Formation of the P1.1 pseudoknot is critical for both the cleavage activity and substrate specificity of an antigenomic trans-acting hepatitis delta ribozyme

Abstract

Hepatitis delta virus RNAs possess self-cleavage activities that produce 2',3'-cyclic phosphate and 5'-hydroxyl termini (i.e. cis-acting delta ribozyme). Trans-acting delta ribozymes have been engineered by removing a junction from the cis version, thereby producing one molecule possessing the substrate sequence and the other the catalytic domain. According to the pseudoknot model, the secondary structure of the delta ribozyme includes a pseudoknot (i.e. P1.1 stem) formed by two base pairs from residues of the L3 loop and J1/4 junction. A collection of 48 P1.1 stem mutants was synthesized in order to provide an original characterization of both the importance and the structure of this pseudoknot in a trans-acting version of the ribozyme. Several structural differences were noted compared to the results reported for cis-acting ribozymes. For example, a combination of two stable Watson-Crick base pairs composing the essential P1.1 stem was demonstrated to be crucial for a significant level of activity, while the cis version required only one base pair. In addition, we present the first physical evidences revealing that the composition of the P1.1 stem affects the substrate specificity for ribozyme cleavage. Depending on the residues forming the J1/4 junction, non-productive ribozyme-substrate complexes can be observed. This phenomenon is proposed to be important for further development of a gene-inactivation system based on delta ribozyme.

Figure 1

Secondary structure and nucleotide sequences of a trans-acting delta ribozyme. S and Rz represent the substrate and ribozyme, respectively. The dotted line represents the J1/2 single-stranded region, which joins the S and Rz molecules in the cis-acting version. The pseudoknot P1.1 is formed by the base pairs G₂₇C₁₂ and G₂₈C₁₁, and is boxed. The stems are numbered P1 to P4. The homopurine base pair (i.e. GG) at the top of the P4 stem is indicated by two large dots, whereas the wobble base pair (i.e. GU) is indicated by a single large dot. The bold arrow indicates the cleavage site.

Figure 2

Cleavage activities of the P1.1 stem mutants as compared to that of the original delta ribozyme. (A) Typical autoradiogram of a 20% denaturing gel for the analysis of the cleavage reaction of three P1.1 stem mutants and the original delta ribozyme. For each ribozyme, aliquots were recovered from the reaction pool after 0, 60 and 120 min. The positions of the bromophenol blue (BPB), the 11 nt substrate (S) and the 4 nt product (P) are indicated. (B) Graphical representation of time courses for the cleavage reactions catalyzed by the original ribozyme (i.e. G₂₇C₁₂/G₂₈C₁₁, closed circles), the A₂₇U₁₂/C₂₈G₁₁ mutant (i.e. forming a 2 bp P1.1 stem, open circles), the C₂₇G₁₂/C₂₈C₁₁ mutant (i.e. forming a 1 bp P1.1 stem, closed squares), and the U₂₇U₁₂/U₂₈U₁₁ mutant (i.e. forming 0 bp and therefore no P1.1 stem, open squares).

Figure 3

Graphical representation of the cleavage activity over time of various substrates by a P1.1 stem mutant and the original delta ribozyme. (A) The ribozyme with the original sequence. (B) The G₂₇C₁₂/C₂₈G₁₁ P1.1 mutant. Both ribozymes were incubated in the presence of the _–4GGGC_–1 (which is the original sequence; circles), the _–4GGCC_–1 (squares) and the _–4CUAA_–1 (diamonds) substrates.

Figure 4

Typical autoradiograms of a 10% PAGE of the L3 loop probing. (A) The ribozyme with the original sequence. (B) The G₂₇C₁₂/C₂₈G₁₁ P1.1 mutant. Both ribozymes were pre-incubated in the presence of the SdC4 analog prior to the addition of MgCl₂. Lanes 1 are aliquots of ribozyme and SdC4 analog removed at 0 min and treated with RNase H in the absence of any oligonucleotide. Lanes 2, 3 and 4 are aliquots removed at 0, 1 and 5 min, respectively, and treated with the RNase H in the presence of an oligonucleotide. The position of the xylene cyanol (XC) is indicated.

Figure 5

Typical autoradiograms of 10% PAGE of Mg²⁺-induced cleavage experiments. (A) The ribozyme possessing the original sequence (i.e. G₂₇C₁₂/G₂₈C₁₁). (B) The ribozyme possessing a P1.1 stem mutated to U₂₇U₁₂/U₂₈U₁₁. The 5′ end-labeled ribozymes were incubated in the presence of substrate, but in the absence of Mg²⁺ (lanes 2 and 3), or in the absence of substrate, but in the presence of Mg²⁺ (lanes 4 and 5). The remaining lanes contain the ribozymes, Mg²⁺ and either the substrate (lanes 6 and 7) or the 3′ product (lanes 8 and 9). Lanes 2, 4, 6 and 8 are incubations of 15 min, while lanes 3, 5, 7 and 9 are incubations of 120 min. Alkaline hydrolysis (lane 1) and RNase T1 hydrolysis (data not shown) of the 5′ end-labeled ribozymes were performed in order to determine the location of the metal ion-induced cleavage products. The locations of the RNase T1 major cuts are indicated on the left. The arrow on the right indicates the location of the G₅₂.

Figure 6

Schematic representation of the minimal folding and catalytic pathway of delta ribozyme. This representation includes the formation of either productive (i.e. with P1.1 stem) or non-productive (i.e. with base pairs between positions –1 and –2 of the substrate and positions 27 and 28 of the J1/4 region) RzS′ complexes. The base pairs that are formed after the formation of the P1 stem are indicated by dotted lines.