Structure and activity of the hairpin ribozyme in its natural junction conformation: effect of metal ions.
The natural form of the hairpin ribozyme consists of a four-way RNA junction of which the single-stranded loop-carrying helices are adjacent arms. The junction can be regarded as providing a framework for constructing the active ribozyme, and the rate of cleavage can be modulated by changing the conformation of the junction. We find that the junction-based form of the hairpin ribozyme is active in magnesium, calcium, or strontium ions, but not in manganese, cadmium, or sodium ions. Using fluorescence resonance energy transfer experiments, we have investigated the global structure of the ribozyme. The basic folding of the construct is based on pairwise helical stacking, so that the two loop-carrying arms are located on opposite stacked helical pairs. In the presence of magnesium, calcium, or strontium ions, the junction of the ribozyme undergoes a rotation into a distorted antiparallel geometry, creating close physical contact between the two loops. Manganese ions induce the same global folding, but no catalytic activity; this change in global conformation is therefore necessary but not sufficient for catalytic activity. Fitting the dependence of the conformation on ionic concentration to a two-state model suggests that cooperative binding of two ions is required to bring about the folding. However, further ion binding is required for cleavage activity. Cobalt hexammine ions also bring about global folding, while spermidine generates a more symmetrical form of the antiparallel structure. Cadmium ions generate a different folded form, interpreted in terms of close loop-loop association while the junction is unfolded. Sodium ions were unable to induce any folding of the ribozyme, which remained slightly parallel. These results are consistent with a folding process induced by the binding of two group IIA metal ions, distributed between the junction and the loop interface.
Walter
F
F
CRC Nucleic Acid Structure Research Group, Department of Biochemistry, The University, Dundee, UK.
Murchie
A I
AI
Thomson
J B
JB
Lilley
D M
DM
D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D002414:Cations, Monovalent; D003035:Cobalt / Q000494:pharmacology; D004735:Energy Transfer; D004789:Enzyme Activation / Q000187:drug effects; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D008969:Molecular Sequence Data; D017860:Nepovirus / Q000235:genetics; D009690:Nucleic Acid Conformation / Q000187:drug effects; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013324:Strontium / Q000494:pharmacology; D013329:Structure-Activity Relationship
D002413:Cations, Divalent; D002414:Cations, Monovalent; D008670:Metals; D016337:RNA, Catalytic; D002104:Cadmium; C030862:cobalt ammonium complex; D003035:Cobalt; D008345:Manganese; D012964:Sodium; D002118:Calcium; D013324:Strontium