Trans-Acting Hepatitis Delta Virus Ribozyme:  Catalytic Core and Global Structure Are Dependent on the 5‘ Substrate Sequence

Abstract

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.

HDV Family of Self-Cleaving Ribozymes

“…126 In the HDV and CPEB3 ribozymes, the upstream sequences have been shown to markedly decrease self-cleavage activity if they are capable of extending the P1 helix by base pairing with nucleotides required to form the P1.1 helix, thereby disrupting active-site formation of the molecule. 55,56,59,98 An identical event is observed among the R2 ribozymes. As the number of alternate base pairs that the upstream sequence can form is increased from zero to three, the rate constants decrease from approximately 80 to 0.30 per hour, respectively.…”

“…Such kinetically trapped alternative conformations can form through improper base-pairing interactions of flanking sequences with the ribozyme core or even within the cores. 50,[55][56][57][58][59][60][61] Alternative conformations result in slow overall cleavage rates, biphasic kinetics, or high fractions of uncleaved ribozymes. Mutations that destabilize these ribozyme-disrupting interactions tend to result in faster cleaving ribozymes.…”

“…2 Several studies have indicated that the catalytic rate of the HDV ribozyme can be modified upon the inclusion of 5 0 and 3 0 flanking sequences. 50,55,56,59,60 For instance, destabilization of the formation of alternate P1 and P3 helices by mutation of G11 to C and removal of the terminal U from the L3 region of genomic HDV enhances the catalytic rate by over three orders of magnitude. 60 The catalytic rates of the CPEB3 ribozymes appear to be affected in a similar manner.…”

Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

“…Briefly, in vitro transcribed, gel-purified mRNA was refolded in the same S30 buffer where we observed bypassing, and probed with either 1 mM Tb 3+ ions as described (Walter et al 2000;Walter 2003, 2005;Jeong et al 2003;Harris et al 2004;Sefcikova et al 2007a,b) or 2 mM of the SHAPE reagent 1M7 (Mortimer and Weeks 2007;Gherghe et al 2008Gherghe et al , 2010Watts et al 2009). These two complementary reagents target accessible 2 ′ -OH groups, either to deprotonate them and cleave the adjacent phosphodiester bond (Tb 3+ ) or to acylate them (1M7).…”

Ribozymes: Analytical Solution of the One-substrate, Two-intermediate Reversible Scheme for Enzyme Reactions

“…Although most ribozyme studies have revealed single turnover behavior [14,16,17], sometimes the data are better fit by bi-exponential kinetics [18].…”