Dr. John Boynton and his collaborator Dr. Nick Gillham study several aspects of chloroplast development in the unicellular, green alga Chlamydomonas. The chloroplast of Chlamydomonas provides ATP and reduced carbon compounds. The chloroplast genome encodes many of the proteins needed and collaborates with proteins encoded in the nucleus that are imported into the chloroplast. Chloroplasts in Chlamydomonas and mitochondria in Saccharomyces are clearly the two premier experimental organisms for studying organelles. This proposal has three main goals. These are the further characterization of mechanisms involved in translational accuracy; translational control of gene expression in chloroplasts, and an examination of the role of recombination in repair in the chloroplast, which is a new area of research for this group. Accuracy of translation in E. coli has been studied for sometime and mutations that alter it have been isolated. For example, mutations in several small ribosomal proteins that bind to the ribosomal RNA alter translational accuracy. Translational accuracy in Chlamydomonas will be studied by the isolation of mutations in S4, which has been one of the key genes studied in E. coli. These mutations will be used in a structure/function study in collaboration with Dr. S. White. One idea that will be examined is whether translational accuracy is determined by sites in S4 that contact RNA based on the crystallized structure of the protein from B. stearothermophilus and rRNA binding data from E. coli. In the last grant period, suppressors of a streptomycin-dependent mutant in S12 were isolated. Some of these are in S4, while the map position on the chloroplast genome of the others is not yet known. These suppressor mutations may define additional components needed for translational accuracy. The genes will be identified by complementation, sequenced, and antibodies produced to allow a comparison with E. coli and yeast. If the suppressor mutations are not ribosomal proteins, the genes will be disrupted using the aadA gene. This gene confers resistance to spectinomycin and streptomycin and its use in Chlamydomonas was developed during the last grant period. Nuclear-encoded genes sr-1 and spr-1 may encode an unspecified ribosomal protein and S5, respectively. Attempts are underway to tag these loci with transforming DNA and to clone them. Finally, mutations that alter translational accuracy in vivo will be studied by in vitro proofreading assay on a poly-U template for leucine or isoleucine incorporation. In the second goal, translational regulation of ribosomal proteins and photosynthetic proteins will be examined. The RNAs for these two groups of proteins are selectively translated. The experiments proposed will begin to address the role of the 5'UTR. Shine-Delgarno-like (SD) sequences do not show the optimal spacing that is observed in prokaryotic genes. This positioning will be examined in a series of experiments. These potential SD sequences can be anywhere from -10 to -114 from the AUG codon. The S4 gene has no SD sequence within 200 basepairs of the AUG. The first questions that will be asked are whether the SD sequences are functional. A construct will be made with promoters and 5'UTRs from four different genes (rps7, atpE, rsp4, and atpB) and a reporter construct with the aadA gene and the 3' end of the rbcL gene. They will be placed in a noncoding region of the chloroplast genome. Both enzyme activity assays and aadA protein levels can be assayed. Ribosome binding will be assayed by extension-inhibition (toeprinting) with ribosomes from E. coli and from Chlamydomonas. Initiator tRNA will be from E. coli. In addition, proteins that bind to the 5'UTR will be identified in binding assays. Binding sites in 5'UTRs will be examined by deletion constructs. In the final part of this section, the difference in translation of ribosomal proteins and photosynthetic proteins when chloroplast protein synthesis is compromised will be studied. Both cis-acting and trans-acting factors will be examined. In the third goal, the biological role of chloroplast recombination will be examined. They suggest that its role is to repair damaged or mutated DNA. Dominant mutations in E. coli RecA will be examined for their effects on chloroplast genomes. They report in a manuscript submitted that Chlamydomonas cells show reduced survival when exposed to DNA damaging agents, decreased repair and recombination when dominant RecA genes are introduced into the chloroplast. They will examine the effects of introducing the gam gene from phage lambda. This protein inhibits the RecBCD enzyme in E. coli. This will be one test of the presence of a RecBCD-like set of genes in Chlamydomonas. In the long term, they would like to identify nuclear genes that encode recombination enzymes by tagging and mutating nuclear genes.
