Regulation of Chlorophyll/Apoprotein Synthesis

Mullet, John

Abstract

The long range goal of this research is to elucidate biochemical mechanisms which regulate the synthesis of chlorophyll-protein in chloroplasts of higher plants. The study of chlorophyll-protein synthesis provides special insight into the general process of membrane protein synthesis and cofactor assembly. This is because chlorophyll is highly fluorescent and provides a sensitive probe into the assembly process. Furthermore, because chlorophyll biosynthesis is light dependent in higher plants, the process can be easily controlled. The crystal structure of bacterial reaction centers containing the subunits L and M have been obtained. Bacterial reaction centers and l and M, are homologous to higher plant photosystem II reaction centers and their subunits, D1 and D2. Therefore, analysis of reaction center chlorophyll-protein synthesis has a strong biophysical and structural basis. Thr proposed research will focus ont he synthesis of D1 and D2, two membrane-bound, chlorophyll-binding proteins which form the reaction center of Photosystem II. D1 and D2 are encoded by the plastid genes psbA and psbD. The accumulation of these proteins is regulated through modulation of RNA stability, translation initiation, translation elongation (ribosome pausing), cofactor binding and activation of a special blue light inducible promoter. Three proteins, RNP95, RNP48 and RNP60, have been identified which bind to the psbA 5'-untranslated RNA. A specific binding site for RBNP95 has been defined. Regulation of psbA mRNA stability and translation initiation may occur through the action of these RNA binding proteins. The gene encoding RNP95 will be cloned and used to examine the role of RNP95 in modulating psbA mRNA stability and translation regulation in response to light intensity, light/dark cycles and chloroplast development. Plastid translation is also regulated at the level of translation elongation during light/dark cycles. This general regulation of elongation may modulate the amount of selective ribosome pausing observed during translation of the chlorophyll-protein D1. The mechanistic basis of light modulated translation elongation will be investigated by extract complementation and translation elongation assays. In addition, the relationship between ribosome pausing, chlorophyll binding, and stabilization of paused chlorophyll-apoprotein translation intermediates will be investigated. The chlorophyll-proteins D1 and D2 are damaged and turnover as a consequence of photochemistry especially at high light. In response, plants activate transcription from a blue light inducible promoter which transcribes psbD, the gene encoding D2. The biochemical mechanisms which regulates the blue light inducible psbD promoter will be investigated. Promoter constructs and DNA binding protein function will be analyzed using an in vitro transcription system. The role of reversible phosphorylation of DNA binding proteins and the blue light signal transduction pathway will be characterized.