Structure, Function, and Evolution of RNA Maturation Enzymes in Plants

University Of Kansas Center For Research Inc, Lawrence KS

Investigators

Abstract

0094288 Peter Gegenheimer The transfer RNA (tRNA) species in a cell or organelle are synthesized as large precursor molecules. Their subsequent maturation is accomplished by enzymes that recognize and break a single, specific bond in each precursor RNA, thus separating the functional tRNA from the nonessential fragments. One of these enzymes, ribonuclease P (RNase P), is present in bacteria and archaea, and in the nuclei, mitochondria, and chloroplasts of all eukaryotic cells. The RNase P enzymes from all known bacteria contain a large RNA molecule and a small protein; it is the RNA component which performs the essential reaction. Although chloroplasts evolved from a cyanobacteria, which possess a bacterial RNase P, the spinach chloroplast enzyme appears to consist exclusively of protein. Recent studies demonstrated that the chloroplast enzyme uses a different catalytic mechanism than the bacterial RNase P. Transfer RNA maturation in chloroplasts is the first example of a single biochemical reaction that is catalyzed in different organisms by a protein or by an RNA enzyme. The goal of this project is to complete the characterization of this extraordinary enzyme. The major polypeptide associated with chloroplast RNase P activity is a novel nuclear-encoded RNA-binding protein that is related to bacterial signal transduction proteins as well as to the superfamily of dinucleotide-binding proteins. Preliminary modeling studies indicated a possible mechanism for proteins of this class to bind tRNA. This project will test this hypothesis by completing the biochemical purification of chloroplast RNase P. By treatment of purified enzyme preparations with antibodies specific for each of the polypeptides in this preparation, the investigators will identify which polypeptides are necessary for RNase P activity. cDNAs encoding these polypeptides, as well as cDNAs able to restore activity to a bacterial strain lacking RNase P, will be isolated and the respective proteins over-expressed and characterized in vitro. The results of these investigations will provide insight into how a single biochemical reaction can be accomplished by an RNA enzyme or by a protein enzyme.