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

Published

Journal Article

Self-cleavage of the genomic and antigenomic ribozymes from hepatitis delta virus (HDV) requires divalent cation for optimal activity. Recently, the HDV genomic ribozyme has been shown to be active in NaCl in the absence of added divalent metal ion at low pH (apparent pKa 5.7). However, we find that the antigenomic ribozyme is 100 to 1000-fold less active under similar conditions. With deletion of a three-nucleotide sequence (C41-A42-A43) unique to the genomic ribozyme, the rate constant for cleavage decreased substantially, while activity of the antigenomic ribozyme was enhanced by introducing a CAA sequence. From the crystal structure, it has been proposed that C41 in this sequence is protonated. To investigate a possible connection between activity at low pH and protonation of C41, mutations were made that were predicted to either eliminate protonation or alter the nature of the tertiary interaction upon protonation. In the absence of added Mg2+, these mutations reduced activity and eliminated the observed pH-rate dependence. Thermal denaturation studies revealed a pH-sensitive structural feature in the genomic ribozyme, while unfolding of the mutant ribozymes was pH-independent. We propose that, in the absence of added Mg2+, protonation of C41 contributes to enhanced activity of the HDV genomic ribozyme at low pH.

Full Text

Duke Authors

Cited Authors

  • Wadkins, TS; Shih, I; Perrotta, AT; Been, MD

Published Date

  • February 2, 2001

Published In

Volume / Issue

  • 305 / 5

Start / End Page

  • 1045 - 1055

PubMed ID

  • 11162113

Pubmed Central ID

  • 11162113

International Standard Serial Number (ISSN)

  • 0022-2836

Digital Object Identifier (DOI)

  • 10.1006/jmbi.2000.4368

Language

  • eng

Conference Location

  • England