Not making the cut: Techniques to prevent RNA cleavage in structural studies of RNase–RNA complexes

Abstract

RNases are varied in the RNA structures and sequences they target for cleavage and are an important type of enzyme in cells. Despite the numerous examples of RNases known, and of those with determined three-dimensional structures, relatively few examples exist with the RNase bound to intact cognate RNA substrate prior to cleavage. To better understand RNase structure and sequence specificity for RNA targets, in vitro methods used to assemble these enzyme complexes trapped in a pre-cleaved state have been developed for a number of different RNases. We have surveyed the Protein Data Bank for such structures and in this review detail methodologies that have successfully been used and relate them to the corresponding structures. We also offer ideas and suggestions for future method development. Many strategies within this review can be used in combination with X-ray crystallography, as well as cryo-EM, and other structure-solving techniques. Our hope is that this review will be used as a guide to resolve future yet-to-be-determined RNase–substrate complex structures.

Keywords

• RNase–RNA complex
• Pre-cleavage
• Scissile-phosphate
• Structure determination
• Enzymatic inhibition
• Nucleic acid recognition