Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

Abstract

Protein synthesis is catalyzed in the peptidyl transferase center (PTC), located in the large (50S) subunit of the ribosome. No high-resolution structure of the intact ribosome has contained a complete active site including both A- and P-site tRNAs. Additionally, though structures of the 50S subunit found no ordered proteins at the PTC, biochemical evidence suggests specific proteins are capable of interacting with the 3′ ends of the tRNA ligands. Here we present structures at 3.5 Å and 3.55 Å resolution of the 70S ribosome in complex with A- and P-site tRNAs that mimic pre- and post-peptidyl transfer states. These structures demonstrate that the PTC is very similar between the 50S subunit and the intact ribosome. Additionally they reveal interactions between ribosomal proteins L16 and L27 and the tRNA substrates, helping to elucidate the role of these proteins in peptidyl transfer.

Structure of BipA in GTP form bound to the ratcheted ribosome

“…While the uL10-bL12 stalk is more defined in the absence of tRNAs/mRNA, the BipA-ribosome-tRNAs/mRNA complex can provide a valuable insight into BipA interaction with tRNA. Superposing the present structure with that of ribosome containing A-, P-and E-site tRNA (34) by aligning the 23S rRNAs shows that the A-and E-site tRNAs take a similar positioning, except for a ∼4-Å shift of the anticodon loop of E-site tRNA following the 30S head swiveling in the present complex (Fig. S6D).…”

“…Furthermore, the distal loop residues (552-555 and beyond 555) appear to touch the hairpin loop of H92 at the PTC. Note that the hairpin loop of H92, so-called "A-loop," binds the 3′-CCA end of A-site aminoacyl-tRNA (aa-tRNA) and helps to position the aminoacyl group in the PTC during the protein synthesis (34). Taken together, these results provide a structural explanation for the biochemical data (5) that these regions (more specifically, Lys427, Lys434, and Arg436 in domain V and His527 and Arg529 in CTD) in BipA make a significant contribution to its association with the ribosome.…”

Structural insights into species-specific features of the ribosome from the pathogen Staphylococcus aureus

“…uL16 protein is positioned in the intersubunit surface and interacts with the A-and P-site transfer RNAs (tRNAs) (superimposed on PDB ID code 2WDK) (6). The N terminus of uL16 SA50S is pointing in the opposite direction compared with E70S, and thus may form a unique network of interactions with the acceptor stem of A-site tRNA (Fig.…”

“…Previous structural studies of the modes of action of antibiotics that inhibit bacterial ribosomes, performed using their complexes with then-available crystallizable ribosomal particles, yielded high-resolution crystal structures. These include the large ribosomal subunit of Deinococcus radiodurans (D50S) and the whole ribosome of Thermus thermophilus (T70S), as well as Escherichia coli (E70S) (5)(6)(7). These studies provided useful clues about the common traits of inhibitory mechanisms of antibiotics, namely binding at ribosomal functional sites, such as the peptidyl transferase center (PTC) or the protein exit tunnel (Fig.…”

Large facilities and the evolving ribosome, the cellular machine for genetic-code translation

“…Indeed, the initial diffracting crystals of the whole ribosome from T. thermophilus (T70S) with mRNA and tRNA molecules diffracted to rather low resolution (Hansen et al 1990). The advances of the brightness and collimation of synchrotron radiation X-ray beams, the installation of advanced detectors and the introduction of cryo-bio-crystallographic techniques (Hope et al 1989) yielded impressive advances in resolution from many crystal forms, including of functional complexes (Yusupov et al 2001;Korostelev et al 2006;Selmer et al 2006;Yusupova et al 2006;Voorhees et al 2009). Also, these techniques enabled structure determination of ribosomes trapped at a specific, albeit not necessarily functional, conformation (Schuwirth et al 2005).…”