Biology subjects

Hamlish, N. X.

Publications and source records attributed to Hamlish, N. X..

2 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↗

bioRxiv2023

Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β2-peptide linkages

Proteins and polypeptides containing extended backbone monomers embody highly desirable structures and functions, but they cannot yet be biosynthesized in cells. There are two challenges at work. First is the ribosome, whose ability to promote rapid bond-forming reactions to and from anything other than an -amino acid or -hydroxy acid is unknown. The second challenge is the absence of orthogonal enzymes that acylate tRNA with extended backbone monomers. Here we describe a general approach to the programmed cellular synthesis of proteins containing extended backbone monomers that circumvents both of these challenges. Rather than relying on direct and uncharacterized reactions of non--amino acid monomers within the ribosomal PTC, we develop a proximity-guided intramolecular rearrangement that effectively edits the protein backbone post-translationally. The method relies on the ability of PylRS-like aminoacyl-tRNA synthetase enzymes to accept diverse -hydroxy acid monomers, including those whose side chains contain masked nucleophiles. Introduction of such an -hydroxy acid monomer into a protein translated in vivo, followed by nucleophile unmasking, sets up a thermodynamically favored and quantitative intramolecular Backbone Extension Acyl Rearrangement (BEAR) reaction that edits the protein backbone to install an extended backbone monomer. In the examples described here, the intramolecular rearrangement converts an -peptide backbone directly into a {beta}-backbone. As far as we know, this report represents the first example in which a much-desired expanded backbone {beta}-amino acid linkage has been introduced site-selectively into a protein in a cell.

synthetic biology↗