Poster abstracts

Poster number 89 submitted by Sharla Wood

Single molecule studies of the c-di-GMP riboswitch

Sharla Wood (Chemistry Department, Wayne State University), Adrian Ferre-dAmare (National Heart, Lung, and Blood Institute, NIH), David Rueda (Chemistry Department, Wayne State University)

Abstract:
Cyclic diguanylate (c-di-GMP) is a bacterial second messenger important for physiologic adaptation and virulence. The class-I c-di-GMP riboswitch is phylogenetically widespread and is thought to mediate pleiotropic genetic responses to the second messenger. Previous crystallographic, solution X-ray scattering and biochemical studies suggest that the aptamer domain of the RNA switches from an extended free state to a compact c-di-GMP-bound conformation in which two helical stacks dock side-by-side. Single molecule fluorescence resonance energy transfer (smFRET) experiments now reveal that the free RNA exists in four distinct populations that differ in their characteristic dwell times in the extended and docked conformations. In the presence of c-di-GMP and Mg2+, a population that is stably docked for >30 minutes becomes predominant. smFRET analysis of riboswitch mutants demonstrates that tertiary interactions distal to the c-di-GMP binding site strongly modulate the population structure of the RNA, even in the absence of the second messenger. These allosteric interactions accelerate ligand recognition by preorganizing the RNA, favoring rapid c-di-GMP binding.

Keywords: riboswitch, single molecule FRET, c-di-GMP