FOLDING OF THE HDV ANTIGENOMIC RIBOZYME PSEUDOKNOT STRUCTURE DEDUCED FROM LONG-RANGE PHOTOCROSSLINKS

A trans-acting system has been designed in order to explore the three- dimensional structure of the antigenomic HDV ribozyme, In this system, the substrate (SANT) is associated by base-pairing to the catalytic R NA (RzANT) forming helix H1. RzANT is able to cleave specifically the RNA substrate as well as a deoxysubstrate analogue containing a single ribocytidine at the cleavage site (position -1), This demonstrates th at such deoxysubstrate analogues are valuable tools for structural stu dies of this ribozyme domain, They form however weak complexes with Rz ANT which is due in part to their ability to fold as stable hairpins u nlike the RNA substrate, Using a set of full deoxy or of mixed deoxy-r ibo substrate analogues site-specific substituted with the photoaffini ty probe deoxy-4-thiouridine, ds(4)U, at a defined position, we were a ble to determine a number of long range contacts between the substrate and the ribozyme core, In particular, crosslinks between substrate po sition -1 and position -2 with residues C15, G19 and C67, thought to b e involved in the ribozyme catalytic site, were detected, A three dime nsional model of the antigenomic ribozyme system, derived from the str ucture proposed by Tanner et al. [Current Biol. (1994) 4, 488-498] for the genomic system was constructed, Apart from residue deletion or in sertion, only minor accommodations were needed to account for all phot ocrosslinks but one which is attributed to an alternative hybridizatio n of the substrate with the ribozyme, This study therefore further sup ports the structure proposed by Tanner ef al, for the pseudoknot model .