Drugging tRNA aminoacylation

<p>Inhibition of tRNA aminoacylation has proven to be an effective antimicrobial strategy, impeding an essential step of protein synthesis. Mupirocin, the well-known selective inhibitor of bacterial isoleucyl-tRNA synthetase, is one of three aminoacylation inhibitors now approved for human or animal use. However, design of novel aminoacylation inhibitors is complicated by the steadfast requirement to avoid off-target inhibition of protein synthesis in human cells. Here we review available data regarding known aminoacylation inhibitors as well as key amino-acid residues in aminoacyl-tRNA synthetases (aaRSs) and nucleotides in tRNA that determine the specificity and strength of the aaRS-tRNA interaction. Unlike most ligand-protein interactions, the aaRS-tRNA recognition interaction represents coevolution of both the tRNA and aaRS structures to conserve the specificity of aminoacylation. This property means that many determinants of tRNA recognition in pathogens have diverged from those of humans—a phenomenon that provides a valuable source of data for antimicrobial drug development.</p

The Mechanism of Pre-transfer Editing in Yeast Mitochondrial Threonyl-tRNA Synthetase

Background: The mechanism of pre-transfer editing by which aaRSs regulate translational fidelity is not well understood. Results: Yeast mitochondrial ThrRS, MST1, hydrolyzes seryl adenylate at the aminoacylation active site more rapidly than the cognate threonyl adenylate. Conclusion: MST1 discriminates against serine and reduces mischarging of threonine tRNA by employing pre-transfer editing. Significance: The mechanism of misactivation and pre-transfer editing of serine by ThrRS is provided

Suppression of Amber Codons in <i>Caulobacter crescentus</i> by the Orthogonal <i>Escherichia coli</i> Histidyl-tRNA Synthetase/tRNA^His Pair

<div><p>While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium <i>Caulobacter crescentus</i>. Based on a previous report that <i>C. crescentus</i> and <i>Escherichia coli</i> tRNA^His have distinctive identity elements, we constructed <i>E. coli</i> tRNA^His_CUA, a UAG suppressor tRNA for <i>C. crescentus</i>. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the <i>C. crescentus</i> xylonate dehydratase), we demonstrated that the <i>E. coli</i> histidyl-tRNA synthetase/tRNA^His_CUA pair enables <i>in vivo</i> UAG suppression in <i>C. crescentus</i>. <i>E. coli</i> histidyl-tRNA synthetase (HisRS) or tRNA^His_CUA alone did not achieve suppression; this indicates that the <i>E. coli</i> HisRS/tRNA^His_CUA pair is orthogonal in <i>C. crescentus</i>. These results illustrate that UAG suppression can be achieved in <i>C. crescentus</i> with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.</p></div

Suppression of a UAG codon of the mCherry gene.

<p>A, A fluorescence (left) and a phase contrast (right) image. The cells contained pRV-lac2-mCherry and pBX-HisRS-tRNA^His_CUA. The scale bar represents 1 µm. B. Images of cells that contained the mutant mCherry gene and pBXMCS-2, which is the empty vector. C. Images of cells expressing the mutant mCherry gene and the <i>E. coli</i> HisRS/tRNA^His_CUA. D, Histogram of the ratio between percentages of cells and fluorescence intensities. CB15N stands for <i>C. crescentus</i> CB15N strain harboring pRV-lac2-mCherryTAG. <i>Eco</i> HisRS+tRNA^His_CUA stands for the same strain containing the additional pBX-HisRS-tRNA^His_CUA plasmid.</p

Suppression of amber codons in <i>C. crescentus</i> by <i>E. coli</i> HisRS/tRNA^His_CUA.

<p>The <i>E. coli</i> HisRS/tRNA^His_CUA pair with the CUA anticodon is orthogonal in <i>C. crescentus</i>. The <i>E. coli</i> pair suppresses an in-frame amber codon in the reporter gene, which allows the expression of the gene while <i>C. crescentus</i> aminoacyl-tRNA synthetase (aaRS)/tRNA pairs are not able to suppress the amber mutation. <i>E. coli</i> HisRS is shown in blue and <i>C. crescentus</i> aminoacyl-tRNA synthetases are shown in green.</p

Mass spectroscopic confirmation of histidine incorporation by the <i>E. coli</i> HisRS/tRNA^His_CUA pair.

