Mapping RNA–Protein Interactions Using Iodine Footprinting

Abstract

Footprinting methods are used to determine the binding site of a protein on an RNA. They are based on the fact that a protein bound to an RNA protects the RNA from cleavage by chemicals or nucleases. The footprinting method described here relies on the ability of iodoethanol to cleave the backbone of RNA when a phosphodiester bond contains sulfur in place of a nonbridging oxygen. A potential advantage of using iodoethanol for cleavage is that one can prepare RNAs that contain selective thiol substitutions such that the resulting cleavage patterns contain fewer bands, making quantification “easier” and the results cleaner. For example, a population of RNAs that only contains nonbridging thiol substitutions 5′ to each adenine can be prepared by including αS ATP in the transcription reaction. In this protocol, all positions on an RNA are surveyed. First, a series of RNAs is synthesized by transcription in the presence of αS ATP, αS CTP, αS UTP, or αS GTP. Each of the selectively substituted RNAs is probed with the binding protein of interest. The portion of the RNA that is not bound by protein is accessible and vulnerable to cleavage by iodoethanol. Finally, the cleavage products are analyzed by gel electrophoresis on denaturing polyacrylamide gels.