A new understanding of the decoding principle on the ribosome

Abstract

During protein synthesis, the ribosome accurately selects transfer RNAs (tRNAs) in accordance with the messenger RNA (mRNA) triplet in the decoding centre. tRNA selection is initiated by elongation factor Tu, which delivers tRNA to the aminoacyl tRNA-binding site (A site) and hydrolyses GTP upon establishing codon-anticodon interactions in the decoding centre. At the following proofreading step the ribosome re-examines the tRNA and rejects it if it does not match the A codon. It was suggested that universally conserved G530, A1492 and A1493 of 16S ribosomal RNA, critical for tRNA binding in the A site, actively monitor cognate tRNA, and that recognition of the correct codon-anticodon duplex induces an overall ribosome conformational change (domain closure). Here we propose an integrated mechanism for decoding based on six X-ray structures of the 70S ribosome determined at 3.1-3.4 Å resolution, modelling cognate or near-cognate states of the decoding centre at the proofreading step. We show that the 30S subunit undergoes an identical domain closure upon binding of either cognate or near-cognate tRNA. This conformational change of the 30S subunit forms a decoding centre that constrains the mRNA in such a way that the first two nucleotides of the A codon are limited to form Watson-Crick base pairs. When U·G and G·U mismatches, generally considered to form wobble base pairs, are at the first or second codon-anticodon position, the decoding centre forces this pair to adopt the geometry close to that of a canonical C·G pair. This by itself, or with distortions in the codon-anticodon mini-helix and the anticodon loop, causes the near-cognate tRNA to dissociate from the ribosome.

Aminoacyl-tRNA synthetase evolution and sectoring of the genetic code

“…We identify two features of translation systems that are relevant. First of all, in the decoding center of the ribosome, proofreading of anticodon base pair attachments to mRNA codons, involving small ribosomal subunit conformational closure enabling EF-Tu and GTP hydrolysis, applies to the second and third anticodon positions only, not the first (wobble) position [43]. For most amino acids, the tRNA wobble position was selected to broaden recognition of mRNA codons, supporting code degeneracy and making tRNAs more readily available for insertion of the encoded amino acid.…”

“…The initial code evolved to utilize any mRNA sequence to synthesize polyglycine, used to stabilize protocells. According to this view, conformational tightening and EF-Tu and GTP proofreading of Watson-Crick base pairing between the anticodon and the codon in the second and third anticodon positions [43] became necessary at the 8→16 letter stage. The 8 letter stage is characterized by resolution of purines and pyrimidines only, but not individual bases, in the first mRNA codon position and the corresponding third tRNA anticodon position.…”

Dynamic basis for dG•dT misincorporation via tautomerization and ionization

“…Indeed, prior studies have shown that DNA polymerases cannot undergo the necessary conformational changes needed for catalysis when dG•dT is in a wobble conformation 7 and all available structures of catalytically active polymerases with bound mismatches within the active site feature WC-like dG•dT or dA•dC geometries 6,7 . Similarly, WC-like rG•rU mismatches have been shown to form in the first and second codon positions of catalytically active ribosomes 9 , in which wobbles are typically rejected 5 , potentially helping to explain translational error hotspots 41 .…”

“…1b) and potentially give rise to spontaneous mutations. Decades later, it is well established that the replicative and translational machineries form a tight grip around the WC geometry to discriminate against mismatches 2–5 . There is also evidence that both tautomeric 6–13 (Fig.…”

Celebrating wobble decoding: Half a century and still much is new

“…As the first recorded nucleoside modification within the sequence of an anticodon, 2 Crick introduced inosine in his 1966 Wobble Hypothesis. 1 While the first two bases of the codon undergo traditional base-pairing without exception, 96 Crick proposed the potential for non-canonical base pairs between the first base of the anticodon (“wobble” position 34) and the third base of the codon, U 34 ○G3, or I 34 ○A3/U3/C3 (Fig. 5).…”