As already mentioned, V1 digestion pattern for pre-cleaved ribozymes shows well-defined bands with different but comparable intensities for each nucleotide between 15 and 26 nucleotides. This pattern is routine for each uncleaved construct. After cleavage, all these bands are replaced by a strong doublet at nt U17–C18 (SBL, LSB and BLS) or nt C18–C19 (LBS) (Figures 2b, 3b, 4b and 4d).
It is noteworthy that V1 digestion pattern of post-cleaved HDV ribozyme reported by Rosenstein & Been [7] also contains a strong doublet U17–C18 and is practically free from cut sites between nt 15 and 26. Interestingly, point mutations in the 3'product P2 region which disrupt P2 lead to a nuclease-digestion pattern typical of uncleaved unmodified ribozyme (cuts in the upstream strand of P2, P3 and l3 between position 16 and 26), while compensatory changes which restore stem P2 of 3'product provide a pattern typical of the 3'-product (cuts at positions 17 and 18) [8]. However, uncleaved LBS exhibits a pattern typical of the uncleaved ribozyme (cuts between position 18 and 26), irrespective of the presence of UUCG which closes nucleotide sequence and forces the formation of stem P2.
Purified large product of ribozyme self-cleavage also exhibits one of the two alternative states for the region between U15 and U26. Note that the product purification procedure includes unwinding the whole structure of RNA by a denaturant and subsequent annealing. After this procedure, the purified products of SBL (Figure 2c) and LBS (LB, Figure 4d) acquire a structure with a doublet at C18 region, while the product of LSB (SB, Figure 3c) regains the structure characteristic of the pre-cleaved form. It should be noted that the product of SBL self-cleavage possesses all elements included in the pseudoknot model. The product of LBS (LB) does not have the S region and therefore cannot form stem P1. The product of LSB (SB) cannot form stem P4 or stem P2 since it lacks L chain. Comparison of Figures 3a and 3c shows that the digestion patterns of the region between G1 and G40 for LSB and SB are almost identical except for very strong cleavages in R region of SB which are very weak in LSB. Thus, it can be suggested that in uncleaved ribozyme SB fragment is folded as a separate domain without direct interaction with P4 and L fragment. Previously we have shown that SB domain forms autonomously [9]. Obviously, stem P1 is present in SB and there is no candidate for forming a lengthy Watson-Creek double-stranded region with a polypyrimidine stretch from U17 to U26. However, the nucleotides of the region seem to be well stacked as they are well accessible to V1 nuclease and are not susceptible to single-strand-specific nucleases. In contrast to P2 and P3–l3, other parts of post-cleaved SBL and BLS exhibited no changes in nuclease digestion patterns.
It should be noted that the most unexpected changes occurred in the susceptibility of l2 to Fe(II)-EDTA cleavage (Figures 4c and 4d). According to any available 3D model of the ribozyme, this part of the molecule may become fully exposed to the solvate.
We have projected our nuclease probing data on 3D structures of both ribozyme forms. Atomic coordinates deposited at the RCSB Protein Data Bank (PDB) [10] under accession codes 1drz and 1cx0 for post-cleaved form [2, 3] and 1sj3, 1sj4, 1sjf, 1vbx, 1vby, 1vbz, 1vc0, 1vc5, 1vc6 and 1vc7 for pre-cleaved form [4] were used. Secondary structure homology between antigenomic and genomic HDV ribozymes was used accordingly to Been and Wickham alignments of both ribozyme sequences [11] in order to make the projection of our antigenomic data on the structure of genomic ribozyme (Figures 5a and 5b, see Figure 6).
Figure 5a shows a nuclease cleavage digestion pattern obtained for pre-cleaved SBL ribozyme projected on the model of pre-cleaved genomic ribozyme [4], and Figure 5b shows a nuclease cleavage pattern obtained for post-cleaved SBL ribozyme projected on the model of post-cleaved genomic form [2]. For comparison, nuclease probing data of the pre-cleaved SBL ribozyme projected on a 3D model of pre-cleaved SBL are given in Figure 5c. The SBL molecule was modeled on the basis of structural data for HDV ribozyme and optimized with Tripos Sybyl V.6 software run on a Silicon Graphics Computer. For this illustration we have selected a model of pre-cleaved SBL possessing stem P2 which formed without L chain as shown in Figure 3c and composed of nt 16–18 and nt 11–13 instead of nt 82–84 (stem P2'). Several models were considered for a possible conformation of the C16 – U32 region of pre-cleaved ribozyme. The model given in Figure 5c was selected because it shows the best fit for the X-ray data, does not contradict RNAse cleavage pattern, and maintains reasonable orientation of catalytic C76 relative to the cut site phosphate and does not use the 3'end part of L chain for the formation of stem P2. The projection of the RNAse cleavage data on a number of 3D models of antigenomic HDV ribozyme can be found at our laboratory site [6].
As seen from Figure 5a, the major cut sites for both single-strand-specific (red balls) and double-strand-specific (orange balls) nucleases located in the vicinity of the self-cleavage site (green ball) are in conflict with the ribozyme model. The area within black circle in the proximity to C24, C25 (C21, C22 in the genomic ribozyme) contains nuclease cut sites identified by RNAse mapping which are inaccessible to nucleases due to their large size. All other cut sites in this figure and all cut sites in Figure 5b are located at the periphery of the molecule and are accessible to nucleases from the outside. From these data it can be concluded that at least in pre-cleaved antigenomic ribozyme nucleotides C24 and C25 (C21, C22 in the genomic ribozyme) are located on the molecule surface and, consequently, cannot form stem P1.1.
For details and discussion see Additional files 1, 2, 3, 4, 5, 6, 7, 8.