Certain RNA sequences from several plant satellite RNAs exhibit autolytic transesterification reactions that result in site-specific cleavage reactions (arrow). The small consensus sequence common in these RNAs can be formed from two (or three) fragments and is composed of three helices (one or more of which can terminate in a hairpin loop) and has been termed a hammerhead. There are nine conserved non-helical nucleoside residues (and one or two base pairs) that appear to be critical for the observed cleavage activity. This project will determine the importance of specific base and carbohydrate functional group available on the thirteen conserved nucleotide residues, some of which will be involved in key interbase hydrogen bonding, base stacking or magnesium interactions necessary for the observed autocatalytic function. The critical functional groups will be identified by preparing a series of """"""""deletion modified"""""""" ribozyme complexes in which specific functional groups located on the base or carbohydrate residues of the conserved nucleotides are selectivity deleted. The functional groups targeted in this study include the purine and pyrimidine ring nitrogen as well as the exocyclic amino and carbonyl groups. The modified fragments will be prepared by chemical syntheses. A total of 14 modified nucleoside phosphoramidites will be prepared for site-specific incorporation of the deletion modifications into the ribozyme or substrate fragment. The relative rates of cleavage will then be determined under both stoichiometric and catalytic conditions. We will compare the effects of the excision ribozyme/substrate structure (CD, thermal stability, and NMR) will allow dissection of the geometry and enable us to propose and active site conformation for the complex. The results of the proposed work will shed light on the process by which certain RNA sequences can adopt a unique secondary/tertiary structure to precisely position specific functional groups to bind Mg2+ and labilize the scissle phosphodiester bond.
| Guo, M J; Hildbrand, S; Leumann, C J et al. (1998) Inhibition of DNA polymerase reactions by pyrimidine nucleotide analogues lacking the 2-keto group. Nucleic Acids Res 26:1863-9 |
| Fu, D J; Rajur, S B; McLaughlin, L W (1994) Activity of the hammerhead ribozyme upon inversion of the stereocenters for the guanosine 2'-hydroxyls. Biochemistry 33:13903-9 |
