Biology subjects

Roe, L. T.

Publications and source records attributed to Roe, L. T..

3 recordsLinked to original sources

bioRxiv2024

Sporadic distribution of a new archaeal genetic code with all TAG codons as pyrrolysine

Numerous genetic codes developed during the evolution of Eukaryotes and three are known in Bacteria, yet no alternative genetic code has been established for Archaea. Some bacterial and archaeal proteins include selenocysteine or pyrrolysine, the 21st and 22nd amino acids, but no evidence establishes the adoption of a genetic code in which a stop codon universally encodes either amino acid. Here, we used proteomics to confirm the prediction that certain Archaea consistently incorporate pyrrolysine at TAG codons, supporting a new archaeal genetic code which we designate Genetic Code 34. This genetic code has 62 sense codons encoding 21 amino acids, and only two stop codons. In contrast with monophyletic genetic code distributions in bacteria, Code 34 occurs sporadically. This, combined with evidence for lateral gene transfer of the code change machinery and anticipated barriers to code reversal, suggests Code 34 arose independently in multiple lineages. TAG codon distribution patterns in Code 34 genomes imply a wide range in time since code switch. We identified many new enzymes containing Pyl residues, raising questions about potential roles of this amino acid in protein structure and function. We used five new PylRS/tRNAPyl pairs from Code 34 archaea to introduce new-to-nature pyrrolysine analogs into proteins in E. coli, demonstrating their utility for genetic code expansion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/615893v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@f363a5org.highwire.dtl.DTLVardef@112f061org.highwire.dtl.DTLVardef@63ad59org.highwire.dtl.DTLVardef@9eddb_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

AcrIF11 is a potent CRISPR-specific ADP-ribosyltransferase encoded by phage and plasmid

Phage-encoded anti-CRISPR (Acr) proteins inhibit CRISPR-Cas systems to allow phage replication and lysogeny maintenance. Most of the Acrs characterized to date are stable stoichiometric inhibitors. While enzymatic Acrs have been characterized biochemically, little is known about their potency, specificity, and reversibility. Here, we examine AcrIF11, a widespread phage and plasmid-encoded ADP-ribosyltransferase (ART) that inhibits the Type I-F CRISPR-Cas system. We present an NMR structure of an AcrIF11 homolog that reveals chemical shift perturbations consistent with NAD (cofactor) binding. In experiments that model both lytic phage replication and MGE/lysogen stability under high targeting pressure, AcrIF11 is a highly potent CRISPR-Cas inhibitor and more robust to Cas protein level fluctuations than stoichiometric inhibitors. Furthermore, we demonstrate that AcrIF11 is remarkably specific, predominantly ADP-ribosylating Csy1 when expressed in P. aeruginosa. Given the reversible nature of ADP-ribosylation, we hypothesized that ADPr eraser enzymes (macrodomains) could remove ADPr from Csy1, a potential limitation of PTM-based CRISPR inhibition. We demonstrate that diverse macrodomains can indeed remove the modification from Csy1 in P. aeruginosa lysate. Together, these experiments connect the in vitro observations of AcrIF11s enzymatic activity to its potent and specific effects in vivo, clarifying the advantages and drawbacks of enzymatic Acrs in the evolutionary arms race between phages and bacteria.

microbiology↗

bioRxiv2022

Orthogonal synthetases for polyketide precursors

The absence of orthogonal aminoacyl-tRNA synthetases that accept non-L--amino acids is the primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers. Here we report PylRS enzymes that accept -hydroxy acids, -thio acids, N-formyl-L--amino acids, and -carboxyl acid monomers (malonic acids) that are formally precursors to polyketide natural products. These monomers are all accommodated and accepted by the translation apparatus in vitro. High-resolution structural analysis of the complex between one such PylRS enzyme and a meta-substituted 2-benzylmalonate derivative reveals an active site that discriminates pro-chiral carboxylates and accommodates the large size and distinct electrostatics of an -carboxyl acid substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose -substituent diverges significantly from the -amine embodied in proteinogenic amino acids. These enzymes could act in synergy with natural or evolved ribosomes to generate diverse sequence-defined non-protein hetero-oligomers.

synthetic biology↗