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

Abstract

Here we employed site-directed cross-linking with the application of tRNA and mRNA analogues bearing an oxidized ribose at the 3'-terminus to investigate mutual arrangement of the main components of translation termination complexes formed on the human 80S ribosome bound with P site deacylated tRNA using eRF1•eRF3•GTP or eRF1 alone. In addition, we applied a model complex obtained in the same way with eRF1•eRF3•GMPPNP. We found that eRF3 content in the complexes with GTP and GMPPNP is similar, proving that eRF3 does not leave the ribosome after GTP hydrolysis. Our cross-linking data allowed determining locations of the 3'-terminus of the P site tRNA relatively the eRF1 M domain and of the mRNA stop signal toward the N domain and the ribosomal decoding site at the nucleotide-peptide resolution level. Our results indicate that locations of these components do not change after peptide release up to post-termination pre-recycling state, and the positioning of the mRNA stop signal remains similar to that when eRF1 recognizes it. Besides, we found that in all the complexes studied eRF1 shielded the N-terminal part of ribosomal protein eS30 from the interaction with the nucleotide adjacent to stop codon observed with pre-termination ribosome free of eRFs. Altogether, our findings brought important information on contacts of the key structural elements of eRF1, tRNA and mRNA in the ribosomal complexes including those mimicking different translation termination steps, thereby providing a deeper understanding of molecular mechanisms underlying events occurring in the course of protein synthesis termination in mammals.

Advances in therapeutic use of a drug-stimulated translational readthrough of premature termination codons

“…The eRF1 further interacts with another release factor – eRF3, and with GTP. This ternary termination complex (eRF1-eRF3-GTP) interacts with the poly(A)-binding protein (PABP) present at the 3′-UTR of mRNA; this leads to efficient hydrolysis of GTP and cleavage of the peptidyl-tRNA bond (Bulygin et al, 2017 ). As a consequence, the newly synthesized polypeptide chain is released from the ribosome.…”

Targeting Translation Termination Machinery with Antisense Oligonucleotides for Diseases Caused by Nonsense Mutations

“…In eukaryotes, two translation termination factors are required for cytoplasmic translation termination, eukaryotic release factor (eRF) 1 and eRF3. eRF1 recognizes all three stop codons (UAA, UAG, and UGA) through direct interaction at the ribosome decoding A site and activates the peptidyl transferase center to trigger hydrolysis of the peptidyl-tRNA, releasing the newly synthesized polypeptide [20,21]. Translational read-through efficiency depends on competition between stop codon recognition by eRF1 and decoding of the stop codon by a near-cognate tRNA [7,8].…”

Two alternative conformations of mRNA in the human ribosome during elongation and termination of translation as revealed by EPR spectroscopy

“…28), and do not affect significantly K d values of the respective mRNA analogues [10] . Model termination complexes, very similar to those used here, have been fruitfully applied for studying various aspects of translation termination by means of site-directed cross-linking [24] , [31] , [32] and chemical footprinting [9] , which justifies the utilization of such complexes assembled on the MR-stop in the current study. The adenosine residue following the UAG stop codon allows the formation of a quadruplet/U-turn structure in the A-site peculiar for termination complexes [7] .…”

“…To obtain 80S ribosomal complexes, where the A site was occupied with eRF1 alone or as eRF1•eRF3•GMPPNP or eRF1•eRF3•GTP, the complex with MR-stop and tRNAs at the P and E sites was incubated at 23 °C for 1 h with either 200 pmol of eRF1 in 1.5 μl or with a mixture containing 200 pmol of eRF1, 200 pmol of eRF3 and 20 mM GTP or GMPPNP in 2.5 μl, pre-incubated for 5 min before use. The eRF1 and eRF3 contents in these 80S ribosomal complexes were examined by dot-blot hybridization with the use of the respective polyclonal rabbit antibodies (#NBP1-57565 and #NBP1-33024 from Novus Biologicals) applied to the fractions of the complexes purified by sucrose density gradient centrifugation as described [9] , [24] . The binding levels of tRNAs targeted to the E site or the A site were examined by nitrocellulose filtration technique using the respective 5′-32P-labeled tRNAs, similar to how it has been done in our previous studies [10] , [11] , [12] .…”