Specific, rapid synthesis of Phe-RNA by RNA

Abstract

RNA 77, derived by selection amplification, accelerates its own conversion to Phe-RNA (relative to randomized RNA) more than 6 x 10(7)-fold, by using amino acid adenylates as substrate. A modified assay system allows measurement of slow rates of aa-RNA formation, which for disfavored amino acid substrates can be more than 10(4)-fold slower than phenylalanine. Thus unlike previously characterized self-aminoacylators, RNA 77 catalysis is highly amino acid selective. Remarkably, both rates of aminoacyl transfer and amino acid specificities are greater for RNA 77 than measured for protein PheRS. These data experimentally support the possible existence of an ancestral amino acid-specific translation system relying entirely on RNA catalysis. RNA 77 itself embodies a possible transitional evolutionary state, in which side-chain-specific aa-RNA formation and anticodon-codon pairing were invested in the same molecule.

Figure 1

Secondary structures of RNA 29 and RNA 77 with lead and S1 cuts shown.

Figure 2

Chromatographic resolution of products of acylation with six aa-AMP–RNA 77 reactions are at pH 7.25 and 0°C with 1–5 mM adenylate. Phe and Tyr reactions are for 0.5 min, all others for 20 min. RNA 29 reactions are similar but terminated at 2 min. The 3′ [³²P] nucleotide was released from aa-RNAs by P1 nuclease digestion and aminoacylated and unacylated forms resolved by cellulose polyethyleneimine chromatography.

Figure 3

Relative acylation kinetics for RNA 77 with six different aa-AMPs. Adenylate concentrations are Phe-AMP (3.3 μM), Tyr-AMP (2.6 μM), Ile-AMP (12 mM), Ala-AMP (5 mM), Gln-AMP (5 mM), and Ser-AMP (7 mM). The symbols are acylated fractions measured by PhosphorImaging, and the lines fitted second-order kinetic curves.

Figure 4

Inhibition of RNA 77 by AMP; determination of K_I acylations were carried out at 12.5 μM Phe-AMP in 0.2 M Mes, pH 6.5 at 0° in the presence of AMP, and as a control on chelation, UMP. At each concentration of AMP and UMP, 0.8 of the nucleotide concentration as CaCl₂ was added to minimize the effects of the nucleotides on free divalents. Symbols are experimental data, and the line is the least-squares best fit to where A₀ is the initial Phe-AMP concentration and I is the inhibitor concentration.

Figure 5

Effect of pH on the measured second-order rate constant (k_2nd) of RNA 77–Hepes was used at pH 7.25, Mes was used from pH 5.0–7.0, and NaOAc was used at pH 4.8. Buffers were 0.2 M and are represented by different symbols. A line with a slope of 1 is drawn near the points.

Figure 6

Determination of K_M for RNA 77 with Phe-AMP reactions were at pH 5.75 and 0° in 0.2 M Mes. The symbols are data, and the line is the least-squares best fit to where A₀ is the initial Phe-AMP concentration.

Figure 7

Apparent saturation levels of aa-RNA 77 at pH 7.25 and different initial adenylate concentrations. Symbols are data and the lines are least-squares fits to kinetics that take account of the second-order RNA-adenylate reaction and the instability of the adenylates.