Cross-backbone templating; ribodinucleotides made on poly(C)

Abstract

G(5')pp(5')G synthesis from pG and chemically activated 2MeImpG is accelerated by the addition of complementary poly(C), but affected only slightly by poly(G) and not at all by poly(U) and poly(A). This suggests that 3'-5' poly(C) is a template for uncatalyzed synthesis of 5'-5' GppG, as was poly(U) for AppA synthesis, previously. The reaction occurs at 50 mM mono- and divalent ion concentrations, at moderate temperatures, and near pH 7. The reactive complex at the site of enhanced synthesis of 5'-5' GppG seems to contain a single pG, a single phosphate-activated nucleotide 2 MeImpG, and a single strand of poly(C). Most likely this structure is base-paired, as the poly(C)-enhanced reaction is completely disrupted between 30 and 37 °C, whereas slower, untemplated synthesis of GppG accelerates. More specifically, the reactive center acts as would be expected for short, isolated G nucleotide stacks expanded and ordered by added poly(C). For example, poly(C)-mediated GppG production is very nonlinear in overall nucleotide concentration. Uncatalyzed NppN synthesis is now known for two polymers and their complementary free nucleotides. These data suggest that varied, simple, primordial 3'-5' RNA sequences could express a specific chemical phenotype by encoding synthesis of complementary, reactive, coenzyme-like 5'-5' ribodinucleotides.

Keywords: RNA gene; coenzyme; cofactor; phenotype; template.

FIGURE 1.

Cellulose TLC resolution of standard reactions. 0, zero time, no incubation. 24, after 24 h at 12°C. 24 h poly(C), 24 h incubation with 5 mM poly(C). 3′–5′ pGpG marker was from Thermo Fisher Scientific.

FIGURE 2.

Initial rate of GppG synthesis with variation in nucleotide substrate concentration. Triangles, with poly(C); circles, without polymer. Rates are those from least squares slopes from lines fitted to GppG measured six times in the initial 8 h at 12°C.

FIGURE 3.

Response of GppG synthesis to poly(C) concentration; triangles are initial velocities from least squares linear fitting to early GppG concentrations versus time.

FIGURE 4.

Initial rates of GppG synthesis, at different ratios of substrate nucleotides, with and without poly(C). Triangles indicate data, poly(C) added; circles denote data without polymer. Dashed lines are calculated, matching the overall velocity and assuming rate ∝ [2MeImpG]*[pG] (curves marked 1:1). Dotted lines are calculated, matching overall velocity and assuming rate ∝ [2MeImpG]²*[pG] (curve marked 2:1), or rate ∝ [2MeImpG]*[pG]² (curve marked 1:2).

FIGURE 5.

Fitted rate constants for chemical and poly(C)-stimulated synthesis of GppG at different temperatures. Dashed line (triangles) poly(C)-stimulated synthesis; dotted line (circles) least squares fit of Eyring–Polanyi equation to chemical reactions.

FIGURE 6.

Polymer specificity; molar GppG synthesized in the reaction, in the presence of poly(U) (hyphens), poly(C) (triangles), poly(A) (squares) and poly(G) (diamonds), as well as with no polymer (circles). Fine dots are points calculated from fitted least squares differential equations, described above.

SCHEME 1.

The number of binding sites for a small stack of nucleotides (below) on a long polymer (above) is almost equal to the total number of nucleotides in the long polymer.

FIGURE 7.

Free nucleotides stack or bind weakly to an added complementary polymer, where further stacking is enhanced by complementary base pairing. The figure shows species at equilibrium. (Top, right axis) Total free stacked N (circles; stacks ≥2 nt); polymer-bound stacked N (triangles). (Leftward axis) Circles indicate mean stack size for free N; diamonds refer to mean stack size for polymer-bound N. (Bottom, right axis) Paired (triangles) or free (circles) nucleotide concentration in polymer. (Left axis) Paired, stacked N/total stacked N (diamonds). Arrows beside curves point to the relevant y-axis.

SCHEME 2.

All possible stacks of three nucleotides containing normal nucleotides (darker lines) and activated nucleotides (lighter lines).

FIGURE 8.

A novel primordial gene, logically related to modern gene expression. (Left) A simple, perhaps even monotonous, sequence in a small, primordial RNA (the archegene) expresses a phenotype by cross-templating a cofactor-like nucleotide RppA molecule, using an activated nucleotide with the primordial leaving group L. The archegene is the predecessor of a later, more evolved genetic system (the RNA Gene, center) which can perform RNA-catalyzed 3′–5′ coenzyme-RNA synthesis (center). Later yet, RNA world translation makes possible modern catalysts: coenzymes bound to peptides (from the Gene, on the right).