Figure 1.
Structure and folding pathway of the antigenomic HDV Rz.
(A) Nucleotide sequence and secondary structure of the HDV Rz antigenomic version used in this study. The ribozyme and the substrate are denoted by Rz and S, respectively. The cleavage site is indicated by the arrow. The harmonized nomenclature using roman numerals and colors for the stems is indicated [2]. The dotted line represents the junction I/II that was removed to generate a trans-acting version. The inset shows the H-bonds involved in the formation of either the trans Watson-Crick/Hoogsteen, in anti conformation, or the trans Watson-Crick, in syn conformation, GU base pair between U23 and G28 of loop III. The atom numbering for both nucleotide bases is shown. A putative magnesium binding site [21] is also shown. (B) Schematic representation of the secondary structure of the mutants capable of halting the folding pathway of HDV Rz at several stable intermediates. The mutated nucleotides are indicated.
Figure 2.
Cleavage activities of all of the ribozymes mutated in positions 23 and 28.
(A) Autoradiogram of the denaturing PAGE gel used for the analysis of the cleavage reactions. In all cases, the ribozymes (100 nM) were incubated for 2 h with trace amounts of 5′-end-labeled substrate (<1 nM). The positions of the bromophenol blue dye (BPB), the 11-nt substrate (S) and the 4-nt product (P) are indicated. “Neg” represents a cleavage reaction performed in the absence of any Rz. (B) Graphical representation of the cleavage percentages for all of the 16 nucleotide combinations examined. The values are means of at least 2 different experiments and the error bars represent the standard deviation (C) Putative H-bond representation and magnesium ion binding site of the wild-type (U23/G28) and four other nucleotide combinations.
Table 1.
Kinetic parameters of the active mutant ribozymes.
Figure 3.
Cleavage activities of the various modified chemical groups of G28.
(A) Schematic representation of the trans-Watson-Crick GU base pair (inset) and of three potential substitutions for G28. (B) Sequence and secondary structure of the original RD mixed ribozyme. The boxed regions represent the DNA parts of the oligonucleotide. (C) Autoradiogram of the denaturing PAGE gel used for the analysis of the cleavage reactions of the RD mixed oligonucleotides containing the modified chemical groups. In each case, the ribozyme (100 nM) was incubated for 2 h with trace amounts of 5′-end-labeled substrate (<1 nM) and the reactions were analyzed on denaturing 20% PAGE gels. Neg represents a cleavage reaction performed in the absence of any Rz. The positions of the bromophenol blue dye (BPB), the 11-nt substrate (S) and the 4-nt product (P) are indicated. (D) Graphical representation of the cleavage percentages for the reactions shown in (C). The values are means of at least 2 different experiments and the error bars represent the standard deviation.
Figure 4.
Chemical probing of the trans Watson-Crick GU base pair.
Various cis-acting mutants were folded in the presence of MgCl2 and then probed in either the absence or presence of either kethoxal (A) or CMCT (B). The RNA samples were reverse transcribed and the reactions then fractionated on 8% denaturing PAGE gels. The accessibility of either G (kethoxal and CMCT) or U (CMCT) was visualized by the presence of bands that downshift one nucleotide as compared to either a G or a U ladder produced using an untreated wild-type ribozyme with either dideoxy ATP (ddA) or dideoxy CTP (ddC) during the reverse transcription step. The positions of xylene cyanol dye (XC) and of both the G and the U nucleotides of the ribozymes are indicated on both gels.
Figure 5.
Global magnesium localization along the HDV Rz’s folding pathway studied by magnesium-induced cleavage.
Different 5′-end-labeled trans-acting mutant ribozymes that halt the folding pathway at each known HDV Rz folding intermediate were folded either in the absence or the presence (+) of SdA-1 substrate or 3′-end product. The probings were then allowed to proceed for 48 h at room temperature in the presence of 50 mM Tris-HCL (pH 8.3) and 20 mM MgCl2. A control reaction without MgCl2 (−) was also performed. The resulting probings were analysed on 8% denaturing PAGE gels. The positions of bromophenol blue dye (BPB) and of the different regions of the Rz are indicated on the right of the gel. The lanes designated “Ladder” and “T1” represent an alkaline hydrolysis and a ribonuclease T1 (RNase T1) mapping of the wild-type version of the ribozyme, respectively. Representative guanosine residues are indicated on the left of the gel.
Figure 6.
MC-sym structure depicting the formation of the trans Watson-Crick GU base pair after the cleavage step.
(A) Representative structure of the post-cleavage HDV ribozyme. (B) Closer view of stereodiagrams of loop III both before (containing a tWH GU base pair, upper panel) and after (containing a tWW GU base pair, lower panel) the cleavage step. The colors are harmonized as in Figure 1.
Figure 7.
Representation of the number of putative structures as a function of constraints used in the modeling performed with MC-Sym.