Observation of Global Changes in Conformation of an RNA Kissing Complex using Single-Molecular-Pair FRET

Abstract

We report on the observation of a change in the bend angle or twist of an RNA kissing complex upon Rop binding. Subtle global changes in molecular structure upon binding are generally difficult to discern using NMR or crystallography. FRET is well suited to observe these changes because of its sensitivity to interdye distance around the Forster radius, typically ≈5nm. For this reason, FRET is often referred to and used as a “molecular ruler” on this length scale. Here we show that for dye pairs that have minimal rotational freedom, FRET can also be used to observe changes in structure for which there is no significant change in distance between the dyes. The R1inv-R2inv kissing complex studied here is derived from the RNA I - RNA II system in E. coli. RNA II is a primer for replication of the ColE1 plasmid; its function is modulated by interaction with RNA I. Rop is known to bind and stabilize kissing complexes. It is also known to bind RNA in a structure, but not in a sequence dependent fashion. It has long been thought that Rop increases the bend of the R1inv-R2inv complex upon binding, but this has never been directly observed. Here we use FRET and modeling to investigate the structural change of this kissing complex upon Rop binding.