We use physical and biochemical methods to understand how RNAs fold. The three main projects in the lab investigate the folding dynamics of group I ribozymes, assembly of the 30S ribosome, and interactions between bacterial small regulatory RNAs (sRNA) and the RNA chaperone Hfq.
In living cells, RNAs fold as soon as they are transcribed. Important questions that remain to be addressed are how intracellular conditions influence the folding pattern of new transcripts, how proteins make the self-assembly of very large RNAs such as the ribosome more accurate, and how incorrectly folded or misassembled RNAs are recognized in the cell and targeted for degradation.
A variety of biophysical and biochemical methods are used to catch RNAs in the act of folding. Time-resolved hydroxyl radical footprinting probes individual tertiary contacts in RNA in milliseconds, yielding “snapshots” of the RNA structure as it forms. This method can also be used to visualize the structures of RNPs inside the cell. Stopped-flow fluorescence spectroscopy, small angle X-ray scattering, and neutron diffraction provide a global picture of RNA dynamics and stability. Single-molecule FRET reveals fluctuations between different RNA conformations and how these motions change as proteins bind. Native polyacrylamide gel electrophoresis is a simple and direct way of separating long-lived intermediates, and can be coupled to a wide range of biochemical methods.