Catalytic core structure of the trans-acting HDV ribozyme is subtly influenced by sequence variation outside the core.
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.
Gondert
Melissa E
ME
Department of Chemistry, The University of Michigan, 930 North University, Ann Arbor, Michigan 48109-1055, USA.
Tinsley
Rebecca A
RA
Rueda
David
D
Walter
Nils G
NG
D015075:2-Aminopurine / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D031541:Fluorescence Resonance Energy Transfer; D003698:Hepatitis Delta Virus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics; D012367:RNA, Viral / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D016337:RNA, Catalytic; D012367:RNA, Viral; D015534:Trans-Activators; D015075:2-Aminopurine; D008274:Magnesium