The hepatitis delta virus (HDV) ribozyme is an RNA enzyme from the human pathogenic HDV. Cations play a crucial role in self-cleavage of the HDV ribozyme, by promoting both folding and chemistry. Experimental studies have revealed limited but intriguing details on the location and structural and catalytic functions of metal ions. Here, we analyze a total of approximately 200 ns of explicit-solvent molecular dynamics simulations to provide a complementary atomistic view of the binding of monovalent and divalent cations as well as water molecules to reaction precursor and product forms of the HDV ribozyme. Our simulations find that an Mg2+ cation binds stably, by both inner- and outer-sphere contacts, to the electronegative catalytic pocket of the reaction precursor, in a position to potentially support chemistry. In contrast, protonation of the catalytically involved C75 in the precursor or artificial placement of this Mg2+ into the product structure result in its swift expulsion from the active site. These findings are consistent with a concerted reaction mechanism in which C75 and hydrated Mg2+ act as general base and acid, respectively. Monovalent cations bind to the active site and elsewhere assisted by structurally bridging long-residency water molecules, but are generally delocalized.

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The hepatitis delta virus (HDV), an infectious human pathogen affecting millions of people worldwide, leads to intensified disease symptoms, including progression to liver cirrhosis upon coinfection with its helper virus, HBV. Both the circular RNA genome of HDV and its complementary antigenome contain a common cis-cleaving catalytic RNA motif, the HDV ribozyme, which plays a crucial role in viral replication. Previously, the crystal structure of the product form of the cis-acting genomic HDV ribozyme has been determined, and the precursor form has been suggested to be structurally similar. In contrast, solution studies by fluorescence resonance energy transfer (FRET) on a trans-cleaving form of the ribozyme have shown significant global conformational changes upon catalysis, while 2-aminopurine (AP) fluorescence assays have detected concomitant local conformational changes in the catalytic core. Here, we augment these studies by using terbium(III) to probe the structure of the trans-acting HDV ribozyme at nucleotide resolution. We observe significant structural differences between the precursor and product forms, especially in the P1.1 helix and the trefoil turn in the single-stranded region connecting P4 and P2 (termed J4/2) of the catalytic core. We show, using terbium(III) footprinting and sensitized luminescence spectroscopy as well as steady-state, time-resolved, and gel-mobility FRET assays on a systematic set of substrates, that the substrate sequence immediately 5' to the cleavage site significantly modulates these local as well as resultant global structural differences. Our results suggest a structural basis for the previously observed impact of the 5' substrate sequence on catalytic activity.

The α subunit of the replicative DNA polymerase III of Escherichia coli is the active polymerase of the 10-subunit bacterial replicase. The C-terminal region of the α subunit is predicted to contain an oligonucleotide binding (OB-fold) domain. In a series of optical tweezers experiments, the α subunit is shown to have an affinity for both double- and single-stranded DNA, in distinct subdomains of the protein. The portion of the protein that binds to double-stranded DNA stabilizes the DNA helix, because protein binding must be at least partially disrupted with increasing force to melt DNA. Upon relaxation, the DNA fails to fully reanneal, because bound protein interferes with the reformation of the double helix. In addition, the single-stranded DNA binding component appears to be passive, as the protein does not facilitate melting but instead binds to single-stranded regions already separated by force. From DNA stretching measurements we determine equilibrium association constants for the binding of α and several fragments to dsDNA and ssDNA. The results demonstrate that ssDNA binding is localized to the C-terminal region that contains the OB-fold domain, while a tandem helix-hairpin-helix (HhH)2 motif contributes significantly to dsDNA binding.