AARS urzymes: experimental biochemistry to map genetic coding

Dr. Charlie Carter of the University of North Carolina at Chapel Hill explores how advances in enzymology and phylogenetics enable biochemical measurements that could map the ancestral development of genetic coding

Chemical reactions in the cell degrade energy-rich food molecules and rearrange their chemical bonds into other molecules needed to build and sustain living things far from chemical equilibrium. These reactions occur spontaneously at rates that cover an extraordinary range, the fastest rising to 1025 times faster than the slowest. (1) All of these reactions need to be sped up by different amounts to maintain proper concentrations of chemical components in the cell. Enzymes do that differential acceleration exquisitely well.

Enzymology and phylogenetics

Enzymes are the nanomachines that transform random chemistry into regulated metabolic pathways that sustain life. Their precisely shaped cavities, called active sites, can distinguish one molecule from another based on how well they fit within the site. That unique talent allows enzymes to speed up reactions by the astronomical amounts needed to synchronize the chemistry of life. It also provides the specificity needed to build networks of metabolic pathways.

Enzymes are proteins that the cell assembles according to genetic patterns (genes). Reading those blueprints is the main flow of information in living cells. Molecular evolution is the study of how enzymes got into their current shape. Phylogenetic studies find successively more distant relatives and use these comparisons to construct family trees. Those trees, in turn, hint at the amino acid sequences of common ancestors. The extensive databases of gene sequences open up access to the recent evolutionary history of enzymes and related regulatory proteins. (2-4)

The remote origins of the proteome, however, pose a greater challenge. Three-dimensional structures must guide the phylogenetic approach. Such analyzes reveal that contemporary enzymes have a hierarchical modularity. Evolutionarily related enzymes use the same active sites to speed up similar chemical reactions. Nature then incorporates these active sites, like Russian matryoshka dolls, into successive layers of supplemental polypeptides that enhance function in mature enzymes and differentiate one family from another. That hierarchy points to the deepest roots of modern enzymes.

(Fig 1.) A system for exploring the experimental biochemistry of ancestral genetic coding.  A. represents the contemporary leucyl-tRNA cognate pair synthetasetRNALeu.  LeuRS and tRNALeu have two domains: a catalytic domain that interacts with the acceptor stem of the tRNA and another domain that interacts with other parts of the tRNA.  B. shows a single domain functional system.  The leucyl-tRNA synthetase urzyme, LeuAC, catalyzes the acylation of the tRNALeu minihelix, facilitating experimental measurement of the spectrum of functional amino acid substrates and minihelixes, as well as the impact of the three base pairs (blue shading) that drive cognate recognition.
(Fig.1.) A system for exploring the experimental biochemistry of ancestral genetic coding.
A. represents the contemporary leucyl-tRNA synthetasetRNALeu kindred couple. LeuRS and tRNALeu they have two domains: a catalytic domain that interacts with the acceptor stem of the tRNA and another domain that interacts with other parts of the tRNA. B. shows a single domain functional system. Urzyme leucyl-tRNA synthetase, LeuAC, catalyzes the acylation of tRNALeu minihelix, facilitating experimental measurement of the spectrum of functional amino acids and minihelix substrates, as well as the impact of the three base pairs (light blue shading) that drive affine recognition.

Urzymology

An important breakthrough was to deconstruct that modular hierarchy and express only the active site module from the two (Class I and II) families of aminoacyl-tRNA synthetase (AARS). (5-7) AARS are the nanomachines that actually translate the genetic code. They are central to how information is read by genes. To our surprise, these small extracts require minimal reengineering to behave like mature contemporary enzymes. They are just weaker and less discriminating. We call them urzymes, contracting the prefix ur (= ‘first’) with enzyme. Their amino acid sequences are also closely related, and their catalytic activities and substrate specificities resemble their full-length counterparts. These two properties make urzymes useful experimental models for the primordial emergence and early evolution of enzymes. (8)

Catalysis by full-length class I AARS depends on two different active site signature sequences, ALTA near the N-terminus and KMSKS at the C-terminus of the catalytic domain. By mutating the polar histidine and lysine residues into these signatures separately and in combination with both full-length leucyl-tRNA synthetase and its urzyme, LeuAC (9) it enabled the measurement of the energy coupling energy by coordinating the impact of the two signatures on catalysis. Surprisingly, the two signatures work synergistically in full-length LeuRS but oppose each other’s effects in urzymes. That result is prima facie evidence that both urzyme catalysis is real and domains deleted to cause urzyme to enforce the coordinated behavior of these signatures in the mature enzyme.

Urzymes allows for experimental studies of how genetic coding began

It has never been obvious how to investigate the molecular ancestry of genetic coding experimentally. Recent developments in urzymology now make this possible (see Fig. 1). While we were developing AARS urzymes, other groups found that full-length AARS could acylate the acceptor stem domains of their cognate tRNAs.

A key step in the development of an experimental system occurred when we recently demonstrated that AARS urzymes also acylate tRNA minihelices. (10) The generation of additional such pairs could be a game changer by facilitating experimental measurements of the ranges of minihelix amino acid and tRNA substrates that can be aminoacylated by different urzymeminihelix cognate pairs.

My previous piece (11) noted that AARStRNA cognate pairs are the computational AND gates that make genetic coding work. The specific coding properties of the urzymeminihelix cognate pairs Fig. 1B can help us understand which catalysts can be made using fewer coding letters. Experimental access to that new kind of information can help us map the growth of the coding alphabet.

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