A guanine nucleobase important for catalysis by the VS ribozyme

Abstract

A guanine (G638) within the substrate loop of the VS ribozyme plays a critical role in the cleavage reaction. Replacement by any other nucleotide results in severe impairment of cleavage, yet folding of the substrate is not perturbed, and the variant substrates bind the ribozyme with similar affinity, acting as competitive inhibitors. Functional group substitution shows that the imino proton on the N1 is critical, suggesting a possible role in general acid-base catalysis, and this in accord with the pH dependence of the reaction rate for the natural and modified substrates. We propose a chemical mechanism for the ribozyme that involves general acid-base catalysis by the combination of the nucleobases of guanine 638 and adenine 756. This is closely similar to the probable mechanism of the hairpin ribozyme, and the active site arrangements for the two ribozymes appear topologically equivalent. This has probably arisen by convergent evolution.

Figure 1

The VS ribozyme. The sequence of the ribozyme in its trans-acting form, comprising the substrate stem-loop (helix I) and the ribozyme (helices II–VI). The site of cleavage is arrowed. The A730 loop within helix VI (shaded) is the putative active site of the ribozyme.

Figure 2

Sequence substitutions at position 638 in the substrate strongly impair cleavage by the VS ribozyme. Products of VS ribozyme cleavage in trans on the substrate with the natural G638, and substitution by A, U or C. Cleavage reactions were performed on radioactively 5′-³²P-labelled substrates under single-turnover conditions at 37°C in standard VS buffer, that is, 50 mM Tris (pH 8.0), 10 mM MgCl₂, 25 mM KCl, 2 mM spermidine. The natural substrate was reacted for 5 min and the three variants for 60 min. Substrate and product were separated by electrophoresis in 20% polyacrylamide gels under denaturing conditions and visualized by phosphorimaging. Tracks: 1, G638A; 2, G638U; 3, G638C and 4, G638 natural sequence substrate.

Figure 3

Comparison of the conformations of the natural and G638A substrates by in-line probing. Versions of natural sequence and G638A substrates with 5′ and 3′ terminal extensions of deoxyribonucleotides were synthesized to improve the electrophoretic resolution of the RNA sections. Radioactively 5′-³²P-labelled substrates were incubated in standard VS buffer at 25°C for 40 h. Cleavages were analysed by electrophoresis in a 20% polyacrylamide gel under denaturing conditions. Tracks 1 and 4, base cleavage of natural and G638A substrates, respectively and tracks 2 and 3, in-line probing analysis of natural and G638A substrates, respectively. The scheme shows the sequence of the natural substrate, with the arrow indicating the position of ribozyme cleavage. The positions of sensitivity to in-line probing are indicated by filled circles, the size of which reflects the extent of cleavage. The open circle shows the position of the phosphodiester linkage that is more sensitive in the G638A substrate; note that this position also exhibits enhanced base cleavage. The shorter fragments migrate as doublets, due to resolution of the cyclic 2′3′-phosphates and their products of hydrolysis during the long incubation.

Figure 4

Affinity and rate of substrate binding in trans to the VS ribozyme. (A) Reaction scheme for the ribozyme cleavage in the presence of the variant substrate. The natural sequence substrate (S_G) binds to the ribozyme (Rz) with an affinity K_S to form a non-covalent complex that undergoes the cleavage reaction at rate k₂. The G638A substrate binds to the ribozyme with an affinity K_I. The variant substrate undergoes a negligible amount of cleavage during the incubation, and therefore simply acts as an inhibitor of the reaction. (B) Cleavage of a natural sequence substrate by VS ribozyme was performed in trans under standard single-turnover conditions in the presence of different concentrations of G638A substrate. Progress curves are shown for reactions carried out in the presence of the following concentrations of G638A substrate: 0 (filled circles), 0.3 (open circles), 0.8 (filled squares), 1.4 (open squares), 2.5 (filled diamonds) and 5 μM (open diamonds). In the inset, the observed rate constants (k_obs) are plotted as a function of G638A substrate concentration and fitted to equation 1, appropriate for competitive inhibition by the variant substrate. (C) Substrate cleavage by VS ribozyme that had been preincubated with G638A variant substrate. Ribozyme (1 μM, with or without 2.8 μM G638A substrate) and substrate were separately incubated followed by mixing together at 0 time. The progress of both reactions is shown: no G638A substrate (open circles); plus G638A substrate (filled circles) and fitted to single exponentials, yielding rates of 0.77±0.03 and 0.28±0.01 min⁻¹. The data for the first 0.6 min are shown expanded in the inset. Note that no lag phase is discernible. (D) A VS cleavage reaction interrupted by addition of G638A substrate. A cleavage reaction was initiated by addition of 10 μl of 1 μM ribozyme to 10 μl of natural sequence substrate, followed by addition of 10 μl of 30 μM G638A substrate, 1 μM ribozyme after 60 s. Progress curves are plotted for the interrupted reaction (filled circles) and one reaction allowed to continue normally (open circles; these data are taken from panel C), and fitted to single exponentials. There is no discernible intermediate phase, and the curves intersect at 64.8 s.