<p>Annotated MS/MS spectra and ions for the HRPLGDR peptide from GroES. H stands for an immonium ion of histidine.</p

DataSheet4_Rational design of the genetic code expansion toolkit for in vivo encoding of D-amino acids.pdf

Once thought to be non-naturally occurring, D-amino acids (DAAs) have in recent years been revealed to play a wide range of physiological roles across the tree of life, including in human systems. Synthetic biologists have since exploited DAAs’ unique biophysical properties to generate peptides and proteins with novel or enhanced functions. However, while peptides and small proteins containing DAAs can be efficiently prepared in vitro, producing large-sized heterochiral proteins poses as a major challenge mainly due to absence of pre-existing DAA translational machinery and presence of endogenous chiral discriminators. Based on our previous work demonstrating pyrrolysyl-tRNA synthetase’s (PylRS’) remarkable substrate polyspecificity, this work attempts to increase PylRS’ ability in directly charging tRNAPyl with D-phenylalanine analogs (DFAs). We here report a novel, polyspecific Methanosarcina mazei PylRS mutant, DFRS2, capable of incorporating DFAs into proteins via ribosomal synthesis in vivo. To validate its utility, in vivo translational DAA substitution were performed in superfolder green fluorescent protein and human heavy chain ferritin, successfully altering both proteins’ physiochemical properties. Furthermore, aminoacylation kinetic assays further demonstrated aminoacylation of DFAs by DFRS2 in vitro.</p

Archaeal Tuc1/Ncs6 Homolog Required for Wobble Uridine tRNA Thiolation Is Associated with Ubiquitin-Proteasome, Translation, and RNA Processing System Homologs

<div><p>While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of <i>Haloferax volcanii</i>) that is essential for maintaining cellular pools of thiolated tRNA^Lys_UUU and for growth at high temperature. When purified from <i>Hfx. volcanii</i>, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA^Lys_UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.</p></div

Multiple amino acid sequence alignment of <i>Hfx. volcanii</i> NcsA (HVO_0580) with ANH superfamily members including proteins of <i>Saccharomyces cerevisiae</i> (ScNcs6, GI:50593215), <i>Homo sapiens</i> (HsNcs6, GI:74713747), <i>Pyrococcus horikoshii</i> (PH1680, GI:14591444; PH0300, GI:14590222), <i>Thermus thermophilus</i> (TTHA0477 or TtuA, GI: 55980446), <i>Salmonella typhimurium</i> (StTtcA, GI:16764998), and <i>Escherichia coli</i> (EcTtcA, GI:85674916).

<p>Conserved residues are highlighted in red, grey and black, with the conserved residues in red of the ATP pyrophosphatase signature PP-motif (SGGXDS) involved in ATP binding (Bork and Koonin, 1994) as well as motifs CXXC and GHXXDD (which act to recognize RNA) present in the TtcA protein family (Jager et al., 2004). Zinc fingers are highlighted in blue boxes, ubiquitin-fold modified lysine residues are in red boxes, and conserved catalytic cysteine residues are indicated by a star. Secondary structural elements predicted for HVO_0580 based on Phyre2 3D homology modeling are highlighted with blue arrows (β-sheets) and green cylinders (α-helices) above the amino acid sequence.</p

Proteins Identified by LC-MS/MS proteomic analysis^a.

a<p>MS-identified proteins with coverage above 25% are reported according to the <i>Hfx. volcanii</i> gene locus tag from the National Center for Biotechnology Information and were unique to samples prepared from strain <i>ΔncsA</i> expressing the FLAG-tagged SAMP1 in tandem with StrepII-tagged NcsA, FLAG-tagged SAMP2 in tandem with StrepII-tagged NcsA, or StrepII-tagged NcsA alone compared to the vector alone. Theoretical molecular mass (M_r) estimated from deduced polypeptide based on <i>Hfx. volcanii</i> DS2 genome sequence.</p