A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits

Abstract

The large ribosomal subunit catalyzes peptide bond formation during protein synthesis. Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol. Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active. Addition of these crystals to solutions containing substrates results in formation of products, which ceases when crystals are removed. When substrates are diffused into large subunit crystals, the subsequent structure shows that products have formed. The CC-puromycin-peptide product is found bound to the A-site and the deacylated CCA is bound to the P-site, with its 3prime prime or minute OH near N3 A2486 (Escherichia coli A2451). Thus, this structure represents a state that occurs after peptide bond formation but before the hybrid state of protein synthesis.

Functional aspects of ribosomal architecture: symmetry, chirality and regulation

“…As such, it is a major participant in the remote interactions that govern the accurate positioning of the tRNA substrates. 8,9,41 The crucial contribution of H69 interactions to productive alignment of the A-site tRNA substrate is demonstrated by the finding that in their absence similar, albeit distinctly different, binding modes were observed within the PTC, [39][40][41] all requiring conformational rearrangements in order to participate in peptide bond formation. 10 Lack of remote interactions could be due to the disorder of H69, as observed in the H50S high-resolution structure, 2,3 or result from the use of tRNA analogues that are too short to reach the upper rim of the PTC cavity, where the components providing the remote interactions reside.…”

“…39 It appears that, in general, the orientations of reactants that are not placed by remote interactions could be incompatible with polypeptide elongation. Furthermore, almost all of the substrateanalogues mimicking only the tRNA 3 0 end were found to necessitate conformational rearrangement in order to participate in peptide bond formation and/or chain elongation.…”

“…10 Lack of remote interactions could be due to the disorder of H69, as observed in the H50S high-resolution structure, 2,3 or result from the use of tRNA analogues that are too short to reach the upper rim of the PTC cavity, where the components providing the remote interactions reside. 39,40 The structures of the substrates bound ribosome 6 and to the large subunit, D50S, 8 indicate that similar to Helix H69, protein L16 is also involved in moderating accurate substrate positioning. This protein is located at the upper rim periphery of the PTC cavity [Plate 1(c)], in a position allowing interactions with the acceptor stem of the A-site tRNA, and these remote interactions appear to be of major importance.…”

“…39 Such a sequence of events is bound to be impractical since it implies that the entire newly formed polypeptide, no matter what its length is, has to be rotated by 180 each time a peptide bond is being formed. Alternatively, the nascent chain exploits free rotations around its main-chain bonds in order to compensate for the 180 rotation of the tRNA 3 0 end carrying the nascent polypeptide.…”

Cross-crystal averaging reveals that the structure of the peptidyl-transferase center is the same in the 70S ribosome and the 50S subunit

“…Insights into the structural basis of mRNA decoding have come largely from structures of the Thermus thermophilus 30S ribosomal subunit and its substrate complexes (1)(2)(3)(4), whereas understanding of the structural basis of peptide bond formation has been derived from structures of the Haloarcula marismortui 50S subunit (Hma50) complexed with substrate, intermediate, and product analogues (5)(6)(7)(8)(9). The structures of the Hma50 complexed with a peptidyl-CCA substrate or with an analogue of the intermediate show that the CCA bound in the P-site interacts with the P-loop, nucleotides 2246-2258 of the 23S rRNA, and that the attacking ␣-NH 2 group of a bound CC-puromycin substrate analogue is hydrogen-bonded to both the 2Ј-OH of the terminal A76 of the peptidyl-CCA P-site substrate and to the N3 of the ribosomal base A2451 (Escherichia coli numbering).…”