Sourav Kumar Patra

Translation of symbols in one chemical language into another defined genetics. Yet, the co-linearity of codons and amino acids is so commonplace an idea that few even ask how it arose. Readout is done by two distinct sets of proteins, called aminoacyl-tRNA synthetases (AARS). AARS must enforce the rules first used to assemble themselves. To understand the roots of translation we must experimentally test the structural codes that the earliest AARS•tRNA cognate pairs used to recognize both amino acid and RNA substrates. We present here new results on five different facets of that problem. (i) The surfaces of structures coded by opposite strands of the same gene have opposite polarities. Core residues in proteins from one strand are surface residues in proteins from the other strand. The complementarity of base pairing thus projects into the proteome. That leads in turn to contrasting amino acid and RNA substrate binding modes. (ii) E. coli reproduces in vivo a nested hierarchy of active excerpts, or “urzymes”, similar to those we had designed as models for ancestral AARS. (iii) A third novel deletion produced in vivo and a new Class II urzyme suggests how to design bidirectional urzyme genes. (iv) Codon middle-base pairing provides a basis to constrain Class I and II AARS family trees. (v) AARS urzymes acylate Class-specific subsets of an RNA library, showing RNA substrate specificity for the first time. Four new tree-building tools augment these results to compose a viable platform for experimental study of the origins of genetic coding.

Protein design plays a key role in our efforts to work out how genetic coding began. That effort entails urzymes. Urzymes are small, conserved excerpts from full-length aminoacyl-tRNA synthetases that remain active. Urzymes require design to connect disjoint pieces and repair naked nonpolar patches created by removing large domains. Rosetta allowed us to create the first urzymes, but those urzymes were only sparingly soluble. We could measure activity, but it was hard to concentrate those samples to levels required for structural biology. Here, we used the deep learning algorithms ProteinMPNN and AlphaFold2 to redesign a set of optimized LeuAC urzymes derived from leucyl-tRNA synthetase. We select a balanced, representative subset of eight variants for testing using principal component analysis. Most tested variants are much more soluble than the original LeuAC. They also span a range of catalytic proficiency and amino acid specificity. The data enable detailed statistical analyses of the sources of both solubility and specificity. In that way, we show how to begin to unwrap the elements of protein chemistry that were hidden within the neural networks. Deep learning networks have thus helped us surmount several vexing obstacles to further investigations into the nature of ancestral proteins. Finally, we discuss how the eight variants might resemble a sample drawn from a population similar to one subject to natural selection.

The reflexive translation of symbols in one chemical language to another defined genetics. Yet, the co-linearity of codons and amino acids is so commonplace an idea that few even ask how it arose. Readout is done by two distinct sets of proteins, called aminoacyl-tRNA synthetases (AARS). AARS must enforce the rules first used to assemble themselves. The roots of translation lie in experimentally testing the structural codes that the earliest AARS•tRNA cognate pairs used to recognize both amino acid and RNA substrates. We review here new results on five different facets of that problem. (i) The surfaces of structures coded by opposite strands of the same gene have opposite polarities. The corresponding proteins then fold up “inside out” relative to one another. The inversion symmetry of base pairing thus projects into the proteome. That leads in turn to contrasting amino acid and RNA substrate binding modes. (ii) E. coli reproduces in vivo the nested hierarchy of active excerpts we had designed as models—protozymes and urzymes—for ancestral AARS. (iii) A third novel deletion produced in vivo and a new Class II urzyme suggest how to design bidirectional urzyme genes. (iv) Codon middle-base pairing provides a basis to constrain Class I and II AARS family trees. (v) AARS urzymes acylate Class-specific subsets of an RNA library, showing RNA substrate specificity for the first time. Four new phylogenetic routines augment these results to compose a viable platform for experimental study of the origins of genetic coding. Significance Statement The origin of genetic coding poses questions distinct from those faced studying the evolution of enzymes since the first cells. Modern enzymes that translate the code range in size from ∼330 to ∼970 amino acids. Ancestral forms cannot have been nearly as complex. Moreover, such primitive enzymes likely could enforce only a much-reduced coding alphabet. Structural and molecular biology data point to a broad sketch of events leading to the code. That research platform will enable us to see how Nature came to store information about the physical chemistry of amino acids in the coding table. That, in turn, allowed searching of a very broad amino acid sequence space. Selection could then learn how to assemble amino acids into functional, reflexive catalysts. Those catalysts had rates and fidelities consistent with bootstrapping the modern coding alphabet. New phylogenetic algorithms need to be developed to fully test that putative sketch experimentally.

