RNA 96 (the RNA Society), May 28 - June 2 1996, Madison, WI

An Update of the RNase P Tertiary Structure : Phylogenetic Perspectives and New Crosslinking Reagents

James M. Nolan, Michael E. Harris, Jiunn-Laing Chen, Bong-Kyeong Oh, James W. Brown*, and Norman R. Pace.

Department of Biology, Indiana University, Bloomington, IN 47405
*Department of Microbiology, North Carolina State University, Raleigh, NC 27695

We are investigating the structure of bacterial RNase P RNA by attaching photoagents to its tRNA substrate. By mapping the many sites in RNase P to which tRNA crosslinks, we have obtained data with which to model the tertiary structure of RNase P RNA. In addition to the photoreactive 5'-GMPS-azido and 3'-alkyl-azido modifications previously utilized for these studies, we have investigated the use of another crosslinking reagent, 6-thio-GMP. Like GMPS, 6-thio-GMP can be specifically incorporated at the 5' end of T7 transcripts, 6-thio-GMP can be specifically photoactivated at 340nm for crosslinking. Alternatively, the unique thiol of 6-thio-GMP-modified RNAs can be reacted with azidophenacyl bromide, for attachment of a photoactive azido group with a 9A spacer in the major groove of the RNA.

The various modified tRNAs were crosslinked to RNase P RNAs from Escherichia coli, Bacillus subtilis, and Thermotoga maritima. These RNase P RNAs differ to some extent in secondary structure, but must share a common core tertiary structure. Thus, any valid model of RNase P tertiary structure that satisfies the secondary structure and crosslinking constraints of one species should also be able to accommodate the constraints imposed upon the RNAs from other species. We are using several strategies to enforce constraints imposed by the crosslinking data from each of the diverse RNase P RNAs studied onto working models of RNase P RNA tertiary structure. In this analysis, we are using the YAMMP RNA modeling package, in collaboration with Steve Harvey at the University of Alabama-Birmingham. For all-atom modeling, we are using MCSYM, in collaboration with Francois Major at the University of Montreal, and Thomas Easterwood at the University of Alabama-Birmingham.