23] Analysis of global conformation of branched RNA species using electrophoresis and fluorescence

Abstract

This chapter describes the analysis of global conformation of branched RNA species using electrophoresis and fluorescence. Branch points are very common features of natural RNA molecules. These include asymmetrical bulges and helical junctions of various kinds. Naturally occurring junctions include perfect three- or four-way junction (3H and 4H junctions, respectively), although these are more frequently complicated by the presence of one or more unpaired bases. The hammerhead ribozyme provides a good example of a more complicated three-way RNA junction, whereas the hairpin ribozyme is based on a perfect four-way junction in the natural viral RNA sequence. The structure of branched nucleic acids is obtained by lower resolution approaches. Lower resolution approaches have proved valuable in gaining insight into the global structures of branched RNA and DNA species and in the study of conformational transitions. The chapter also discusses a combined approach that takes information from a number of techniques such as comparative gel electrophoresis and fluorescence resonance energy transfer (FRET) in combination with other methods where possible, including chemical probing, functional group modification, cryoelectron microscopy, and molecular modeling.