Codon-dependent translation connects late chemical evolution to biological evolution, which is the basis for phylogenetic inference and genetics. Aminoacyl-tRNA syntheses (aaRS) actually translate the code. Experimental study of ancestral aaRS is thus deeply and broadly valuable to contemporary molecular biology, genetics, biophysics, and biochemistry. Previous work created and extensively validated a new approach to accessing events and processes associated with the origins of translation. The experimental approach uses Urzymes, which are constructs developed from invariant cores of protein superfamilies'. Urzymes representing ancestral forms of Class I and II (aaRS) have ~15% of the mass but retain 60% of the catalytic proficiency of contemporary aaRS. The approach also introduced new bioinformatics to show that Class I and II aaRS descended from opposite strands of the same gene. Thus, codon middle-base pairing of multiple sense/antisense alignments () probes earlier phylogenetic relationships than those accessible using ancestral gene reconstruction. This proposal will exploit these new tools to address three questions: (I) What molecules and processes gave rise to the highly evolved sophistication of Class I and II Urzymes? values for 94-residue Class I and II aaRS Urgenes will distinguish the order in which the two strands specialized, breaking the constraint of sense/antisense coding, and radiated to give subclasses and then a full canonical set of coded amino acids. Catalysis by 46-aa ATP binding sites from a designed sense/antisense gene have been validated by mutagenesis. We will use thermodynamic cycles to measure coupling between 46mer active-site residues and attempt to construct them using reduced amino acid alphabets. (II) How do Urzyme structures and functions differ from contemporary enzymes? tRNA specificities and long-range energetic coupling will be measured in Urzymes and in Class II HisRS. NMR studies will determine whether the Uryzmes are folded, molten globular or intrinsically disordered; the temperature dependence of stability and catalysis will verify the resulting conclusions. (III) What biological roles could the Urzymes have carried out in the course of implementing the genetic code? A novel method will use construction of aaRS knockouts to test whether Urzymes can complement aaRS knockouts or whether they are toxic in vivo and an in vitro translation system will be developed, to eventually recapitulate steps in the development of the canonical genetic code from defined components. Previous results imply these are all doable.

Public Health Relevance

Results will shift many paradigms in phylogenetics and the origins of translation, protein folding, catalytic activity, specificity, and allostery. Urzymes represent deletion of ~85% of contemporary enzyme masses, and can be viewed as molecular 'knockouts' useful for applied biomedical applications such as enhancing gene therapy by reducing masses of genes too large to embed in viral vectors, and their measurable activities are a key baseline for mechanistic analysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM078227-09A1
Application #
8964980
Study Section
Genetic Variation and Evolution Study Section (GVE)
Program Officer
Bender, Michael T
Project Start
2006-08-01
Project End
2019-05-31
Budget Start
2015-09-08
Budget End
2016-05-31
Support Year
9
Fiscal Year
2015
Total Cost
$355,721
Indirect Cost
$117,174
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Wills, Peter R; Carter Jr, Charles W (2018) Insuperable problems of the genetic code initially emerging in an RNA world. Biosystems 164:155-166
Carter Jr, Charles W; Wills, Peter R (2018) Hierarchical groove discrimination by Class I and II aminoacyl-tRNA synthetases reveals a palimpsest of the operational RNA code in the tRNA acceptor-stem bases. Nucleic Acids Res 46:9667-9683
Carter Jr, Charles W; Wills, Peter R (2018) Interdependence, Reflexivity, Fidelity, Impedance Matching, and the Evolution of Genetic Coding. Mol Biol Evol 35:269-286
Potempa, Marc; Lee, Sook-Kyung; Kurt Yilmaz, Nese et al. (2018) HIV-1 Protease Uses Bi-Specific S2/S2' Subsites to Optimize Cleavage of Two Classes of Target Sites. J Mol Biol 430:5182-5195
Carter Jr, Charles W (2017) Coding of Class I and II Aminoacyl-tRNA Synthetases. Adv Exp Med Biol 966:103-148
Carter Jr, Charles W (2017) High-Dimensional Mutant and Modular Thermodynamic Cycles, Molecular Switching, and Free Energy Transduction. Annu Rev Biophys 46:433-453
Carter Jr, Charles W; Chandrasekaran, Srinivas Niranj; Weinreb, Violetta et al. (2017) Combining multi-mutant and modular thermodynamic cycles to measure energetic coupling networks in enzyme catalysis. Struct Dyn 4:032101
Sapienza, Paul J; Li, Li; Williams, Tishan et al. (2016) An Ancestral Tryptophanyl-tRNA Synthetase Precursor Achieves High Catalytic Rate Enhancement without Ordered Ground-State Tertiary Structures. ACS Chem Biol 11:1661-8
Carter Jr, Charles W; Wolfenden, Richard (2016) tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry. RNA Biol 13:145-51
Carter Jr, Charles W (2016) An Alternative to the RNA World. Nat Hist 125:28-33

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