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1. Atomistic simulations of the Escherichia coli ribosome provide selection criteria for translationally active substrates

   https://doi.org/10.1038/s41557-023-01226-w  

   Watson, Zoe L.; Knudson, Isaac J.; Ward, Fred R.; Miller, Scott J.; Cate, Jamie H. D.; Schepartz, Alanna; Abramyan, Ara M. (June 2023, Nature Chemistry)

   Abstract As genetic code expansion advances beyondl-α-amino acids to backbone modifications and new polymerization chemistries, delineating what substrates the ribosome can accommodate remains a challenge. TheEscherichia coliribosome tolerates non-l-α-amino acids in vitro, but few structural insights that explain how are available, and the boundary conditions for efficient bond formation are so far unknown. Here we determine a high-resolution cryogenic electron microscopy structure of theE. coliribosome containing α-amino acid monomers and use metadynamics simulations to define energy surface minima and understand incorporation efficiencies. Reactive monomers across diverse structural classes favour a conformational space where the aminoacyl-tRNA nucleophile is <4 Å from the peptidyl-tRNA carbonyl with a Bürgi–Dunitz angle of 76–115°. Monomers with free energy minima that fall outside this conformational space do not react efficiently. This insight should accelerate the in vivo and in vitro ribosomal synthesis of sequence-defined, non-peptide heterooligomers. 

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2. Fine-Tuning of Alanyl-tRNA Synthetase Quality Control Alleviates Global Dysregulation of the Proteome

   https://doi.org/10.3390/genes11101222  

   Kelly, Paul; Kavoor, Arundhati; Ibba, Michael (October 2020, Genes)

   null (Ed.)

   One integral step in the transition from a nucleic acid encoded-genome to functional proteins is the aminoacylation of tRNA molecules. To perform this activity, aminoacyl-tRNA synthetases (aaRSs) activate free amino acids in the cell forming an aminoacyl-adenylate before transferring the amino acid on to its cognate tRNA. These newly formed aminoacyl-tRNA (aa-tRNA) can then be used by the ribosome during mRNA decoding. In Escherichia coli, there are twenty aaRSs encoded in the genome, each of which corresponds to one of the twenty proteinogenic amino acids used in translation. Given the shared chemicophysical properties of many amino acids, aaRSs have evolved mechanisms to prevent erroneous aa-tRNA formation with non-cognate amino acid substrates. Of particular interest is the post-transfer proofreading activity of alanyl-tRNA synthetase (AlaRS) which prevents the accumulation of Ser-tRNAAla and Gly-tRNAAla in the cell. We have previously shown that defects in AlaRS proofreading of Ser-tRNAAla lead to global dysregulation of the E. coli proteome, subsequently causing defects in growth, motility, and antibiotic sensitivity. Here we report second-site AlaRS suppressor mutations that alleviate the aforementioned phenotypes, revealing previously uncharacterized residues within the AlaRS proofreading domain that function in quality control. 

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3. Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

   https://doi.org/10.1038/s41467-021-25663-8  

   van der Stel, Anne-Xander; Gordon, Emily R.; Sengupta, Arnab; Martínez, Allyson K.; Klepacki, Dorota; Perry, Thomas N.; Herrero del Valle, Alba; Vázquez-Laslop, Nora; Sachs, Matthew S.; Cruz-Vera, Luis R.; et al (December 2021, Nature Communications)

   null (Ed.)

   Abstract Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC–ribosome complex captures a single L-Trp molecule to undergo termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite. 

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4. Ribosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro

   https://doi.org/10.1038/s41467-020-18001-x  

   Lee, Joongoo; Schwarz, Kevin J.; Kim, Do Soon; Moore, Jeffrey S.; Jewett, Michael C. (August 2020, Nature Communications)

   Abstract Ribosome-mediated polymerization of backbone-extended monomers into polypeptides is challenging due to their poor compatibility with the translation apparatus, which evolved to use α-L-amino acids. Moreover, mechanisms to acylate (or charge) these monomers to transfer RNAs (tRNAs) to make aminoacyl-tRNA substrates is a bottleneck. Here, we rationally design non-canonical amino acid analogs with extended carbon chains (γ-, δ-, ε-, and ζ-) or cyclic structures (cyclobutane, cyclopentane, and cyclohexane) to improve tRNA charging. We then demonstrate site-specific incorporation of these non-canonical, backbone-extended monomers at the N- and C- terminus of peptides using wild-type and engineered ribosomes. This work expands the scope of ribosome-mediated polymerization, setting the stage for new medicines and materials. 

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5. Charting the sequence-activity landscape of peptide inhibitors of translation termination

   https://doi.org/10.1073/pnas.2026465118  

   Baliga, Chetana; Brown, Tyler J.; Florin, Tanja; Colon, Sarah; Shah, Vallari; Skowron, Kornelia J.; Kefi, Amira; Szal, Teresa; Klepacki, Dorota; Moore, Terry W.; et al (March 2021, Proceedings of the National Academy of Sciences)

   Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api’s activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineeredapigene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide. 

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