Conformational control of translation termination on the 70S ribosome

Abstract

Translation termination ensures proper lengths of cellular proteins. During termination, release factor (RF) recognizes a stop codon and catalyzes peptide release.Conformational changes in RF are thought to underlie accurate translation termination.If true, the release factor should bind the A-site codon in inactive (compact) conformation(s), but structural studies of ribosome termination complexes have only captured RFs in an extended, active conformation. Here, we identify a hyper-accurate RF1 variant, and present crystal structures of 70S termination complexes that suggest a structural pathway for RF1 activation. In the presence of blasticidin S, the catalytic domain of RF1 is removed from the peptidyl-transferase center, whereas the codonrecognition domain is fully engaged in stop-codon recognition in the decoding center.RF1 codon recognition induces decoding-center rearrangements that precede accommodation of the catalytic domain. Our findings suggest how structural dynamics of RF1 and the ribosome coordinate stop-codon recognition with peptide release, ensuring accurate translation termination.

Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release

“…2 D ; 14 nM RF1), at a concentration approximately sevenfold below the lowest K D of RF1 for a modified stop codon—i.e., UAP. Under both conditions, RF1 rate constants were nearly identical for 70S complexes formed with UAA [420 nM RF1: k cat = (6.0 ± 0.1) × 10 −2 s −1 ; 14 nM RF1: k cat = (1.9 ± 0.1) × 10 −2 s −1 ] and UAG [420 nM RF1: k cat = (6.1 ± 0.1) × 10 −2 s −1 ; 14 nM RF1: k cat = 2.0 × 10 −2 s −1 ], which is in line with earlier reports ( 22 , 44 – 46 ). The rates of release on UAP [420 nM RF1: k cat = (4.7 ± 0.1) × 10 −2 s −1 ; 14 nM RF1: k cat = 1.6 × 10 −2 s −1 ] and UPG codons [420 nM RF1: k cat = (5.2 ± 0.1) × 10 −2 s −1 ; 14 nM RF1: k cat = (2.0 ± 0.1) × 10 −2 s −1 ] were similar to UAA and UAG, respectively.…”

“…However, mutational studies also identified other amino acids that were involved in stop codon recognition, implicating that the tripeptide anticodon alone is not sufficient for providing translation termination ( 20 ). Remarkably, mutations altering the codon specificity of RFs were only identified outside of the potential codon recognition site ( 20 – 22 ).…”

Ribosome inhibition byC9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

“…The effects of GR n or PR n on peptide bond formation in E. coli ribosome were tested in an in vitro assay that reacts the 70S ribosome loaded with [ 35 S]-fMet-tRNA fMet in the P site with puromycin, an A-site tRNA mimic, and measures the release of the resulting dipeptide containing formylmethionine covalently bonded to puromycin, as described (Svidritskiy and Korostelev, 2018). E. coli tRNA fMet (Chemical Block) was aminoacylated with [ 35 S]-N-formyl-L-methionine (Perkin Elmer) as described (Lancaster and Noller, 2005).…”

“…E. coli tRNA fMet (Chemical Block) was aminoacylated with [ 35 S]-N-formyl-L-methionine (Perkin Elmer) as described (Lancaster and Noller, 2005). 70S ribosomes were prepared from E. coli (MRE600) as described (Svidritskiy and Korostelev, 2018). 70S ribosomes were stored in the ribosome-storage buffer (20 mM Tris-HCl, pH 7.0; 100 mM NH 4 Cl; 20 mM MgCl 2 ) at -80°C.…”

“…30S and 50S ribosomal subunits were prepared from MRE600 E. coli as described (Svidritskiy and Korostelev, 2018) and stored in Buffer A (20 mM Tris-HCl, pH 7, 10.5 mM MgCl 2 , 100 mM NH 4 Cl, 0.5 mM EDTA, 6 mM β-mercaptoethanol) at -80°C. E. coli EF-Tu, fMet-tRNA fMet and Val-tRNA Val (tRNA Val from Chemical Block) were prepared as described (Loveland et al, 2017).…”