The CCA-end of P-tRNA Contacts Both the Human RPL36AL and the A-site Bound Translation Termination Factor eRF1 at the Peptidyl Transferase Center of the Human 80S Ribosome

Abstract

We have demonstrated previously that the E-site specific protein RPL36AL present in human ribosomes can be crosslinked with the CCA-end of a P-tRNA in situ. Here we report the following: (i) We modeled RPL36AL into the structure of the archaeal ortholog RPL44E extracted from the known X-ray structure of the 50S subunit of Haloarcula marismortui. Superimposing the obtained RPL36AL structure with that of P/E tRNA observed in eukaryotic 80S ribosomes suggested that RPL36AL might in addition to its CCA neighbourhood interact with the inner site of the tRNA elbow similar to an interaction pattern known from tRNA•synthetase pairs. (ii) Accordingly, we detected that the isolated recombinant protein RPL36AL can form a tight binary complex with deacylated tRNA, and even tRNA fragments truncated at their CCA end showed a high affinity in the nanomolar range supporting a strong interaction outside the CCA end. (iii) We constructed programmed 80S complexes containing the termination factor eRF1 (stop codon UAA at the A-site) and a 2',3'-dialdehyde tRNA (tRNAox) analog at the P-site. Surprisingly, we observed a crosslinked ternary complex containing the tRNA, eRF1 and RPL36AL crosslinked both to the aldehyde groups of tRNAox at the 2'- and 3'-positions of the ultimate A. We also demonstrated that, upon binding to the ribosomal A-site, eRF1 induces an alternative conformation of the ribosome and/or the tRNA, leading to a novel crosslink of tRNAox to another large-subunit ribosomal protein (namely L37) rather than to RPL36AL, both ribosomal proteins being labeled in a mutually exclusive fashion. Since the human 80S ribosome in complex with P-site bound tRNAox and A-site bound eRF1 corresponds to the post-termination state of the ribosome, the results represent the first biochemical evidence for the positioning of the CCA-arm of the P-tRNA in close proximity to both RPL36AL and eRF1 at the end of the translation process.

Keywords: A-site stop codon; CCA-end; P-tRNA; RPL36AL/tRNAox/eRF1 ternary complex on the human 80S ribosome.; abnormally low pK for Lys-53 of human RPL36AL; conformational change of eRF1 upon binding to the ribosome; crosslinking; eRF1; effect of the eRF1/eRF3 complex on the crosslinking of eRF1 in the human 80S ribosome; human s80S ribosomes; recombinant human RPL36AL.

Fig. (1)

Model of the L36AL protein and of its interaction with tRNA. (A), ribbon representation of the 3-D structure of human RPL36AL (fragment 1-94) modeled by homology with the crystallographic structure of the archaeal counterpart RPL44E of the 60S ribosomal subunit from Haloarcula marismortui. The post-translational modifications (including the methylated Q51) [7], and the consensus pattern 61Kx(TorV)KKxxL(KorR)xxC72 (numbering of human RPL36AL) of the L44e family of r-proteins are colored pink and cyan, respectively. The 49GG50 dipeptide of the GGQ motif is highlighted in green. A zinc ion represented by a cadmium colored yellow is also shown. Fragment 86-94 corresponding to the nucleotide binding motif 2 (NBD2) common to all eukaryotic RPL7 [29] and RPL36A/RPL44 is shown in wheat. (B), overlaid structures of tRNAPhe (PDB ID 1JGQ) colored grey and of human RPL36AL (blue). The GGQ motif is shown in red, the 3’ terminal CCA trinucleotide of tRNA in pink, and the side chain of Lys-53 in green. (C), NBD2 [29] located in the C-terminal region of the eukaryotic RPL7 family (ortholog of the bacterial L30 protein) is conserved in the C-terminal region of the eukaryotic RPL36A/RPL44 family. The organisms are: Arabidopsis thaliana (arb. thal.), Drosophila melanogaster (droso.), Saccharomyces cerevisiae (s. cer.), Solanum tuberosum (sol. tub.). The bottom line labels residues as either strictly conserved (*), highly conserved (:) or weakly conserved (.). The alignment was generated with the program ClustalX.

Fig. (2)

Kinetic measurement of RPL36AL:tRNA interaction. (A), His-tagged RPL36AL was immobilized on the surface of NTA sensor chip at low density resonance units (RU). Various concentrations of tRNAAsp76 (0, 1, 2, 4, 5 and 6 nM) were run over the chip surface. (B), the same experiment with tRNAAsp71.

Fig. (3)

Crosslinking of recombinant human RPL36AL with periodate-oxidized full length tRNA (tRNAAsp76ox, lane 1), or tRNAox species that were shortened by one (tRNAAsp75ox, lane 2), two (tRNAAsp74ox, lane 3), three (tRNAAsp73ox, lane 4) or four nucleotides (tRNAAsp72ox, lane 5) from the 3’-end. The incubation mixtures were applied onto a 10% polyacrylamide gel run by 8 M Urea electrophoresis and colored with Stains all. The “a” bands represent the tRNAox species crosslinked with recombinant L36AL, while the lower bands represent the tRNA analogues, as verified by routinely performed control experiments on the full length tRNA molecule (tRNAAsp76), or the truncated tRNA species.

Fig. (4)

Complexes 1 and 2 designed for crosslinking of Human 80S ribosome with P-tRNAAsp76ox in the absence or in the presence of human eRF1 bound to an A-site UAA stop codon.

Fig. (5)

Crosslinking of endogenous ribosomal protein L36AL in human 80S ribosomes with full length or truncated tRNAox species in the absence or in the presence of the translation termination factor eRF1. (A) Complexes 2: 80S ribosomes incubated in the presence of eRF1 with [5'-32P]tRNAAsp76ox, [5'-32P] tRNAAsp75ox, [5'-32P]tRNAAsp74ox or [5'-32P]tRNAAsp73ox (lanes 1-4, respectively). The controls are: (i) crosslinking in ribosomal complex 1 with [5'-32P]tRNAAsp76ox, in the absence of eRF1 (lane 1*); (ii) crosslinking of [5'- 32P]tRNAAsp76ox with eRF1 in the absence of 80S ribosomes (lane k). (B) 80S ribosomes incubated with [5'-32P]tRNAAsp75ox at pH 5.0, 6.0, 7.0, 7.5, 8.0 and 9.0 (lanes 1-6, respectively) with a free adjacent A-site. Sodium borohydride (NaBH4) was used to trap the fraction of unprotonated reactive ε-amino group that is responsible for the nucleophilic addition to the aldehyde in order to form the Schiff base. (C) Same experiment as in (A) with 80S ribosomes carrying [5'-32P]tRNAAsp76ox at the P-site and eRF1 at the A-site incubated at pH 7.5 and 8.0, respectively (lanes 2) with the following controls. Lanes 1, crosslinking in ribosomal complex 1 with [5'-32P]tRNAAsp76ox, in the absence of eRF1 at pH 7.5 and 8.0, respectively. Lanes k, crosslinking of [5'-32P]tRNAAsp76ox with eRF1 in the absence of 80S ribosomes at pH 7.5 and 8.0, respectively (lanes k). For details see Materials and Methods.

Fig. (6)

Structure of the ternary RPL36AL-tRNAox-eRF1 covalent complex.

Fig. (7)

Analysis in 10% SDS-PAGE of crosslinked ribosomal complexes 1-5 obtained in the presence of [5’-32P]tRNAAsp76ox (lanes 1-5, respectively). Autoradiogram of the gel. The positions of tRNA analogues and crosslinking products are marked.

Fig. (8)

Reaction scheme of the Schiff base formation and its in situ reduction with sodium borohydride (NaBH4) or sodium cyanoborohydride (NaBH3CN). With both reducing agents, the overall crosslinking reaction is rate-limited by the formation of the imminium moiety, not by its in situ reduction.

Fig. (9)

Plot of the molar fraction of crosslinking as a function of pH. Two data sets from two separate experiments of Fig. (5B) were used to obtain this graph. The data were fitted with Mathematica to the function inscribed in the figure with the following features : the dashed lines defines the 95% confidence intervals for the prediction of a single value, whereas the continous lines and the gray-shaded region define the 95% confidence intervals for the prediction of the average curve with pK = 6.9 ± 0.2.

Fig. (10)

Peptidyl-tRNA hydrolase assay of the recombinant human L36AL protein using N-acetyl[3H]Phe-tRNAPhe as a substrate. The percent of residual N-acetyl[3H]Phe-tRNAPhe precipitable in trichloracetic acid was measured as a function of the concentration of the recombinant human RPL36AL. As a control, the activity of E. coli peptidyl-tRNA hydrolase (Pth) as a function of enzyme concentration is also shown.

Fig. (11)

Model for the interactions between the ribosomal protein L36AL, the CCA-arm of P-site bound tRNA and the translation termination factor eRF1 bound to an A-site stop codon.

Fig. (12)

Multiple sequence alignment of eRF1s in the region encompassing the GGQ motif common to all class-1 translation termination factors and the corresponding region of rPs of the RPL36A/RPL44E family from different eukaryotic organisms. The top line labels residues as either strictly conserved (*), highly conserved (:) or weakly conserved (.). The alignment was generated with the program ClustalX. XENTR, Xenopus tropicalis; CAEEL, Caenorhabditis elegans.