Ribosome structure and dynamics by smFRET microscopy

Composed of both RNA and protein components, the ribosome is one of the largest macromolecular machines in life responsible for the production of all protein. Interestingly, the major catalytic center of the ribosome (the peptidyl transferase center) and much of the binding regions for both mRNA and tRNA are composed of RNA making the ribosome one of the most complex and widely studied ribozymes. Further, large-scale conformational rearrangements throughout the ribosome are required for proper function making the ribosome a riboswitch as well. Recent advances in single-molecule biophysics have significantly augmented our understanding of ribosome function as both a ribozyme and riboswitch. Here, we discuss single-molecule Förster resonance energy transfer and its application to the study of the ribosome. Also, we describe how these experiments are designed from sample preparation to data acquisition and analysis. The general approach and methods described here can be generally applied to many other biological systems.