The Escherichia coli large ribosomal subunit at 7.5 A resolution.

BACKGROUND: In recent years, the three-dimensional structure of the ribosome has been visualised in different functional states by single-particle cryo-electron microscopy (cryo-EM) at 13-25 A resolution. Even more recently, X-ray crystallography has achieved resolution levels better than 10 A for the ribosomal structures of thermophilic and halophilic organisms. We present here the 7.5 A solution structure of the 50S large subunit of the Escherichia coli ribosome, as determined by cryo-EM and angular reconstitution. RESULTS: The reconstruction reveals a host of new details including the long alpha helix connecting the N- and C-terminal domains of the L9 protein, which is found wrapped like a collar around the base of the L1 stalk. A second L7/L12 dimer is now visible below the classical L7/L12 'stalk', thus revealing the position of the entire L8 complex. Extensive conformational changes occur in the 50S subunit upon 30S binding; for example, the L9 protein moves by some 50 A. Various rRNA stem-loops are found to be involved in subunit binding: helix h38, located in the A-site finger; h69, on the rim of the peptidyl transferase centre cleft; and h34, in the principal interface protrusion. CONCLUSIONS: Single-particle cryo-EM is rapidly evolving towards the resolution levels required for the direct atomic interpretation of the structure of the ribosome. Structural details such as the minor and major grooves in rRNA double helices and alpha helices of the ribosomal proteins can already be visualised directly in cryo-EM reconstructions of ribosomes frozen in different functional states.

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 7.5 A 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 ribosomal subunit, using an unfiltered version of the recently published 50 S reconstruction at 7.5 A resolution. At this resolution, the EM density shows a well-defined network of fine structural elements, in which the major 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 are provided by intra and inter-rRNA crosslinking data, as well as by tertiary 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 determined by immuno-electron microscopy. The published X-ray or NMR structures of seven 50 S ribosomal proteins or RNA-protein complexes were incorporated into the EM density. The 3D locations of cross-link and foot-print sites to the 23 S rRNA from tRNA bound to the ribosomal A, P or E sites were correlated with the positions of the tRNA molecules directly observed in earlier reconstructions of the 70 S ribosome at 13 A or 20 A. 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 were all found to lie within or close to the internal tunnel connecting the peptidyl transferase region with the presumed peptide exit site on the solvent side of the 50 S subunit. The post-transcriptionally modified bases in the 23 S rRNA form a cluster close to the peptidyl transferase area. The minimum conserved core elements of the secondary structure of the 23 S rRNA form a compact block within the 3D structure and, conversely, the points corresponding to the locations of expansion segments in 28 S rRNA all lie on the outside of the structure.

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 variations 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 the insertions were introduced into an E. coli strain lacking all seven rrn operons. In four of the five cases the cells were viable and 50 S subunits could be isolated; only the insertion in h63 was lethal. The modified subunits were examined by cryo-electron microscopy (cryo-EM), with a view to locating extra electron density corresponding to the insertion elements. The results 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 modelling studies based on cryo-EM reconstructions of E. coli ribosomes. The insertion 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 at a position corresponding precisely to that predicted from the E. coli modelling studies. In the region of h9, the difference between the E. coli and H. marismortui secondary structures is ambiguous, and the extra electron density corresponding to the insertion was seen at a location intermediate between the position of the nearest helix in the atomic structure and that in the modelled structure. In 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.