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.

One key question in protein biosynthesis is how the ribosome couples mRNA and tRNA movements to prevent disruption of weak codon-anticodon interactions and loss of the translational reading frame during translocation. Here we report the complete path of mRNA on the 70S ribosome at the atomic level (3.1-A resolution), and we show that one of the conformational rearrangements that occurs upon transition from initiation to elongation is a narrowing of the downstream mRNA tunnel. This rearrangement triggers formation of a network of interactions between the mRNA downstream of the A-site codon and the elongating ribosome. Our data elucidate the mechanism by which hypermodified nucleoside 2-methylthio-N6 isopentenyl adenosine at position 37 (ms(2)i(6)A37) in tRNA(Phe)(GAA) stabilizes mRNA-tRNA interactions in all three tRNA binding sites. Another network of contacts is formed between this tRNA modification and ribosomal elements surrounding the mRNA E/P kink, resulting in the anchoring of P-site tRNA. These data allow rationalization of how modification deficiencies of ms(2)i(6)A37 in tRNAs may lead to shifts of the translational reading frame.

Guanine-rich nucleic acid sequences challenge the replication, transcription, and translation machinery by spontaneously folding into G-quadruplexes, the unfolding of which requires forces greater than what most polymerases can exert1,2. Eukaryotic cells host numerous helicases capable of unfolding G-quadruplexes3. The molecular basis for helicase recognition and unfolding of G-quadruplexes remains poorly understood. DHX36 (RHAU, G4R1), a member of the DEAH/RHA family of helicases, binds both DNA and RNA G-quadruplexes with uniquely high affinity4,5,6, is consistently found bound to G-quadruplexes in cells7,8, and is a major source of G-quadruplex unfolding activity in HeLa cell lysates6. DHX36 is a multi-functional helicase implicated in G-quadruplex-mediated transcriptional and post-transcriptional regulation, and is essential for heart development, hematopoiesis, and embryogenesis in mice9,10,11,12. Here, we report the co-crystal structure of bovine DHX36 bound to a DNA with a G-quadruplex and a 3’ single-stranded (ssDNA) segment. We show that the N-terminal DHX36-specific motif folds into a DNA-binding-induced α-helix that together with the OB-fold-like subdomain selectively binds parallel G-quadruplexes. Comparison with our unliganded and ATP-analog-bound DHX36 structures, together with single-molecule FRET analysis, suggests that G-quadruplex binding alone induces rearrangements of the helicase core, which by pulling on its ssDNA tail, drive G-quadruplex unfolding by one residue at a time.

Discrimination of tRNA on the ribosome occurs in two consecutive steps: initial selection and proofreading. Here we propose a proofreading mechanism based on comparison of crystal structures of the 70S ribosome with an empty A site or with the A site occupied by uncharged cognate or near-cognate tRNA. We observe that ribosomal proteins S13, S19, L16, L25, L27 and L31 are actively involved in the proofreading of tRNA. We suggest that proofreading begins with the monitoring of the entire anticodon loop of tRNA by nucleotides from 16S rRNA (helices 18 and 44) of the small subunit and 23S rRNA (helix 69) of the large subunit with involvement of magnesium ions. Subsequently, the elbow region is scanned by rRNA (helices 38 and 89) and proteins from the large subunit determining whether to accommodate the acceptor end of tRNA in the peptidyl transferase center or not.