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Translation initiation is a highly regulated, multi-step process that is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a dedicated initiator fMet-tRNA fMet bind the small (30S) ribosomal subunit. Specific binding of fMet-tRNA fMet in the peptidyl (P) site is mediated by the inspection of the fMet moiety by initiation factor IF2 and of three conserved G-C base pairs in the tRNA anticodon stem by the 30S head domain. Tandem A-minor interactions form between 16S ribosomal RNA nucleotides A1339 and G1338 and tRNA base pairs G30-C40 and G29-C41, respectively. Swapping the G30-C40 pair of tRNA fMet with C-G (called tRNA fMet M1) reduces discrimination against the noncanonical start codon CUG in vitro, suggesting crosstalk between the gripping of the anticodon stem and recognition of the start codon. Here, we solved electron cryomicroscopy structures of Escherichia coli 70S initiation complexes containing the fMet-tRNA fMet M1 variant paired to the noncanonical CUG start codon, in the presence or absence of IF2 and the non-hydrolyzable GTP analog GDPCP, alongside structures of 70S initiation complexes containing this tRNA fMet variant paired to the canonical bacterial start codons AUG, GUG, and UUG. We find that the M1 mutation weakens A-minor interactions between tRNA fMet and 16S nucleotides A1339 and G1338, with IF2 strengthening the interaction of G1338 with the tRNA minor groove. These structures suggest how even slight changes to the recognition of the fMet-tRNA fMet anticodon stem by the ribosome can impact the start codon selection., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest regarding the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ribosomal incorporation of β-amino acids into nascent peptides is much less efficient than that of the canonical α-amino acids. To overcome this, we have engineered a tRNA chimera bearing T-stem of tRNAGlu and D-arm of tRNAPro1, referred to as tRNAPro1E2, which efficiently recruits EF-Tu and EF-P. Using tRNAPro1E2 indeed improved β-amino acid incorporation. However, multiple/consecutive incorporations of β-amino acids are still detrimentally poor. Here, we attempted fine-tuning of the anticodon arm of tRNAPro1E2 aiming at further enhancement of β-amino acid incorporation. By screening various mutations introduced into tRNAPro1E2, C31G39/C28G42 mutation showed an approximately 3-fold enhancement of two consecutive incorporation of β-homophenylglycine (βPhg) at CCG codons. The use of this tRNA made it possible for the first time to elongate up to ten consecutive βPhg's. Since the enhancement effect of anticodon arm mutations differs depending on the codon used for β-amino acid incorporation, we optimized anticodon arm sequences for five codons (CCG, CAU, CAG, ACU and UGG). Combination of the five optimal tRNAs for these codons made it possible to introduce five different kinds of β-amino acids and analogs simultaneously into model peptides, including a macrocyclic scaffold. This strategy would enable ribosomal synthesis of libraries of macrocyclic peptides containing multiple β-amino acids., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Angiogenin, an RNase-A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases and epigenetic inheritance 1-10 . After activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, resulting in translation repression 11-15 . However, the catalytic activity of isolated angiogenin is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle 3,16-23 . Here we identify these mechanisms using biochemical assays and cryogenic electron microscopy (cryo-EM). Our study reveals that the cytosolic ribosome is the activator of angiogenin. A cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic centre, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S-angiogenin structures capture how tRNA substrate is directed by the ribosome into angiogenin's active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, the abundance of which increases during cellular stress 24-27 . These results may facilitate the development of therapeutics to treat cancer and neurodegenerative diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The ribosome brings 3'-aminoacyl-tRNA and 3'-peptidyl-tRNAs together to enable peptidyl transfer by binding them in two major ways. First, their anticodon loops are bound to mRNA, itself anchored at the ribosomal subunit interface, by contiguous anticodon:codon pairing augmented by interactions with the decoding center of the small ribosomal subunit. Second, their acceptor stems are bound by the peptidyl transferase center, which aligns the 3'-aminoacyl- and 3'-peptidyl-termini for optimal interaction of the nucleophilic amino group and electrophilic ester carbonyl group. Reasoning that intrinsic codon:anticodon binding might have been a major contributor to bringing tRNA 3'-termini into proximity at an early stage of ribosomal peptide synthesis, we wondered if primordial amino acids might have been assigned to those codons that bind the corresponding anticodon loops most tightly. By measuring the binding of anticodon stem loops to short oligonucleotides, we determined that family-box codon:anticodon pairings are typically tighter than split-box codon:anticodon pairings. Furthermore, we find that two family-box anticodon stem loops can tightly bind a pair of contiguous codons simultaneously, whereas two split-box anticodon stem loops cannot. The amino acids assigned to family boxes correspond to those accessible by what has been termed cyanosulfidic chemistry, supporting the contention that these limited amino acids might have been the first used in primordial coded peptide synthesis.

The anticodon modifications of transfer RNAs (tRNAs) finetune the codon recognition on the ribosome for accurate translation. Bacteria and archaea utilize the modified cytidines, lysidine (L) and agmatidine (agm 2 C), respectively, in the anticodon of tRNA Ile to decipher AUA codon. L and agm 2 C contain long side chains with polar termini, but their functions remain elusive. Here we report the cryogenic electron microscopy structures of tRNAs Ile recognizing the AUA codon on the ribosome. Both modifications interact with the third adenine of the codon via a unique C-A geometry. The side chains extend toward 3' direction of the mRNA, and the polar termini form hydrogen bonds with 2'-OH of the residue 3'-adjacent to the AUA codon. Biochemical analyses demonstrated that AUA decoding is facilitated by the additional interaction between the polar termini of the modified cytidines and 2'-OH of the fourth mRNA residue. We also visualized cyclic N 6 -threonylcarbamoyladenosine (ct 6 A), another tRNA modification, and revealed a molecular basis how ct 6 A contributes to efficient decoding., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The frequency of errors upon decoding of messenger RNA by the bacterial ribosome is low, with one misreading event per 1 × 10 4 codons. In the universal genetic code, the AUN codon box specifies two amino acids, isoleucine and methionine. In bacteria and archaea, decoding specificity of the AUA and AUG codons relies on the wobble avoidance strategy that requires modification of C34 in the anticodon loop of isoleucine transfer RNA Ile CAU (tRNA Ile CAU ). Bacterial tRNA Ile CAU with 2-lysylcytidine (lysidine) at the wobble position deciphers AUA while avoiding AUG. Here we report cryo-electron microscopy structures of the Escherichia coli 70S ribosome complexed with elongation factor thermo unstable (EF-Tu) and isoleucine-tRNA Ile LAU in the process of decoding AUA and AUG. Lysidine in tRNA Ile LAU excludes AUG by promoting the formation of an unusual Hoogsteen purine-pyrimidine nucleobase geometry at the third position of the codon, weakening the interactions with the mRNA and destabilizing the EF-Tu ternary complex. Our findings elucidate the molecular mechanism by which tRNA Ile LAU specifically decodes AUA over AUG., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)