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Tang, G. Q.

Publications and source records attributed to Tang, G. Q..

4 recordsLinked to original sources
bioRxiv2025

Escherichia coli transforms the leucyl-tRNA synthetase gene in vivo into primordial genes

E. coli deletes CP1 and ABD domains from [~]40% of plasmids containing leucyl-tRNA synthetase if and only if both active-site signatures are mutated. Shortened ORFs occurred in all reading frames but form three discrete sets in the same frame. One had only the AVGA signature. Two longer ones both retained the same 24-residue segment containing the AMSAS signature. Large pre-steady-state bursts and steady-state acylation assays confirm that they encode active tRNA synthetases. In these respects, the ORFs resemble models for ancestral Class I aminoacyl-tRNA synthetases (AARS). Both signatures thus appear necessary and sufficient for aminoacylation. Growth at 4{degrees} C produced most of the middle-sized ORF, which is incompatible with the others and appears to result from a distinct mechanism. Three different linkages between the two parts of the active site acylate tRNA minihelix at similar rates. That result greatly expands the sequence space of active ancestral AARS. Widely spaced active-site mutants thus trigger deletions of modules acquired as full-length AARS evolved from simpler catalysts. We propose that the deletions survive because they limit mischarging due to disrupted coupling of active-site residues to domain motion. Such deletions may thus be a general phenomenon, opening broad access to primordial gene discovery. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/647346v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1d87333org.highwire.dtl.DTLVardef@1733f25org.highwire.dtl.DTLVardef@136a74borg.highwire.dtl.DTLVardef@12137ce_HPS_FORMAT_FIGEXP M_FIG Schematic rationale for formation and selection of LeuRS urzyme-like deletions. A. Full length LeuRS uses a complex allosteric network of interactions between Dom A (CP1), Dom B (ABD) and WT active-site catalytic histidine and lysine residues. B. Creation of the double mutant corrupts the allosteric effects of the two domains (faded colors). This creates a cytotoxic protein. C. Deletion of the domains whose functions have been corrupted by disrupting the allosteric network produces variants lacking the inactivated domains. These variants have considerably less cytotoxicity. They likely also resemble evolutionary precursors of the full-length protein. This may represent a general phenomenon for double mutant multi-domain protein genes. C_FIG

evolutionary biology↗
bioRxiv2024

Genomic database furnishes a spontaneous example of a functional Class II glycyl-tRNA synthetase urzyme

The chief barrier to studies of how genetic coding emerged is the lack of experimental models for ancestral aminoacyl-tRNA synthetases (AARS). We hypothesized that conserved core catalytic sites could represent such ancestors. That hypothesis enabled engineering functional "urzymes" from TrpRS, LeuRS, and HisRS. We describe here a fourth urzyme, GlyCA, detected in an open reading frame from the genomic record of the arctic fox, Vulpes lagopus. GlyCA is homologous to a bacterial heterotetrameric Class II GlyRS-B. Alphafold2 predicted that the N-terminal 81 amino acids would adopt a 3D structure nearly identical to the HisRS urzyme (HisCA1). We expressed and purified that N-terminal segment. Enzymatic characterization revealed a robust single-turnover burst size and a catalytic rate for ATP consumption well in excess of that previously published for HisCA1. Time-dependent aminoacylation of tRNAGly proceeds at a rate consistent with that observed for amino acid activation. In fact, GlyCA is actually 35 times more active in glycine activation by ATP than the full-length GlyRS-B -subunit dimer. ATP-dependent activation of the 20 canonical amino acids favors Class II amino acids that complement those favored by HisCA and LeuAC. These properties reinforce the notion that urzymes represent the requisite ancestral catalytic activities to implement a reduced genetic coding alphabet.

evolutionary biology↗
bioRxiv2023

Domain Acquisition by Class I Aminoacyl-tRNA Synthetase Urzymes Coordinated the Catalytic Functions of HVGH and KMSKS Motifs

Leucyl-tRNA synthetase (LeuRS) is a Class I aminoacyl-tRNA synthetase (aaRS) that catalyzes synthesis of leucyl-tRNAleu for codon-directed protein synthesis on the ribosome. Class I aaRS, which were key to the evolution of genetic coding, contain two discrete signature sequences, HIGH and KMSKS, that participate in transition-state stabilization by the entire eleven-enzyme Class I aaRS superfamily. Combinatorial mutagenesis and thermodynamic cycle analyses of these catalytic signatures in full-length Pyrococcus horikoshii LeuRS and the 129-residue urzyme ancestral model generated from it (LeuAC) provide quantitative insight into the evolutionary gain of function induced by acquisition of the anticodon-binding (ABD) and multiple insertion modules in the catalytic domain. The free energy coupling terms, {Delta}({Delta}G{ddagger}), are small and unfavorable for LeuAC, but large and favorable for LeuRS. Thus, the ABD and other insertion modules induce strong cooperativity between the two signature sequences, which are uncoupled in LeuAC. These results further substantiate the authenticity of LeuAC urzyme catalysis and implicate domain motion in catalysis by the full-length LeuRS. Most importantly, the implication that backbone elements of secondary structures achieve a major portion of the overall transition-state stabilization by LeuAC is also consistent with coevolution of the genetic code and metabolic pathways necessary to produce histidine and lysine sidechains. Bullet PointsO_LIThe LeuRS HVGH and KMSKS signature motifs are energetically coupled by -1.6 kcal/mole. C_LIO_LIThe same motifs are anti-coupled by +0.8 kcal/mole in the 129 residue urzyme, LeuAC. C_LIO_LIAncestral Class I aaRS did not require either histidine or lysine for catalysis. C_LI

biochemistry↗