Direct localization by cryo-electron microscopy of secondary structural elements in Escherichia coli 23 S rRNA which differ from the corresponding regions in Haloarcula marismortui

Insertions were introduced by a two-step mutagenesis procedure into each of five double-helical regions of Escherichia coli 23 S rRNA, so as to extend the helix concerned by 17 bp. The helices chosen were at sites within the 23 S molecule (h9, h25, h45, h63 and h98) where significant length variatio ns between different species are known to occur. At each of these positions , with the exception of h45, there are also significant differences between the 23 S rRNAs of E. coli and Haloarcula marismortui. Plasmids carrying th e insertions were introduced into an E, coli strain lacking all seven rm op erons. In four of the five cases the cells were viable and 50 S subunits co uld be isolated; only the insertion in h63 was lethal. The modified subunit s were examined by cryo-electron microscopy (cryo-EM), with a view to locat ing extra electron density corresponding to the insertion elements. The res ults were compared both with the recently determined atomic structure of H. marismortui 23 S rRNA in the 50 S subunit, and with previous 23 S rRNA mod elling studies based on cryo-EM reconstructions of E. coli ribosomes. The i nsertion element in h45 was located by cryo-EM at a position corresponding precisely to that of the equivalent helix in H. marismortui. The insertion in h98 (which is entirely absent in H. marismortui) was similarly located a t a position corresponding precisely to that predicted from the E, coli mod elling studies. In the region of h9, the difference between the E, coli and H. marismortui secondary structures is ambiguous, and the extra electron d ensity corresponding to the insertion was seen at a location intermediate b etween the position of the nearest helix in the atomic structure and that i n the modelled structure. Ln the case of h25 (which is about 50 nucleotides longer in H. marismortui), no clear extra cryo-EM density corresponding to the insertion could be observed. (C) 2001 Academic Press.