Abstract

Translation of specific mRNAs of the semiautonomous genetic systems of mitochondria and plastids is controlled by the products of nuclear genes. Previous studies have characterized a particularly complex example of this control pertaining to translation of the chloroplast psbC mRNA in the unicellular eukaryotic alga Chlamydomonas reinhardtii . psbC translation is controlled by interactions between its 547 nucleotides 5 ' untranslated region and the product of the nuclear loci TBC1, TBC2 and TBC3. This thesis extends our understanding of psbC translational control from the genetic level to the biochemical level. RNA structures involved in translation and its control by the nuclear gene products were identified using chemical and enzymatic probing experiments, oligonucleotide hybridization probing experiments, circular dichroism, and reverse transcription inhibition experiments. Results strongly support a large stem-loop secondary structure that was proposed over 13 years ago. Additional evidence indicates this structure forms a pseudoknot structure by hybridization of its apical loop with a proximal sequence. A mutation that removes two bulges from the stem of this structure drastically alters its structure. Evidence supporting 6 smaller stem-loop structures was obtained. Sequence elements and RNA structures in the psbC 5 ' untranslated region that interact with factors encoded or controlled by TBC1, TBC2 and TBC3 were identified by in vivo dimethyl sulfate protection analysis. Control by TBC1 and TBC3 is mediated by specific sequences that are dispersed throughout the 5 ' untranslated region. The TBC2 mutant has a DMS protection within the coding sequence. Models of translational control based on these findings are proposed