Molecular genetic analysis of light-regulated translation in Chlamydomonas reinhardtii

The biogenesis of the photosynthetic apparatus within the chloroplast requires the close cooperation of the nuclear and chloroplast genomes. Synthesis of the proteins required to assemble Photosystem II of the chloroplast is keyed to light. Genetic analysis in the unicellular green algae Chlamydomonas reinhardtii has predicted that nuclear encoded translational activators act in the chloroplast on the 5{dollar}spprime{dollar} untranslated regions of chloroplast encoded mRNAs to affect light regulated translation of these messages. Biochemical analysis has identified several proteins that seem to fit the role of translational activator. In this thesis, several nuclear mutants of C. reinhardtii that affect translation of specific chloroplast mRNAs are analyzed in order to develop a further understanding of the mechanisms involved in this process. The F35 mutation is used to define initiation of translation as a key point for regulation of the chloroplast encoded psbA mRNA. The reduction in accumulation of putative translational activators that correlates with loss of translational initiation supports the role suggested for these RNA binding proteins. Elongation is also defined as a possible step at which translation can be regulated, as shown for the psbD mRNA in the nac1-18 mutant. This mutant, though apparently affected only in elongation, also shows an effect on the putative translational activators. Additionally, out of a library of 100,000 transformants, a set of five nuclear mutants that affect psbA translation were identified. All five of these mutants seem to affect translational initiation of psbA, suggesting that this is the primary regulatory step. One of these mutants, hf149, brings together the genetic and biochemical data regarding translational activators. hf149 does not translate the psbA mRNA because of an apparent lack of translational initiation. In this mutant, one of the putative translational activators (RB47) does not accumulate at any detectable level, suggesting that the loss of this protein directly results in loss of translational initiation. The identification of RB47 as a member of the poly(A) binding protein family of proteins supports the role that RB47 is presumed to play in the chloroplast.