Deletion of internal sequence on the HDV-ribozyme: elucidation of functionally important single-stranded loop regions

Abstract

In elucidating functionally important single-stranded loop regions derived mainly from three models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several internal deletion variants of the HDV133 molecule (654-786 nt on genomic RNA) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among variants, the HDV133DI-1 (deletion of 701-718 nt) and HDV133DI-3 (deletion of 740-752 nt) ribozyme could maintain their self-cleavage activity, despite at reduced level. However, the activity could be regained in both mutants by some extent under partially denaturing conditions. These results suggest that the above two single-stranded RNA loop regions in HDV ribozyme are not part of the catalytic core but might be involved in the stability of the molecule. In contrast, deletion mutants such as HDV133DI-2 (deletion of 696-722 nt), HDV88DI-1 (deletion of 701-718 nt), HDV88DI-2 (deletion of 696-722 nt), and HDV88DI-4 (deletion of 733-760 nt) abolished catalytic activity. These results suggest that the remaining single-stranded regions of bases between 726-731 and 762-766 in the HDV88 ribozyme may be the potential regions to interact with Mg2+ ions.

Systematic substitution of individual bases in two important single‐stranded regions of the HDV ribozyme for evaluation of the role of specific bases

“…The possible role of the essential nucleotides in ribozymes can be considered either to involve interactions with metal ions suggested as real catalyst recently [18,19] and/or maintenance the tertiary structure of the molecule, both of which are required for formation of the active conformation. Our previous results of internal deletion mutants [14], point and compensation mutations at stem regions [15] and random mutations [16] on HDV genomic ribozyme suggested that single-stranded regions, bases from 726-731 (SSrA) and 762-766 (SSrB), in the pseudoknot-like model (Fig. 1A), play an important role for its self-cleavage reaction.…”

“…The plasmid pUHD88 was used in the present study and the construction of the vector has been reported previously [14]. It contains the genomic HDV sequence from nucleotide (nt) 683 to 770, which has self-cleavage activity.…”

“…Results of deletion mutagenesis showed that stem-loop regions between bases 701-718 and 740 752 are structurally important but are not the catalytic core since variants HDV133DI-1 (deletion of bases 701 718; the numbering of bases used here is that of Makino et al [13]) and HDV 88DI-3 (deletion of bases 740 752) continue to exhibit self-cleavage activity [14]. Our results of point and compensation mutations at stem regions on pseudoknot structure of HDV ribozyme suggested that stem I and stem III are essential for catalytic activity.…”

3-D models of the antigenomic ribozyme of the hepatitis delta agent with eight new contacts suggested by sequence analysis of 188 cDNA clones

“…Contacts between A-79 and other parts of the molecule may help to position C-76. Suh and colleagues (20) demonstrated that a genomic ribozyme 133 nucleotides in length retained activity when bases C-13 to C-30 were deleted, suggesting that A-78 (the genomic analog of A-79) makes at least some important contacts with nucleotides in helix I, helix IV or the G-rich junction element. Of these possibilities, an interaction with helix I appears to be most likely and has been built into our three-dimensional versions of the axehead model (see Fig.…”

“…Both comparative sequence analysis and a variety of biochemical studies indicate that the genomic and antigenomic ribozymes have closely related, but not identical, structures. Several models for each ribozyme have been proposed, tested, refined (14,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) and reviewed (13). Despite the progress that has been made, more work is needed to establish the details ofthe active structures.…”