. is supported by the DFG and the state of Hessen (BMRZ). H.S. is a member of the DFG-funded cluster of excellence: macromolecular complexes. A.R. was supported by SPP 1258: Sensory and regulatory RNAs in Prokayrotes, J.W. and H.S. are supported by SFB902: Molecular Principles of RNA-based regulation.
Acknowledgments
We thank Dr. Boris Fürtig and Anna Wacker for helpful discussion and Elke Stirnal and Dr. Christian Richter for technical support.