USE OF PHOTOAFFINITY CROSS-LINKING AND MOLECULAR MODELING TO ANALYZE THE GLOBAL ARCHITECTURE OF RIBONUCLEASE-P RNA

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, but comparativel y little is understood about its 3-D structure. In this analysis, orie ntation and distance constraints between elements within the Escherich ia coli RNase P RNA-pretRNA complex were determined by intra- and inte rmolecular crosslinking experiments. A molecular mechanics-based RNA s tructure refinement protocol was used to incorporate the distance cons traints indicated by crosslinking, along with the known secondary stru cture of RNase P RNA and the tertiary structure of tRNA, into molecula r models. Seven different structures that satisfy the constraints equa lly well were generated and compared by superposition to estimate heli x positions and orientations. Manual refinement within the range of co nformations indicated by the molecular mechanics analysis was used to derive a model of RNase P RNA with bound substrate pretRNA that is con sistent with the crosslinking results and the available phylogenetic c omparisons.