James W. Brown

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

1993 RNA Processing Meetings, Cold Spring Harbor, N. Y.


Michael Harris, Arun Malhotra*, James Brown, James Nolan, Bong-Keong Oh, Steven Harvey*, and Norman Pace. Dept. of Biology, Indiana University and *Dept. of Biochemistry, University of Alabama at Birmingham.

Ribonuclease P (RNase P), an endonuclease involved in processing precursor tRNA, contains an RNA component that is catalytically active. Although much of the secondary structure of this RNA enzyme has been determined, its three-dimensional structure remains largely unexplored. In this analysis, three-dimensional structural constraints were determined by intramolecular crosslinking experiments. This information, along with the known secondary structure and insights gained from intermolecular crosslinking to tRNA (Nolan et. at., Oh and Pace, These Abstracts), was used to construct molecular models of RNase P RNA tertiary structure.

Circularly permuted (cp) RNase P RNAs were used to place photoactivated crosslinking agents at defined sites. These circularly permuted RNAs contain the native RNA sequence; however, the 5' and 3' termini are relocated elsewhere in the secondary and thus the tertiary structure. Relocation of the 5' and 3' ends allowed us to use straightforward, high-yield termini modification techniques to attach crosslinking reagents. Circularly permuted RNaseP RNAs were assayed for catalytic properties in order to determine whether they accurately reflect the structure of the native ribozyme. All cpRNaseP RNAs tested were accurate and efficient catalysts of the pre-tRNA cleavage reaction, with kcat and KM values similar to native RNase P RNA.

For 5' modification, cpRNase P RNAs were transcribed in the presence of guanosine monophosphorothioate (GMPS), which is incorporated only as the initiating nucleotide during in vitro transcription. An azidopheriacyl group was then conjugated to the unique sulfur located on the 5' phosphate. Exposure to UV light converts the azido group into a highly reactive nitrene and can result in intramolecular crosslinking. Individual crosslinked species (lariats) were identified by gel electrophoresis. 1000-fold dilution of the crosslinking reaction had no effect on the yield of crosslinks (3-10%), indicating the intramolecular nature of the reaction. The location of the crosslinked nucleotides was determined by reverse transcriptase primer-extension with individual crosslinked species as templates.

Molecular mechanics computer modeling was used to generate structures which incorporate the known secondary structure along with the positional constraints indicated by crosslinking and phylogenetic considerations. This procedure involves imposing structural constraints on all unfolded RNase P RNA chain via energy minimization and dynamics. Multiple equivalent models were generated and compared by superimposition in order to assess tile resolution of tile structure and to identify regions which were not yet well constrained by the available information. A model of the tertiary structure of RNase P RNA from E. coli will be presented.

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