Catalytic Core Structure of the trans-Acting HDV Ribozyme Is Subtly Influenced by Sequence Variation Outside the Core

Abstract

The human pathogenic hepatitis delta virus (HDV) employs a unique self-cleaving catalytic RNA motif, the HDV ribozyme, during double-rolling circle replication. Fluorescence spectroscopy, circular dichroism, terbium(III) footprinting, and X-ray crystallography of precursor and product forms have revealed that a conformational change accompanies catalysis. In addition, fluorescence resonance energy transfer (FRET) has previously been used on a trans-acting HDV ribozyme to demonstrate surprisingly significant catalytic and global conformational effects of substrate analogues with varying 5' sequences, which reside as dangling overhangs outside the catalytic core. Here, we use the fluorescent guanine analogue 2-aminopurine (AP) in nucleotide position 76, immediately downstream of the catalytically involved C75, to monitor the relative structural effects of these substrate analogues on the ribozyme's trefoil turn of the catalytic core. Steady-state and time-resolved AP fluorescence spectroscopies show that the binding of each substrate analogue induces a unique local conformation with a specific AP76 stacking equilibrium. Binding of the 3' product results in a relative increase in AP fluorescence, suggesting that AP76 becomes more unstacked upon catalysis. These local conformational changes are kinetically concomitant with global conformational changes monitored by FRET. Finally, the rate constant of the local conformational change upon 3' product binding is fast and independent of 3' product concentration yet Mg2+ dependent. Our results demonstrate that the trefoil turn of the HDV ribozyme catalytic core is in a state of dynamic equilibrium not captured by static crystal structures and is highly sensitive to the identity of the 5' sequence and Mg2+ ions.

Fluorescence intensity decays of 2-aminopurine solutions: Lifetime distribution approach

“…However, resolution of measured 2AP intensity decays as a weighted sum of multiple discrete lifetimes is difficult to interpret. For example, some reports have indicated 4 or 5 discrete lifetimes for 2AP in a single sample, and the component lifetime values themselves can vary widely between data sets [19,46]. Furthermore, multi-exponential decays may hide sources of heterogeneity such as by superimposition of heterogeneous decays comprising individual lifetimes close to one another.…”

Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme

“…There are also conceptual challenges in defining where folding ends and the chemical step begins. For HDV and hammerhead ribozymes, there are examples of mutations distant from the active site contributing to changes in k obs in constructs that are thought to be limited by the rate of the chemical step (54)(55)(56). Do such mutants cleave slowly simply because f R(1) is low?…”

Inactivation of the Ribozyme with Fluorescent Dyes

“…1,2 Omitting the oligonucleotides reduces the observed catalytic rate constant (kobs) by 10 3 to 10 6 -fold, indicating that the deoxyoligonucleotide effector is necessary for its full catalytic activity. The activator helix formed by the PBS and the oligo effector is connected by a 5nt "linker" region to the substrate-binding internal guide sequence and stabilizes a long-range base-paring interaction between the 5 nuclotides of the linker and those closer to the catalytic core.…”

“…3,4 Fluorescence resonance energy transfer (FRET) which is distance-dependent interaction between electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon, has been used to get the information for the conformational change of RNA. [5][6][7][8] It is prerequisite to monitor the catalytic activity of ribozymes after the dye labeling for the fluorescence measurement because it can affect its catalytic activity. In this research, RNA species was prepared by labeling two different terminal sites of a kinase RNA with two fluorescent dyes such as a donor (Cy3) and an acceptor (Cy5).…”