Structural Analysis of the Multisynthetase Complex

University Of Mississippi Medical Center, Jackson MS

Investigators

Abstract

0090539 Norcum Aminoacyl-tRNA synthetases control fidelity of protein biosynthesis by accurate pairing of amino acids and transfer RNAs. Nine of these enzymes and three auxiliary proteins are isolated from multicellular eukaryotes as a macromolecular complex. The major biological function of the particle is likely organization of the translational machinery. One of its constituents is the precursor of a cytokine released during apoptosis and so the multisynthetase complex may also link biological signaling, programmed cell death and inflammation. This project will test and refine the three domain model of the rabbit reticulocyte multisynthetase complex. Its three-dimensional structure will be calculated from electron microscopic images of frozen, unstained samples. The reference volume obtained by random conical reconstruction methods will be refined by projection mapping. Toward the overall goal of mapping the full protein topography within the particle, this project will focus on a subset selected to provide a broad test of the working model. Using natural tRNAs or in vitro transcripts of synthetic tRNA genes as specific probes, at least one enzyme within each domain will be located. Work will begin with the arginyl-, leucyl- and glutaminyl-tRNA synthetases. Electron microscopy will provide general protein locations, which will be refined by difference mapping of control and labeled structures. Probes will be visualized directly or by combination of chemical labeling with reporter molecules. So that positions within at least one domain are confirmed and oriented, electron microscopy will be combined with fluorescence resonance energy transfer measurement of distances between pairs of bound fluorophore-labeled tRNAs. Work will begin with the "base", which contains leucyl-, isoleucyl- and bifunctional glutamyl-/prolyl-tRNA synthetases. This project's results are fundamental to understanding eukaryotic protein biosynthesis at the molecular level and to elucidation of principles underlying intracellular organization. The information obtained will also provide information about differences between prokaryotic and eukaryotic protein biosynthesis, a mechanism of autoimmunity, and a route to cell death.