The 3D arrangement of the 23 S and 5 S rRNA in the Escherichia coli 50 S ribosomal subunit based on a cryo-electron microscopic reconstruction at 7.5angstrom resolution

The Escherichia coli 23 S and 5 S rRNA molecules have been fitted helix by helix to a cryo-electron microscopic (EM) reconstruction of the 50 S riboso mal subunit, using an unfiltered version of the recently published 50 S rec onstruction at 7.5 Angstrom resolution. At this resolution, the EM density shows a well-defined network of fine structural elements, in which the majo r and minor grooves of the rRNA helices can be discerned at many locations. The 3D folding of the rRNA molecules within this EM density is constrained by their well-established secondary structures, and further constraints ar e provided by intra and inter-rRNA crosslinking data, as well as by tertiar y interactions and pseudoknots. RNA-protein cross-link and foot-print sites on the 23 S and 5 S rRNA were used to position the rRNA elements concerned in relation to the known arrangement of the ribosomal proteins as determin ed by immune-electron microscopy. The published X-ray or NMR structures of seven 50 S ribosomal proteins or RNA-protein complexes were incorporated in to the EM density. The 3D locations of cross-link and foot-print sites to t he 23 S rRNA from tRNA bound to the ribosomal A, P or E sites were correlat ed with the positions of the tRNA molecules directly observed in earlier re constructions of the 70 S ribosome at 13 Angstrom or 20 Angstrom. Similarly , the positions of cross-link sites within the peptidyl transferase ring of the 23 S rRNA from the aminoacyl residue of tRNA were correlated with the locations of the CCA ends of the A and P site tRNA. Sites on the 23 S rRNA that are cross-linked to the N termini of peptides of different lengths wer e all found to lie within or close to the internal tunnel connecting the pe ptidyl transferase region with the presumed peptide exit site on the solven t side of the 50 S subunit. The post-transcriptionally modified bases in th e 23 S rRNA form a cluster close to the peptidyl transferase area. The mini mum conserved core elements of the secondary structure of the 23 S rRNA for m a compact block within the 3D structure and, conversely, the points corre sponding to the locations of expansion segments in 28 S rRNA all lie on the outside of the structure. (C) 2000 Academic Press.