Aminoacylating urzymes challenge the RNA world hypothesis

Abstract

We describe experimental evidence that ancestral peptide catalysts substantially accelerated development of genetic coding. Structurally invariant 120-130-residue Urzymes (Ur = primitive plus enzyme) derived from Class I and Class II aminoacyl-tRNA synthetases (aaRSs) acylate tRNA far faster than the uncatalyzed rate of nonribosomal peptide bond formation from activated amino acids. These new data allow us to demonstrate statistically indistinguishable catalytic profiles for Class I and II aaRSs in both amino acid activation and tRNA acylation, over a time period extending to well before the assembly of full-length enzymes and even further before the Last Universal Common Ancestor. Both Urzymes also exhibit ∼60% of the contemporary catalytic proficiencies. Moreover, they are linked by ancestral sense/antisense genetic coding, and their evident modularities suggest descent from even simpler ancestral pairs also coded by opposite strands of the same gene. Thus, aaRS Urzymes substantially pre-date modern aaRS but are, nevertheless, highly evolved. Their unexpectedly advanced catalytic repertoires, sense/antisense coding, and ancestral modularities imply considerable prior protein-tRNA co-evolution. Further, unlike ribozymes that motivated the RNA World hypothesis, Class I and II Urzyme·tRNA pairs represent consensus ancestral forms sufficient for codon-directed synthesis of nonrandom peptides. By tracing aaRS catalytic activities back to simpler ancestral peptides, we demonstrate key steps for a simpler and hence more probable peptide·RNA development of rapid coding systems matching amino acids with anticodon trinucleotides.

Keywords: Aminoacyl tRNA Synthesis; Aminoacyl tRNA Synthetase; Catalytic RNA; Enzyme Catalysis; Enzyme Proficiency; Molecular Evolution; Origin of Translation; Protein Synthesis; tRNA.

FIGURE 1.

Superposition of four Class I and four Class II aaRS catalytic domains (65% transparent). Invariant cores (120–130 residues) are shown as ribbons inside differently colored surfaces, and active site residues are shown in CPK spheres.

FIGURE 2.

Models for tRNA TrpRS, HisRS Urzyme complexes, derived from crystal structures of intact components. Orientations are similar to those in Fig. 1. A, TrpRS. Model shows the putative Urzyme·tRNA^Trp complex, derived from the complex between human TrpRS and tRNA^Trp (PDB ID 2AZX) and that of Bacillus stearothermophilus TrpRS (PDB ID 1MAU). The N- and C-terminal β-α-β crossovers are colored sky blue and hot pink, respectively. The specificity helix is orange. tRNA acceptor stem determinants and matching active-site side chains are indicated. B, HisRS. Model for interaction with tRNA^His was derived from the HisRS crystal structure (PDB ID 2EL9) together with a Rhodopseudomonas palustris ProRS·tRNA^Pro complex (S. Cusack and T. Crepin, unpublished observation). Motifs 1, 2, and 3 are hot pink, sky blue, and yellow, respectively. Active-site side chains (Glu-115, Arg-116, Gln-118, Arg-121, and Arg-123) that interact with RNA acceptor stem determinants are shown as sticks.

FIGURE 3.

tRNA Acylation by TrpRS (A) and HisRS (B) Urzymes. Autoradiograms document aminoacylation of cognate tRNAs by TrpRS Urzyme (A) and HisRS-1, HisRS-2, and HisRS-4 (11) Urzymes (B). WT HisRS and HisRS-3 are similar and omitted to save space. Michaelis-Menten plots document saturation of with respective cognate tRNAs. The thin layer chromatographic assay is described in Ref. . Standard errors are given in Table 1.

FIGURE 4.

Rates of ³²P_i exchange with cognate amino acids by Class I (TrpRS, LeuRS) and Class II (HisRS) aaRS Urzymes, catalytic domains, and native enzymes, compared with uncatalyzed and fully catalyzed rates. A, rate accelerations estimated from experimental data for single substrate (red) and bi-substrate (black, bold) reactions adapted to include uncatalyzed and catalyzed rates of bi-substrate reactions of the ribosome (12), amino acid activation (16), and kinases (40). Second-order rate constants (black bars) were converted into comparable units by multiplying by 0.002 m, which is the ATP concentration used to assay the catalysts shown in B. B, experimental rate accelerations estimated from steady-state kinetics as k_cat/K_m for a series of catalysts derived from Class I and Class II aaRSs (5, 6, 11). Vertical scales in A and B are the same. Histograms in B were normalized by subtracting the logarithm of the uncatalyzed rate of amino acid activation in (AAact; A). Red bars denote Class I tryptophanyl- and leucyl-tRNA synthetase constructs, blue bars denote Class II histidyl-tRNA synthetase constructs, and green denotes the ribozymal catalyst (18) for comparison. LeuRS Urzyme is the work of M. Collier, and the TrpRS catalytic domains made by recombining the TrpRS Urzyme with either CP1 or with the anticodon-binding domain are the work of L. Li (both unpublished observations).

FIGURE 5.

Quantitative comparison of relative transition state stabilization energies. A, comparisons outlined under “Experimental Procedures” represented as multidimensional thermodynamic cycles. Edges represent contributions to the overall rate acceleration that can be attributed to each predictor in third through sixth columns of Table 2. B, transition state stabilization free energies from steady-state k_cat/K_m values for activation (open) and acylation (shaded) by Urzymes and full-length contemporary enzymes. C, regression model relating transition state stabilization free energies to contributions from Urzymes themselves, evolutionary enhancements in the full-length enzymes, whether the reaction is amino acid activation or tRNA acylation, and the two-way interaction between enhancements and activation (Table 2). The aaRS Class distinction does not contribute significantly.

FIGURE 6.

Schematic diagram of likely peptide participation with RNA in the origin and evolution of codon-directed protein synthesis (adapted from Ref. 3). Although not required, we assume that the two vertical axes are more or less aligned, that increases in fidelity are accompanied by increases in information, and vice versa, for schematic simplicity. The consensus (18, 19) order of appearance of modern aaRSs, tRNAs, and LUCA is reinforced by the quantitative analysis of rate data summarized in Table 2, Fig. 3, and Fig. 4. Prior existence of sense/antisense genes coding for catalytically active Class I and II closed loops (41) is based on circumstantial evidence discussed under “Results and Discussion.” Initiation of a peptide·RNA World by complementary peptide and RNA hairpins follows a proposal by Carter and Kraut (36).