ANALYSIS OF THE TERTIARY STRUCTURE OF THE RIBONUCLEASE-P RIBOZYME-SUBSTRATE COMPLEX BY SITE-SPECIFIC PHOTOAFFINITY CROSS-LINKING

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA m aturation, is a ribonucleoprotein containing a catalytic RNA. The seco ndary structure of this ribozyme is well-established, and a low-resolu tion model of the three-dimensional structure of the ribozyme-substrat e complex has been proposed based on site-specific crosslinking and ph ylogenetic comparative data [Harris ME et al., 1994 EMBO J 13:3953-396 3]. However, several substructures of that model were poorly constrain ed by the available data. In the present analysis, additional constrai nts between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking expe riments. Circularly permuted RNase P RNAs were used to position an azi dophenacyl photoactive crosslinking agent specifically at strategic si tes within the ribozyme-substrate complex. Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circ ular and linear RNA standards. Crosslinked species generally retained significant catalytic activity, indicating that the results reflect th e native ribozyme structure. The crosslinking results support the gene ral configuration of the structure model and predicate new positions a nd orientations for helices that were previously poorly constrained by the data set. The expanded library of crosslinking constraints was us ed, together with secondary and tertiary structure identified by phylo genetic sequence comparisons, to refine significantly the model of RNa se P RNA with bound substrate pre-tRNA. The crosslinking results and d ata from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme.