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The ribosome is a ribonucleoprotein complex found in all domains of life. Its role is to catalyze protein synthesis, the messenger RNA (mRNA)-templated formation of amide bonds between α-amino acid monomers. Amide bond formation occurs within a highly conserved region of the large ribosomal subunit known as the peptidyl transferase center (PTC). Here we describe the step-wise design and characterization of mini-PTC 1.1, a 284-nucleotide RNA that recapitulates many essential features of the Escherichia coli PTC. Mini-PTC 1.1 folds into a PTC-like structure under physiological conditions, even in the absence of r-proteins, and engages small molecule analogs of A- and P-site tRNAs. The sequence of mini-PTC 1.1 differs from the wild type E. coli ribosome at 12 nucleotides that were installed by a cohort of citizen scientists using the on-line video game Eterna. These base changes improve both the secondary structure and tertiary folding of mini-PTC 1.1 as well as its ability to bind small molecule substrate analogs. Here, the combined input from Eterna citizen-scientists and RNA structural analysis provides a robust workflow for the design of a minimal PTC that recapitulates many features of an intact ribosome.

 

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.

 

The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-l-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio andN-formyl-l-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and am-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.

 

Genome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduce intron-containing ribosomes into theE. coligenome and perform targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering. Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome’s translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. This work sets the stage to engineer mutant ribosomes that polymerize abiological monomers with diverse chemistries and expands the scope of genome engineering for precise editing and evolution of repetitive DNA sequences.

 

Abstract Summary Chemsearch is a cross-platform server application for developing and managing a chemical compound library and associated data files, with an interface for browsing and search that allows for easy navigation to a compound of interest, similar compounds or compounds that have desired structural properties. With provisions for access control and centralized document and data storage, Chemsearch supports collaboration by distributed teams. Availability and implementation Chemsearch is a free and open-source Flask web application that can be linked to a Google Workspace account. Source code is available at https://github.com/gem-net/chemsearch (GPLv3 license). A Docker image allowing rapid deployment is available at https://hub.docker.com/r/cgemcci/chemsearch. 

The ribosome translates the genetic code into proteins in all domains of life. Its size and complexity demand long-range interactions that regulate ribosome function. These interactions are largely unknown. Here, we apply a global coevolution method, statistical coupling analysis (SCA), to identify coevolving residue networks (sectors) within the 23S ribosomal RNA (rRNA) of the large ribosomal subunit. As in proteins, SCA reveals a hierarchical organization of evolutionary constraints with near-independent groups of nucleotides forming physically contiguous networks within the three-dimensional structure. Using a quantitative, continuous-culture-with-deep-sequencing assay, we confirm that the top two SCA-predicted sectors contribute to ribosome function. These sectors map to distinct ribosome activities, and their origins trace to phylogenetic divergences across all domains of life. These findings provide a foundation to map ribosome allostery, explore ribosome biogenesis, and engineer ribosomes for new functions. Despite differences in chemical structure, protein and RNA enzymes appear to share a common internal logic of interaction and assembly.