Primordial aminoacyl-tRNA synthetases preferred minihelices to full-length tRNA.

Aminoacyl-tRNA synthetases (AARS) and tRNAs translate the genetic code in all living cells. Little is known about how their molecular ancestors began to enforce the coding rules for the expression of their own genes. Schimmel et al. proposed in 1993 that AARS catalytic domains began by reading an 'operational' code in the acceptor stems of tRNA minihelices. We show here that the enzymology of an AARS urzyme•TΨC-minihelix cognate pair is a rich in vitro realization of that idea. The TΨC-minihelixLeu is a very poor substrate for full-length Leucyl-tRNA synthetase. It is a superior RNA substrate for the corresponding urzyme, LeuAC. LeuAC active-site mutations shift the choice of both amino acid and RNA substrates. AARS urzyme•minihelix cognate pairs are thus small, pliant models for the ancestral decoding hardware. They are thus an ideal platform for detailed experimental study of the operational RNA code.

Base Pairing Promoted the Self-Organization of Genetic Coding, Catalysis, and Free-Energy Transduction.

How Nature discovered genetic coding is a largely ignored question, yet the answer is key to explaining the transition from biochemical building blocks to life. Other, related puzzles also fall inside the aegis enclosing the codes themselves. The peptide bond is unstable with respect to hydrolysis. So, it requires some form of chemical free energy to drive it. Amino acid activation and acyl transfer are also slow and must be catalyzed. All living things must thus also convert free energy and synchronize cellular chemistry. Most importantly, functional proteins occupy only small, isolated regions of sequence space. Nature evolved heritable symbolic data processing to seek out and use those sequences. That system has three parts: a memory of how amino acids behave in solution and inside proteins, a set of code keys to access that memory, and a scoring function. The code keys themselves are the genes for cognate pairs of tRNA and aminoacyl-tRNA synthetases, AARSs. The scoring function is the enzymatic specificity constant, kcat/kM, which measures both catalysis and specificity. The work described here deepens the evidence for and understanding of an unexpected consequence of ancestral bidirectional coding. Secondary structures occur in approximately the same places within antiparallel alignments of their gene products. However, the polar amino acids that define the molecular surface of one are reflected into core-defining non-polar side chains on the other. Proteins translated from base-paired coding strands fold up inside out. Bidirectional genes thus project an inverted structural duality into the proteome. I review how experimental data root the scoring functions responsible for the origins of coding and catalyzed activation of unfavorable chemical reactions in that duality.

HetMM: A Michaelis-Menten model for non-homogeneous enzyme mixtures.

The Michaelis-Menten model requires its reaction velocities to come from a preparation of homogeneous enzymes, with identical or near-identical catalytic activities. However, this condition is not always met. We introduce a kinetic model that relaxes this requirement, by assuming there are an unknown number of enzyme species drawn from a probability distribution whose standard deviation is estimated. Through simulation studies, we demonstrate the method accurately discriminates between homogeneous and heterogeneous data, even with moderate levels of experimental error. We applied this model to three homogeneous and three heterogeneous biological systems, showing that the standard and heterogeneous models outperform respectively. Lastly, we show that heterogeneity is not readily distinguished from negatively cooperative binding under the Hill model. These two distinct attributes-inequality in catalytic ability and interference between binding sites-yield similar Michaelis-Menten curves that are not readily resolved without further experimentation. Our user-friendly software package allows homogeneity testing and parameter estimation.

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 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.