The role of phosphate groups in the VS ribozyme-substrate interaction

Abstract

The VS ribozyme trans-cleavage substrate interacts with the catalytic RNA via tertiary interactions. To study the role of phosphate groups in the ribozyme-substrate interaction, 18 modified substrates were synthesized, where an epimeric phosphorothioate replaces one of the phosphate diester linkages. Sites in the stem-loop substrate where phosphorothioate substitution impaired reaction cluster in two regions. The first site is the scissile phosphate diester linkage and nucleotides downstream of this and the second site is within the loop region. The addition of manganese ions caused recovery of the rate of reaction for phosphorothioate substitutions between A621 and A622 and U631 and C632, suggesting that these two phosphate groups may serve as ligands for two metal ions. In contrast, significant manganese rescue was not observed for the scissile phosphate diester linkage implying that electrophilic catalysis by metal ions is unlikely to contribute to VS ribozyme catalysis. In addition, an increase in the reaction rate of the unmodified VS ribozyme was observed when a mixture of magnesium and manganese ions acted as the cofactor. One possible explanation for this effect is that the cleavage reaction of the VS ribozyme is rate limited by a metal dependent docking of the substrate on the ribozyme.

Figure 1

(a) The VS ribozyme intermolecular substrate. The residues involved in a kissing interaction with stem–loop V of the ribozyme are shown with black circles. (b) The rearranged or ‘shifted’ structure of the VS ribozyme substrate. (c) The trans-cleaving VS ribozyme used in this study. Lower case nucleotides represent nucleotides added to facilitate transcription. Helix Ia is formed by base pairing of residues C619, G618 and U617 of the trans-cleaving substrate with G640, C641 and G642 of the ribozyme.

Figure 2

(a) The substrates used in this study with either fluorescent (X = b) or radiolabelled (X = c) 5′-termini are: substrate 1 containing the natural VS ribozymes sequence (23 nt); substrate 2 G618C (23 nt); substrate 3 U617C, G616 (24 nt); substrate 4 U617C, G616, U615 (25 nt). Substrates were either radioactively (X = c) or fluorescently labelled (X = b). The modified substrates used in this study are fluorescently labelled variants of substrate 2, where a phosphorothioate internucleoside linkage (d) replaces one of the internucleoside phosphate diester bonds between G620 and G638.

Figure 3

Evaluation of the consequences of replacement of phosphate diester with a phosphorothioate internucleoside linkage in a fluorescent VS ribozyme substrate at the position designated. UM is the unmodified substrate 2. Rates of reaction (above) and % products at end point (below) were determined under pseudo first-order conditions (10 nM substrate, 75 nM ribozyme (except for G620psA621, where 375 nM ribozyme was employed), 25 mM MgCl₂, 25 mM KCl and 40 mM Tris–HCl, pH 8.0) as described in the Materials and Methods section. All data are the average of at least three independent experiments. Note that for G620psA621, the concentration of ribozyme employed to produce a measurable rate of reaction was increased from 75 nM to 375 nM, but all rate constants have been normalized with respect to ribozyme concentration to allow for this.

Figure 4

Reaction of unmodified substrate 2 (UM) and phosphorothioate containing substrates U631psC632, A622psG623 and A621psA622 under pseudo first-order conditions (10 nM substrate, 75 nM ribozyme, 25 mM MgCl₂, 25 mM KCl and 40 mM Tris–HCl, pH 8.0). The reactions have been allowed to proceed for 60 min and then heated to 90°C for 1 min and after cooling to 37°C, a further aliquot of pre-activated ribozyme was added and the reaction was then monitored again. An arrow indicates the denaturation point. In the case of UM, further reaction was stimulated by this procedure and the amount of further reaction approached that predicted if a simple two component equilibrium of active and inactive conformers is present. In the case of the phosphorothioate modified substrates, the amount of further reaction stimulated is much lower than predicted based upon simple equilibrium of conformers and suggests the presence of a non-cleavable or very poor substrate phosphorothioate diastereoisomer.

Figure 5

Summary of the sites within the VS ribozyme substrate where the substitution of epimeric phosphorothioate internucleoside diesters produces a change in the properties of the substrate. Residues involved in the proposed kissing interaction with stem–loop V are circled.