A pH-sensitive RNA tertiary interaction affects self-cleavage activity of the HDV ribozymes in the absence of added divalent metal ion

Self-cleavage of the genomic and antigenomic ribozymes from hepatitis delta virus (HDV) requires divalent cation for optimal activity. Recently, the H DV genomic ribozyme has been shown to be active in NaCl in the absence of a dded divalent metal ion at low pH (apparent pK(a) 5.7). However, we find th at the antigenomic ribozyme is 100 to 1000-fold less active under similar c onditions. With deletion of a three-nucleotide sequence (C41-A42-A43) uniqu e to the genomic ribozyme, the rate constant for cleavage decreased substan tially, while activity of the antigenomic ribozyme was enhanced by introduc ing a CAA sequence. From the crystal structure, it has been proposed that C 41 in this sequence is protonated. To investigate a possible connection bet ween activity at low pH and protonation of C41, mutations were made that we re predicted to either eliminate protonation or alter the nature of the ter tiary interaction upon protonation. In the absence of added Mg2+, these mut ations reduced activity and eliminated the observed pH-rate dependence. The rmal denaturation studies revealed a pH-sensitive structural feature in the genomic ribozyme, while unfolding of the mutant ribozymes was pH-independe nt. We propose that, in the absence of added Mg2+, protonation of C41 contr ibutes to enhanced activity of the HDV genomic ribozyme at low pH. (C) 2001 Academic Press.