Plant PolIII Biochemistry

University Of Missouri-Columbia, Columbia MO

Investigators

Abstract

Elegant studies of human and yeast RNA polymerase III (Pol III) transcription machineries have contributed extensively to our current understanding of the mechanism and regulation of gene expression. In contrast, the plant Pol III machinery is virtually undescribed. An in vitro transcription system from whole cell extracts of Arabidopsis thaliana suspension cell cultures has been developed that will permit purification and biochemical analysis of the plant Pol III machinery. This project has the following objectives:1) To assemble a biochemically defined in vitro plant Pol III transcription system capable of transcribing tRNA and other genes; of particular interest are the plant TFIIIC subunits, which are highly divergent between yeast and human, and that differ dramatically in structure and the capacity to acetylate chromatin; and 2) To define whether the plant TFIIIC has histone acetyltransferase (HAT) activity and if so, to determine whether such activity is important for Pol III dependent gene transcription in vitro and in vivo. Approaches that will be used include: 1) capturing the Pol III machinery on tRNALys1 gene fragments immobilized on magnetic beads; and 2) expressing and purifying Pol III machinery from transformed suspension cells. Components will be reconstituted into a robust in vitro system capable of transcribing tRNA genes. A variety of functional assays, in combination with reconstitution experiments, will be used to define the minimal plant Pol III machinery, and the properties conferred on the complex by accessory factors. Components of the plant Pol III and transcription factor complexes will be assayed for HAT activity, known to be important for modifying chromatin. If HAT activity is found, the responsible proteins and domains will be identified, mutated to eliminate the HAT activity, and the mutated proteins expressed in transgenic A. thaliana to assess the consequences of HAT inactivation on in vivo activity of Pol III and on plant growth and development. The results of this project are expected to significantly advance current understanding of gene expression in plants and other eukaryotes, supplying important information and tools for both further basic and applied research in the plant sciences. The project will support educational experiences for undergraduates and for a postdoctoral fellow, and will contribute to the environment responsible for training students for professional careers. These opportunities will be enhanced by institutional and other federal programs also intended to increase the diversity of underrepresented scientists in the workforce.