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-thiouridin
e 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 co
mplex furthermore elicits, upon Mg2+ addition, a catalytic activity similar
to that exhibited by the complexes cross-linked at the distal ends of eith
er stem I or stem III or of the non-substituted bipartite complex. This sho
ws that the reaction mechanism is fully compatible with a strong structural
constraint between stems I and II and that sodium ions at high concentrati
on (400 mM) are able to promote a proper folding of hammerhead ribozymes. N
one of the multiple cross-links formed within the ribozyme core (probe in p
osition 16.1 or 1.1) was found catalytically active. The cross-link pattern
s 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 stru
cture, some of them (16.1 to U4 and 2.1) definitely can not, suggesting tha
t additional alternative inactive conformations exist in solution. (C) Soci
ete francaise de biochimie et biologie moleculaire / Elsevier, Paris.