The contribution of 2'-hydroxyls to the cleavage activity of the Neurospora VS ribozyme

Abstract

We have used nucleotide analog interference mapping and site-specific substitution to determine the effect of 2'-deoxynucleotide substitution of each nucleotide in the VS ribozyme on the self-cleavage reaction. A large number of 2'-hydroxyls (2'-OHs) that contribute to cleavage activity of the VS ribozyme were found distributed throughout the core of the ribozyme. The locations of these 2'-OHs in the context of a recently developed helical orientation model of the VS ribozyme suggest roles in multi-stem junction structure, helix packing, internal loop structure and catalysis. The functional importance of three separate 2'-OHs supports the proposal that three uridine turns contribute to local and long-range tertiary structure formation. A cluster of important 2'-OHs near the loop that is the candidate region for the active site and one very important 2'-OH in the loop that contains the cleavage site confirm the functional importance of these two loops. A cluster of important 2'-OHs lining the minor groove of stem-loop I and helix II suggests that these regions of the backbone may play an important role in positioning helices in the active structure of the ribozyme.

Figure 1

(A) Secondary structure of G11. G11, a deletion construct of the VS ribozyme (16) consists of six helices (15) and undergoes a site-specific cleavage reaction at the phosphodiester indicated by the arrowhead. The minimal substrate domain is boxed. A 3 bp kissing interaction forms between the circled nucleotides in loops I and V, as indicated (18). In the absence of magnesium, the substrate exists in an inactive conformation; in the presence of magnesium and the kissing interaction, the secondary structure of the substrate is rearranged to form the active conformation (19,36). (B) Alternative representation of the secondary structure of G11 that accommodates structural and functional constraints (21,22). The cleavage site is indicated by the arrowhead.

Figure 2

NAIM analysis of the effects of 2′-deoxynucleotide substitution on G11 self-cleavage. (A) Populations of dNMPαS-substituted G11 were allowed to self-cleave for 10 min, purified, iodine treated and electrophoresed on 8% polyacrylamide–8 M urea gels. Representative autoradiograms are shown. Sites of inhibition are indicated with arrowheads and enhancements with diamonds. Blue symbols indicate sites of phosphorothioate only effects (21) and red symbols indicate new effects that are due to the absence of the 2′-OH. Red circles indicate important 2′-OHs in stem–loop I whose substitutions cause a small (<1.5-fold) but reproducible effect. (B) The results of the dNMPαS NAIM analysis were quantified using ImageQuant. The means of the ratios of the normalized band intensities in the cleaved to uncleaved fractions are represented graphically on a log scale. Dashed lines indicate 1.5-fold inhibitions or enhancements. Ratios that are not significantly different from 1 at the 95% confidence level as determined by Student’s t-test are shown as black bars; those that are significantly different from 1 and are >1.5-fold in magnitude are shown as colored bars, corresponding to the colors in (A). C634–C637 are significantly different from 1 but <1.5-fold in magnitude and therefore were also investigated in a different assay (see Fig. 3) and are marked with red circles. The P value for each significant effect was between 0.008 and 2.6 × 10^–10, with most effects having a P value of <1 × 10^–3.

Figure 3

The effect of site-specific 2′-deoxynucleotide substitution in stem–loop I on cleavage by a trans-acting ribozyme. (A) Substrates for trans cleavage. An oligonucleotide corresponding to nucleotides 608–645 of G11 was synthesized in either an all-ribonucleotide version (ribo) or with 2′-deoxynucleotide substitutions (d) as indicated by the boxed sequences. The symbols next to each boxed substitution correspond to the graph in (B). (B) Time course of cleavage of stem–loop I substrates by Rz646. The substrates shown in (A) were incubated in the presence of 200 nM Rz646, a trans-acting ribozyme consisting of nucleotides 646–783 of G11. The fraction cleaved is plotted as a function of time. The curves represent the fit to a first order exponential equation. (C) Pseudo first order rate constants of cleavage were obtained by fitting time courses of cleavage to a first order exponential equation and the average is given with the standard deviation for each substrate used.

Figure 4

(A) Superimposed on the secondary structure of G11 (15) are nucleotides that display an inhibitory effect on self-cleavage when substituted with 2′-deoxynucleotides in a NAIM experiment (filled arrowheads) and those that display a stimulatory effect (open diamonds). Nucleotides that were tested using site-specific 2′-deoxynucleotide substitutions in a trans cleavage assay and which display inhibitory effects are indicated by filled circles. The cleavage site is indicated by the arrow. (B) Important 2′-OHs are highlighted on the alternative representation of the secondary structure model. Symbols are as in (A).