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-crosslinks 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 Mg2+ 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 Mg2+. 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.
Bravo
C
C
Laboratoire de Photobiologie Moléculaire, Institut Jacques-Monod, CNRS, Université Paris 7, France.
Woisard
A
A
Fourrey
J L
JL
Laugâa
P
P
Favre
A
A
D002021:Buffers; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D010777:Photochemistry; D011487:Protein Conformation; D015347:RNA Probes; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*
D002021:Buffers; D015347:RNA Probes; D016337:RNA, Catalytic