RECENT APPROACHES TO PROBE FUNCTIONAL-GROUPS IN RIBONUCLEASE-P RNA BYMODIFICATION INTERFERENCE

Modification interference is a powerful method to identify important f unctional groups in RNA molecules. We review here recent developments of techniques to screen for chemical modifications that interfere with (i) binding of (pre-)tRNA to bacterial RNase P RNA or (ii) pre-tRNA c leavage by this ribozyme. For example, two studies have analyzed posit ions at which a substitution of sulfur for the pro-Rp oxygen affects t RNA binding [1] or catalysis [2]. The results emphasize the functional key role of a central core element present in all known RNase P RNA s ubunits. The four sulfur substitutions identified in one study [2] to inhibit the catalytic step also interfered with binding of tRNA to E. coli RNase P RNA [1]. This suggests that losses in binding energy due to the modification at these positions affect the enzyme-substrate and the enzyme-transition state complex. In addition, the two studies hav e revealed, for the first time, sites of direct metal ion coordination in RNase P RNA. The potentials, limitations and interpretational ambi guities of modification interference experiments as well as factors in fluencing their outcome are discussed.