Figure 5

Effect of functional group changes at position 638 on the rate of VS ribozyme cleavage. Variant substrates were synthesized with alternative nucleotide bases or analogs at position 638 as shown and radioactively 5′-³²P labelled. These were subjected to cleavage in trans by VS ribozyme under single-turnover conditions for 30 min at 37°C in standard buffer. Substrate and product were separated by electrophoresis in 20% polyacrylamide gels under denaturing conditions, and visualized by phosphorimaging. Tracks 1, natural substrate before cleavage; tracks 2–8, incubation with VS ribozyme; track 2, natural substrate; track 3, substitution by adenine; track 4, substitution by inosine; track 5, substitution by 2-aminopurine; track 6, substitution by 2,6-diaminopurine and track 7, substitution by purine.

Figure 6

Calculated pH dependence of the cleavage reaction of the VS ribozyme as a function of base pK_A, assuming general acid–base catalysis. The fractions of protonated acid f_A, unprotonated base f_B and their product f_A·f_B have been calculated and plotted as a function of pH, following the approach of Bevilacqua (2003). The shaded sections are the regions of pH not accessible to experimental study. Reaction rate should be proportional to the fraction of ribozyme in the appropriate state of protonation, that is, f_A·f_B. Note that in these graphs, f_A and f_B are plotted on a log₁₀ scale (left), whereas f_A·f_B is plotted on a linear scale (right). (A) Plot for pK_A values of 5.5 and 9 for the acid and base, respectively. This might correspond to the natural ribozyme, assuming that the acid is an adenine with an elevated pK_A and the base is a guanine with a slightly reduced pK_A. The predicted reaction rate profile is a broad bell shape, with a maximum close to neutrality. (B) Plot for pK_A values of 5.5 and 5 for the acid and base, respectively. This situation could emerge if the guanine were replaced by a nucleobase of significantly lower pK_A (e.g. adenine or DAP). The reaction rate is predicted to exhibit a marked increase at low pH, with a maximum that is just detectable for these values.

Figure 7

pH dependence of observed rates for VS ribozyme cleavage reactions in trans with the natural and modified substrates. Cleavage reaction rates were measured under single-turnover conditions in 50 mM MES, HEPES or TAPS of required pH containing 200 mM MgCl₂ and 25 mM KCl. All rates are averages of ⩾3 measurements, and the error bars indicate one standard deviation. All data have been fitted to a double-ionization model in which there is a requirement for one protonated and one deprotonated form (equation 2), from which apparent pK_A values have been determined. (A) The pH dependence of the rate of cleavage of the natural sequence substrate. The data are well fitted by the double-ionization model model, giving apparent pK_A values of 5.2±0.1 and 8.4±0.1, and an intrinsic rate of 6.6 min⁻¹. (B) The pH dependence of the rate of cleavage of the G638I substrate. The data (filled circles) are fitted (continuous line) to the double-ionization model model, giving pK_A values of 4.8±0.1 and 8.2±0.1, and an intrinsic rate of 0.29 min⁻¹. The data (open circles) and fit (broken line) for the natural sequence substrate (scaled to the intrinsic rate of the G638I reaction) reveal significant shifts of reaction pK_A values for the modified substrate. (C) The data for four substrates plotted as the log₁₀ of the observed rate as a function of pH. The grid facilitates estimation of the gradients. Natural sequence substrate, circles; G638I substrate, triangles; G638DAP, squares and G638A, diamonds. (D) The pH dependence of the rate of cleavage of the G638DAP substrate. The reaction is accelerated at lower pH values, and a clear maximum is observed at pH 5. The data have been fitted to single- (broken line; equation 3) and double (unbroken line)-ionization models. The fit to the double-ionization model gives pK_A values of 4.6±0.2 and 5.6±0.1 and an intrinsic rate of 0.15 min⁻¹. (E) The K_M for the G638DAP substrate. Rates of cleavage by the trans-acting VS ribozyme were measured as a function of ribozyme concentration over a range of buffer pH. Each rate was plotted and fitted to equation 4, from which values of K_M and k₂ were calculated. The data for pH 5 are shown. The variation of K_M over the range of pH between 5 and 8 is plotted (inset).

Figure 8

Probable catalytic mechanisms, and a strong similarity between the VS and hairpin ribozymes. (A) Two catalytic mechanisms based on the participation of G638 and A756 in general acid–base catalysis. In one possibility (upper), the guanine acts as the general base deprotonating the 2′-OH, whereas the adenine protonates the 5′-oxyanion leaving group. In the other (lower), the roles of the two nucleobases are reversed. At the present time we have no way to distinguish these possibilities; the pH dependence predicted on the basis of the two models would be identical. (B) The VS and hairpin ribozymes may be closely similar in catalytic mechanism. The active geometry of both is generated by loop–loop interaction, and the topological organization of scissile phosphate and the putative catalytic nucleobases is identical in both ribozymes. The hairpin ribozyme utilizes the upper catalytic mechanism shown in (A).