James W. Brown

Associate Professor & Undergraduate Coordinator
Department of Microbiology, NC State University

RNA Processing Meeting (the RNA Society), May 24-29 1994, Madison, WI

Global Architecture of the Ribonuclease P RNA-pretRNA Complex

Michael E. Harris*, James M. Nolan*, Arun Malhotra**, James W. Brown*, Stephen C. Harvey**, and Norman R. Pace*

*Department of Biology and Institute for Molecular and Cellular Biology Indiana University
**Department of Biochemistry and Genetics Schools of Medicine and Dentistry University of Alabama at Birmingham

Bacterial Ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein enzyme whose RNA component is catalytically active. The secondary structure of this ribozyme is well established, but comparatively little is understood about its three-dimensional structure. In this analysis, distance constraints between elements within the Escherichia coli RNase P RNA-pretRNA complex were determined by intra- and inter-molecular crosslinking experiments. We have previously used end-modified tRNA as well as circularly permuted (cp) tRNA as specific photoaffinity structural probes (Burgin and Pace, (1990) EMBO J. 9, 4111-4118; Nolan (1993) Science 261, 762-765). In this study eight different circularly permuted RNAs derived from E. coli RNase P RNA and one additional cptRNA were used to position a photo-activated crosslinking reagent at defined sites within the ribozyme-substrate complex. A molecular mechanics based RNA structure refinement protocol was used to incorporate the distance constraints indicated by crosslinking, along with the known secondary structure of RNase P RNA and the tertiary structure of tRNA, into molecular models. Seven different equivalent structures were generated and compared by superimposition in order to assign probable helix positions and orientations. Manual refinement within the range of conformations indicated by the molecular mechanics analysis was used to derive a first-order model of RNase P RNA with bound substrate pretRNA that is consistent with the crosslinking results and the available phylogenetic comparative analysis.

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