Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome

Abstract

Translation termination in eukaryotes is mediated by release factors: eRF1, which is responsible for stop codon recognition and peptidyl-tRNA hydrolysis, and GTPase eRF3, which stimulates peptide release. Here, we have utilized ribose-specific probes to investigate accessibility of rRNA backbone in complexes formed by association of mRNA-and tRNA-bound human ribosomes with eRF1•eRF3•GMPPNP, eRF1•eRF3•GTP, or eRF1 alone as compared with complexes where the A site is vacant or occupied by tRNA. Our data show which rRNA ribose moieties are protected from attack by the probes in the complexes with release factors and reveal the rRNA regions increasing their accessibility to the probes after the factors bind. These regions in 28S rRNA are helices 43 and 44 in the GTPase associated center, the apical loop of helix 71, and helices 89, 92, and 94 as well as 18S rRNA helices 18 and 34. Additionally, the obtained data suggest that eRF3 neither interacts with the rRNA ribosephosphate backbone nor dissociates from the complex after GTP hydrolysis. Taken together, our findings provide new information on architecture of the eRF1 binding site on mammalian ribosome at various translation termination steps and on conformational rearrangements induced by binding of the release factors.

Exploring contacts of eRF1 with the 3′-terminus of the P site tRNA and mRNA stop signal in the human ribosome at various translation termination steps

“…In this study for the assembly of model human 80S ribosomal termination complexes, we utilized a set of short mRNA analogues containing triplet UUC or GAC targeted to the P site by cognate tRNA Phe or tRNA Asp /tRNA Asp ox , respectively, and stop codon immediately after this triplet, which occurred at the A site upon the P site tRNA binding. These complexes differed from each other by ligands bound to 80S ribosomes associated with mRNA analogue and tRNA/tRNA Asp ox and were similar to those applied earlier in various studies of eRF1 interactions during translation termination [5][6][7]16,31]. Complex 1 comprised eRF1•eRF3•GMPPNP and resembled the state of stop codon recognition with the difference that the P site tRNA was deacylated; peptidyl-tRNA could not be applied here since the approach used in this study required free 3′-terminus of tRNA capable of oxidation to a dialdehyde, which can form zero-length cross-links with proteins.…”

“…A widely accepted idea is that after GTP hydrolysis eRF3•GDP leaves the ribosome, and only then eRF1 promotes hydrolysis of peptidyl-tRNA (e.g., see [12,14]), although another possibility has been considered that eRF3 might remain bound at the ribosomal complex after GTP hydrolysis [15]. Our earlier data on chemical footprinting of rRNA backbone in various model human 80S ribosomal termination complexes have provided information on particular rRNA sites undergoing rearrangements upon eRFs binding and pointed out that eRF3 does not leave the ribosomal complex after GTP hydrolysis [16].…”

“…Ribosomal complexes containing mRNA analogue with stop codon targeted to the A site, P site tRNA/tRNA Asp ox and release factors (or without factors) were obtained and purified according to [16]. At first, the respective ternary ribosome complexes with mRNA analogue and the tRNA/tRNA Asp ox were assembled by incubation of 80S ribosomes (0.5 μM) with the above ligands taken at 5 μM concentration in buffer A at 20°C for 40 min (where specified, one of the ligands was omitted in control experiments).…”

“…The cross-linked 80S ribosomal termination complexes were purified from unbound ligands by centrifugation in 15-30% sucrose (w/w) linear gradient in buffer A (rotor SW40, 17h, 21,800 rpm, 4 o C). The content of eRF3 in the purified complexes was examined by dot blot assay utilizing rabbit polyclonal anti-eRF3 antibodies (1/2000 dilution) (Novus Biologicals), a method employed for examination of eRF1 content in similar complexes [16]. Amounts of eRF3 in the complexes were calculated by scanning of the respective signals displayed on nitrocellulose membrane with application of the ChemiDoc XRS imaging system (Bio-Rad) using a calibration curve obtained by means of definite amounts of eRF3 blotted to the same membrane.…”

Exploring accessibility of structural elements of the mammalian 40S ribosomal mRNA entry channel at various steps of translation initiation

“…Footprinting with BzCN has been fruitfully applied for the determination of 28S rRNA sites undergoing conformational rearrangements upon the binding of ribosome to SBP2, a pivotal protein in the synthesis of selenoproteins [39], and upon interaction with rp uL2, which is involved in maintaining the proper local structure of the ribosomal catalytic center due to the hydroxylation at H216 [40]. Recently with the use of this approach we have revealed rRNA nucleotides implicated in conformational changes induced by binding of release factors to terminating human ribosomes [41].…”