Structure of the bacterial ribosome at 2 Å resolution

High-resolution maps and models of the bacterial ribosome provide new chemical insights into protein synthesis, and should enable the development of robust tools for cryo-EM structure modeling and ref...

elifesciences.org

Backbone Sensitivity - Cate & Schepartz Groups

Researchers in the Cate and Schepartz Groups at the University of California, Berkeley, and the Lawrence Berkeley National Laboratory, published in the <em>Journal of the American Chemical Society</em...

americanpeptidesociety.org

Co-Translational Incorporation of (R)- and (S)-β2-Hydroxy Acids In Vitro: A Structural and Biochemical Study on the E. coli Ribosome

Engineering the translation apparatus to accept backbone-modified amino acid analogues would enable the programmed synthesis of sequence-defined biopolymers with tunable properties. β-Hydroxy acids ar...

pubs.acs.org

Co-Translational Incorporation of (R)- and (S)-β2-Hydroxyacids In Vivo: Directed Evolution of Efficient Aminoacyl-tRNA Synthetases

Expanding the genetic code of living cells with noncanonical monomers (ncMs) relies on engineered aminoacyl-tRNA synthetases (aaRS) and their cognate tRNAs. Conventional aaRS engineering strategies rely on translation-dependent selection systems, limiting their utility for ncMs that are poorly accommodated by the native translational machinery. To address this limitation, we recently developed START, a translation-independent platform that selects Methanomethylophilus alvus pyrrolysyl-synthetase (MaPylRS) mutants based on their ability to acylate cognate tRNAMaPyl. START uses barcoded tRNAs to encode the identity of distinct aaRS mutants in a library. Acylation by active aaRS mutants protects the corresponding tRNAs from periodate oxidation, and their identity is retrieved subsequently through sequencing. START was previously applied to genetically encode noncanonical α-amino acids. Here, we successfully applied START to engineer MaPylRS mutants capable of acylating tRNAMaPyl with diverse non-α-amino acid substrates with good efficiency and fidelity, including (R) and (S) enantiomers of a β2-hydroxy acid, a β2-amino acids, and a malonate. Several mutants exhibit notable polyspecificity across noncanonical backbones while maintaining selectivity against their α-amino acid counterparts. Using these novel enzymes, we demonstrate the ribosomal incorporation of both (R)- and (S)-β2-hydroxy acids into a luciferase reporter protein expressed in Escherichia coli with good efficiency and fidelity. These results imply that highly active engineered aaRS/tRNA pairs can overcome the recently established limitations of EF-Tu with respect to non-α-amino acid substrates. The engineered MaPylRS mutants also enabled the successful incorporation of both (R)- and (S)-β2-hydroxy acids into a protein expressed in mammalian cells, demonstrating for the first time that eukaryotic translation can accommodate non-α-backbones.

pubs.acs.org

doi.org

In conversation with...

Engaging discussions with biological and life sciences professionals about their career journeys and the various opportunities available to graduate students and postdocs.

asbmb.org

You Can Fix Your DNA... Starting Now (feat. Nobel Prize Winner)

YouTube video by Cleo Abram

youtu.be

In conversation with...

Engaging discussions with biological and life sciences professionals about their career journeys and the various opportunities available to graduate students and postdocs.

asbmb.org

Tick-borne flavivirus exoribonuclease-resistant RNAs contain a double loop structure - Nature Communications

Many flaviviruses generate specialized non-coding RNAs that resist degradation by host exonucleases, promoting viral infection. Here, the authors characterize an RNA element in Powassan virus, highlig...

nature.com

Escherichia coli ribosomes support translation of (R) and (S) β2-hydroxyacids in vitro: a structural and biochemical study

The ribosomal incorporation of backbone-modified amino acid analogs into peptides and proteins enables the programmed synthesis of sequence-defined biopolymers with tunable properties. However, the su...

chemrxiv.org

A miniature CRISPR-Cas10 enzyme confers immunity by an inverse signaling pathway

Microbial and viral co-evolution has created immunity mechanisms involving oligonucleotide signaling that share mechanistic features with human anti-viral systems. In these pathways, including CBASS a...

biorxiv.org