| Because of its endosymbiotic origin, the chloroplast protein synthesis machinery generally resembles that of prokaryotes. However, there are some striking differences, such as the lack of functional Shine-Dalgarno (SD) sequences in many chloroplast mRNAs. In Escherichia coli, the SD sequence, located about seven nucleotides upstream of the initiation codon, base pairs with a complementary sequence at the 16S rRNA 3' end to facilitate ribosome positioning and initiation. Previous studies have shown that the site of chloroplast initiation is fixed, and the sequence of the initiation codon contributes to translational efficiency. In vivo site-directed mutagenesis in the unicellular green alga Chlamydomonas reinhardtii was used to investigate other chloroplast mRNA sequences required for efficient initiation. The three nucleotides immediately upstream of the initiation codon (the --1 triplet) and the --1 nucleotide in particular were found to have a profound effect on the efficiency of translation under certain conditions. This sequence is also known to be important for translation in E. coli and for prokaryotic translation in vitro. Site-directed mutagenesis was used to test in vivo two hypotheses. First, the --1 triplet might base pair with a region of the 16S rRNA known as the 530 loop. Second, the --1 nucleotide might base pair with the nucleotide downstream of the initiator tRNA anticodon, forming an extended codon-anticodon interaction. --1 triplet mutations as well as compensatory mutations in the anticodon loop of the initiator tRNA suggested that the extended codon-anticodon interaction, as opposed to the 530 loop interaction, occurs in chloroplast translation initiation, providing the first in vivo evidence in support of this hypothesis.; Another distinctive feature of chloroplast translation is the requirement for nucleus-encoded translational activators. The possibility that MCD2, a factor involved in Chlamydomonas chloroplast petD mRNA stability, might also play a role in translation of petD mRNA or other chloroplast mRNAs was investigated using molecular and genetic analyses. |
