A short fragment of 23S rRNA containing the binding sites for two ribosomal proteins, L24 and L4, is a key element for rRNA folding during early assembly

Previously we described an in vitro selection variant abbreviated SERF (in vitro selection from random rRNA fragments) that identifies protein binding sites within large RNAs, With this method, a small rRNA fragment derived f rom the 23S rRNA was isolated that binds simultaneously and independently t he ribosomal proteins L4 and L24 from Escherichia coli, Until now the rRNA structure within the ternary complex L24-rRNA-L4 could not be studied due t o the lack of an appropriate experimental strategy. Here we tackle the issu e by separating the various complexes via native gel-electrophoresis and an alyzing the rRNA structure by in-gel iodine cleavage of phosphorothioated R NA. The results demonstrate that during the transition from either the L4 o r L24 binary complex to the ternary complex the structure of the rRNA fragm ent changes significantly. The identified protein binding sites are in exce llent agreement with the recently reported crystal structure of the 50S sub unit, Because both proteins play a prominent role in early assembly of the large subunit, the results suggest that the identified rRNA fragment is a k ey element for the folding of the 23S RNA during early assembly. The introd uced in-gel cleavage method should be useful when an RNA structure within m ixed populations of different but related complexes should be studied.