Time-Resolved and Dynamic Studies of Riboswitches

Abstract

Riboswitches are a class of regulatory RNA molecules widely distributed mostly across prokaryotic organisms. The discovery of the first riboswitches, by the Breaker lab and others, opened the door to a previously unimagined layer of genetic regulation. The majority of riboswitches undergo large scale conformational rearrangements upon binding target metabolites in their aptamer domain. Riboswitches are found predominantly in the 5' untranslated region of a number of genes. As a nascent strand is being transcribed by an RNA polymerase, the switch can fold to either occlude the expression platform or to expose it. The former leads to termination of the transcript, while the latter leads to gene expression. It is hypothesized that the concentration of the riboswitch's target metabolite governs gene expression. Thus, if the switch has bound its target metabolite prior to moving past the critical terminator stem, gene expression is regulated. Conversely, if the polymerase moves past the terminator stem before binding the target metabolite, the riboswitch and metabolite will not have time to reach equilibrium before an on/off decision is made. Accordingly, the mechanism of regulation may be highly dependent on the kinetic on-rate. For these reasons, understanding the dynamics of the riboswitch both on a global and local level is critical to determining the molecular mechanism by which riboswitches regulate gene expression. NMR T1, T2, NOE, cross-correlation, and dispersion experiments would be presented to delineate the site-specific dynamics involved in riboswitch function. These local dynamics measurements are combined with other biophysical techniques that we use to track global structural changes on a wide range of time-scales. These studies would likely provide a glimpse of the conformational switching necessary for riboswitch function.