The hypothesis that conserved core catalytic sites could represent ancestral aminoacyl-tRNA synthetases (AARS) drove the design of functional TrpRS, LeuRS, and HisRS ‘urzymes’. We describe here new urzymes detected in the genomic record of the arctic fox, Vulpes lagopus. They are homologous to the α-subunit of bacterial heterotetrameric Class II glycyl-tRNA synthetase (GlyRS-B) enzymes. AlphaFold2 predicted that the N-terminal 81 amino acids would adopt a 3D structure nearly identical to our designed HisRS urzyme (HisCA1). We expressed and purified that N-terminal segment and the spliced open reading frame GlyCA1–2. Both exhibit robust single-turnover burst sizes and ATP consumption rates higher than those previously published for HisCA urzymes and comparable to those for LeuAC and TrpAC. GlyCA is more than twice as active in glycine activation by adenosine triphosphate as the full-length GlyRS-B α₂ dimer. Michaelis–Menten rate constants for all three substrates reveal significant coupling between Exon2 and both substrates. GlyCA activation favors Class II amino acids that complement those favored by HisCA and LeuAC. Structural features help explain these results. These minimalist GlyRS catalysts are thus homologous to previously described urzymes. Their properties reinforce the notion that urzymes may have the requisite catalytic activities to implement a reduced, ancestral genetic coding alphabet.

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 tRNA ^Gly 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 α-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.

Amino acyl tRNA synthetases or aaRSs play a key role in assuring the precision of protein translation. They are highly specific for their cognate amino acid and cognate tRNA substrates during protein synthesis, utilizing ATP to ensure that proper assignments are made between amino acid and anticodon. Specific aaRS for each amino acid are present in all cells. We describe a new zymography technique to qualitatively visualize and semi-quantitatively determine the amino acid activation capacity of each type of aaRS molecule by indirect colorimetric detection of released pyrophosphates during the formation of aminoacyl-AMP. Protein samples containing aaRS are subjected to Native PAGE, followed by incubation in buffer containing cognate amino acid and ATP for sufficient time to generate pyrophosphates (PPi) which are then converted to inorganic phosphates by pyrophosphatase treatment. Finally, the generated and localized phosphates around the aaRS protein inside the gel can be visualized after staining by ammonium molybdate and malachite green solution. This technique has been validated by inspecting the substrate specificities of specific aaRSs. This zymography technique is sufficiently sensitive to detect and authenticate activities of much (i.e., ~10 ^-5 -fold) less active aaRS “Urzymes”, to study alteration of activities of aaRS by various intrinsic or extrinsic factors and to screen aaRS-specific antimicrobial drugs.

Macrophomina phaseolina, a necrotrophic fungal pathogen is known to cause charcoal rot disease in food crops, pulse crops, oil crops and cotton and fibre crops. Necrotrophic fungi survive on dead plant tissue. It is well known that reactive oxygen species (ROS) are produced by the host plant during plant–pathogen interaction. However, it is still unclear how M. phaseolina can overcome the ROS-induced cellular damage. To mimic the invasion of M. phaseolina inside the plant cell wall, we developed solid substrate fermentation where M. phaseolina spore suspension was inoculated on a wheat bran bed and incubated for vegetative growth. To analyse the secretome of M. phaseolina after different day interval, its secretory material was collected and concentrated. Both superoxide dismutase (SOD) and catalase were detected in the secretome by zymogram. The presence of SOD and catalase was further confirmed by liquid chromatography based mass spectrometry. The physicochemical properties of M. phaseolina catalase in terms of stability towards pH, temperature, metal ions and chaotropic agent and inhibitors indicated its fitness at different environmental conditions. Apart from the production of catalase in SSF, the studies on this particular microorganism may also have significance in necrotrophic fungal pathogen and their susceptible host plant interaction.