Structures of deacylated tRNA mimics bound to the E site of the large ribosomal subunit

Abstract

During translation, tRNAs cycle through three binding sites on the ribosome: the A, the P, and the E sites. We have determined the structures of complexes between the Haloarcula marismortui large ribosomal subunit and two different E site substrates: a deacylated tRNA acceptor stem minihelix and a CCA-acceptor end. Both of these tRNA mimics contain analogs of adenosine 76, the component responsible for a large proportion of E site binding affinity. They bind in the center of the loop-extension of protein L44e, and make specific contacts with both L44e and 23S rRNA including bases that are conserved in all three kingdoms of life. These contacts are consistent with the footprinting, protection, and cross-linking data that have identified the E site biochemically. These structures explain the specificity of the E site for deacylated tRNAs, as it is too small to accommodate any relevant aminoacyl-tRNA. The orientation of the minihelix suggests that it may mimic the P/E hybrid state. It appears that the E site on the 50S subunit was formed by only RNA in the last common ancestor of the three kingdoms, since the proteins at the E sites of H. marismortui and Deinucoccus radiodurans large subunits are not homologous.

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

“…Our results suggest that the conformation of the PTC-CCA interface in the two Tth70 crystal forms is identical to that of the current 2.8 Å 70S model (20) and to that observed earlier in the Hma50 structure (7). Therefore, we conclude that the PTC-CCA interface adopts the same conformation in the functionally equivalent 70S ribosome as in the 50S subunit.…”

“…The 3.7 Å-resolution map obtained by cross-crystal averaging confirmed the existence of the triple base pair at the heart of the PTC and also contained density for a metal ion coordinated by these bases. The PTC triple base pair is formed among the bases G2447, A2451, and G2061, and it has been previously seen in the Hma50 model (5,7,9). In that model the hydrogen-bonding network was stabilized by a metal ion positioned between the bases G2447 and G2061.…”

“…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).…”

Symmetry at the active site of the ribosome: structural and functional implications

“…Its location within the large subunit allows direct or indirect interactions with almost all large ribosomal-subunit components possessing functional relevance and with all the factors involved in the elongation process. The 39-ends of the A-and PtRNAs bind to the PTC, whereas the 39-end of the E-site tRNA contacts the neighborhood of nucleotide A2433 at the edge of the SymR in the structure of T70S complexed with three tRNA molecules (Yusupov et al, 2001), but not in the structure of H50S complexed with E-site tRNA (Schmeing et al, 2003). The non-symmetrical extensions radiating from the SymR interact with regions that carry out central roles in the ribosome function.…”

The Structure of the Yeast Mitochondrial Large Ribosomal Subunit

“…The extension of bL33 is stabilized by a change in the rRNA path prior to helix 82-ES1, that is in itself partially stabilized by the unique protein mL59. Thus the E site of ribosomes from archaea (35), bacteria (9) and mitochondria (this work) all bind the terminal A76 in an essentially identical manner by intercalating between two purines of rRNA, while at the same time the rest of the E site has diverged among these classes of ribosomes.…”