A Y form of hammerhead ribozyme trapped by photo-cross-links retains full cleavage activity

The conformation in solution of a small bipartite I-III hammerhead ribozyme has been deduced from the photo-cross-links formed between cleavable ribo-deoxysubstrates appropriately substituted with the probe deoxy-4-thiouridine and ribozyme residues. The ribozyme-substrate complex is able to adopt a...

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Bibliographic Details
Published in:Biochimie 1999-03, Vol.81 (3), p.201-212
Main Authors: Bravo, Catherine, Woisard, Anne, Fourrey, J.-L., Laugâa, Philippe, Favre, Alain
Format: Article
Language:English
Subjects:
Buffers
cleavage activity
hammerhead ribozyme
Hydrolysis
Kinetics
Models, Molecular
Nucleic Acid Conformation
Osmolar Concentration
photo-cross-links
Photochemistry
Protein Conformation
RNA Probes
RNA, Catalytic - chemistry
RNA, Catalytic - metabolism
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Summary:The conformation in solution of a small bipartite I-III hammerhead ribozyme has been deduced from the photo-cross-links formed between cleavable ribo-deoxysubstrates appropriately substituted with the probe deoxy-4-thiouridine and ribozyme residues. The ribozyme-substrate complex is able to adopt a Y-like structure with stems I and II in close proximity in the presence of 400 mM Na + only. Indeed, a cross-link joining stem I (1.6) to loop II (AL2.4) forms in significant amount under these conditions. This cross-linked complex furthermore elicits, upon Mg 2+ addition, a catalytic activity similar to that exhibited by the complexes cross-linked at the distal ends of either stem I or stem III or of the non-substituted bipartite complex. This shows that the reaction mechanism is fully compatible with a strong structural constraint between stems I and II and that sodium ions at high concentration (400 mM) are able to promote a proper folding of hammerhead ribozymes. None of the multiple cross-links formed within the ribozyme core (probe in position 16.1 or 1.1) was found catalytically active. The cross-link patterns nevertheless indicate a higher flexibility of the core in Na + than in Mg 2+. While most of the cross-links can be accommodated by the Y solution structure, some of them (16.1 to U4 and 2.1) definitely can not, suggesting that additional alternative inactive conformations exist in solution.
ISSN:0300-9084
1638-6183
DOI:10.1016/S0300-9084(99)80053-4