Determination of hepatitis delta virus ribozyme N(-1) nucleobase and functional group specificity using internal competition kinetics

Abstract

Biological catalysis involves interactions distant from the site of chemistry that can position the substrate for reaction. Catalysis of RNA 2'-O-transphosphorylation by the hepatitis delta virus (HDV) ribozyme is sensitive to the identity of the N(-1) nucleotide flanking the reactive phosphoryl group. However, the interactions that affect the conformation of this position, and in turn the 2'O nucleophile, are unclear. Here, we describe the application of multiple substrate internal competition kinetic analyses to understand how the N(-1) nucleobase contributes to HDV catalysis and test the utility of this approach for RNA structure-function studies. Internal competition reactions containing all four substrate sequence variants at the N(-1) position in reactions using ribozyme active site mutations at A77 and A78 were used to test a proposed base-pairing interaction. Mutants A78U, A78G, and A79G retain significant catalytic activity but do not alter the specificity for the N(-1) nucleobase. Effects of nucleobase analog substitutions at N(-1) indicate that U is preferred due to the ability to donate an H-bond in the Watson-Crick face and avoid minor groove steric clash. The results provide information essential for evaluating models of the HDV active site and illustrate multiple substrate kinetic analyses as a practical approach for characterizing structure-function relationships in RNA reactions.

Keywords: Enzyme kinetics; Internal competition; Ribozyme.

Fig. 1

The theoretical framework that defines internal competition kinetics can be described by two dimensional free energy landscapes for the competitive reactions of four substrates at the start of the reaction (left; f = 0), at an intermediate time during the reaction (middle; 1 > f > 1), and after completion (right; f = 1). In each panel, the relative concentrations of each competitve substrate and product are shown on the left and right of the free energy diagram, respectivly. Due to differences in activation energy (ΔG‡) for the reactions of the competitive substrates, at intermediate times during the reaction the ratio of the substrates and products will be offset from their initial and final values, respectively. The faster reacting substrates becomes enriched in the product population (P2/P1 > 1), while the slower reacting species is enriched in the residual unreacted substrate population (S2/S1 < 1). These ratios change as a function of f according to the relative rate constant k_rel which is defined by the differences in activation energy (ΔG‡)..

Fig. 2

(A). The secondary structure of the trans, antigenomic ribozyme used in this kinetic analysis, where the cleavage site is indicated by a bolt. This cleavage reaction requires the presence of at least a single nucleotide 5′ of the scissile phosphate and the identity of the upstream sequence at N(-1) impacts the catalytic efficiency of the ribozyme. (B). Single turnover kinetic analysis of N(-1) substrate variation using individual substrate reactions and direct data fitting. U(-1) results in fast cleavage by the HDV ribozyme, while a G(-1) mutation reduces catalytic activity by 25-fold. The single turnover rate constants (s⁻¹) for the N(-1) substrate variants are presented in the inset and the error in the final decimal place from > 3 independent reactions is presented in parenthesis.

Fig. 3

(A). PAGE analysis of internal competition reactions containing four alternative HDV ribozyme substrates, varying in nucleobase composition at the N(-1) position. The length of these substrates were altered by the addition of 1–3 uridine residues to the 3′ end of the RNA in order to resolve these species by PAGE. The U(-1) substrate is depleted early in the time course, whereas the G(-1) substrate that reacts with the slowest rate constant accumulates at intermediate times relative to the other substrates. The A(-1) and C(-1) substrates are depleted from the residual substrate population with similar apparent kinetics. Thus, the qualitative changes in the band intensities from PAGE analysis correspond to expectations from measurement of individual reaction time courses. (B) and (C). To control for the effect of addition of 3′ uridine residues, two substrate populations were examined. The first population consisted of U9, G10, A11, and C12 the second of U9, A10, C11, and G12, where the N(-1) base identity is indicated with its associated substrate sequence length. The k_rel valued measured by internal competition for both populations containing mixtures of four substrates (grey bars) correspond with k_rel ratios (k_rel = k_(obs)exp/k_(obs)ref) calculated from reactions containing individual substrates (shaded bars).

Fig. 4

(A). Effects of additional 3′ U residues on k_obs were calculated by direct data fitting of individual substrate, single turnover kinetic timecourses. These effects are minimal for most substrates, a (> 2-fold). The largest effect (~3.5 fold) was observed for C12 relative to the C9 reference substrate. Thus, for most substrates the observed k_rel is accurate; however, the structural changes introduced to allow resolution by PAGE can introduce inaccuracies that require correction. (B). The observed k_rel values decline or increase with increasing f, before converging at an k_rel of 1. This effect results from the fact that at higher fractions of reaction, the substrate ratio measurement becomes dominated by the contributions from residual unreacted substrate. Restricting the analysis to fractions of reaction progress where all substrates are undergoing reaction can avoid potential inaccuracies from this effect. For this analysis, population reaction progress from f = 0.2 – 0.4 were analyzed. (C). The range of time for which the experimental substrate population is undergoing reaction (f = 0.2 – 0.8). Large differences in observed rate constants between individual substrate variants and the reference substrate can introduce inaccuracies due to signal to noise limitations resulting from the relative abundance of the reference substrate. The A(-1) substrate undergoes reaction with kinetics such that its concentration changes over a range that overlaps most with the other substrates in the population and is the optimal reference for this set of substrates.

Fig. 5

(A). k_rel,obs values measured by internal competition for substrate populations containing all four natural nucleotides at the N(-1) position corrected for 3′ U addition and normalized to U9.

Fig. 6

(A). Analysis of nucleobase analogs designed to probe the functional importance of hydrogen bond donors and acceptors in the major and minor groove at N(-1) in the HDV ribozyme substrate. (B). k_rel values for N(-1) substrate variants determined by internal competition have been corrected for 3′U addition and normalized to U9. The k_rel valued measured by internal competition for both populations containing mixtures of four substrates (grey bars) correspond with k_rel ratios (k_rel ratio = k_(obs)exp/k_(obs)ref) calculated from reactions containing individual substrates (shaded bars).

Scheme 1