STRUCTURE OF AN ANTI-HIV-1 HAMMERHEAD RIBOZYME COMPLEX WITH A 17-MER DNA SUBSTRATE-ANALOG OF HIV-1 GAG RNA AND A MECHANISM FOR THE CLEAVAGEREACTION - 750 MHZ NMR AND COMPUTER EXPERIMENTS

The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. Th e ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides , and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with t wo DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems as sume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed t hat the A-family RNA residues at the core expand the A-family initiate d at the core into the DNA stems because of the large free energy requ irement for the formation of A/B junctions. Assignments of the base H8 /H6 protons and H1' of the 47 residues were made by a NOESY walk. In a ddition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The e xtracted NMR data along with available crystallographic data, were use d to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, th e base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediat e region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on th e derived structure, and consistent with biochemical data in the liter ature, is proposed. The complex between the anti HIV-1 gag ribozyme an d its abortive DNA substrate manifests in the detection of a continuou s track of Ave A.T base pairs; this suggests that the interaction betw een the ribozyme and its DNA substrate is stronger than the one observ ed in the case of the free ribozyme where the bases in stem I and stem III regions interact strongly with the ribozyme core region (Sarma, R . H., et al. FEBS Letters 375, 317-23, 1995). The complex formation pr ovides certain guidelines in the design of suitable therapeutic ribozy mes. if the residues in the ribozyme stem regions interact with the co nserved core, it may either prevent or interfere with the formation of a catalytically active tertiary structure.