FUNCTION OF 3'-NONCODING SEQUENCES AND STOP CODON USAGE IN EXPRESSIONOF THE CHLOROPLAST PSAB GENE IN CHLAMYDOMONAS-REINHARDTII

The rate of mRNA decay is an important step in the control of gene exp ression in prokaryotes, eukaryotes and cellular organelles. Factors th at determine the rate of mRNA decay in chloroplasts are not well under stood. Chloroplast mRNAs typically contain an inverted repeat sequence within the 3' untranslated region that can potentially fold into a st em-loop structure. These stem-loop structures have been suggested to s tabilize the mRNA by preventing degradation by exonuclease activity, a lthough such a function in vivo has not been clearly established. Seco ndary structures within the translation reading frame may also determi ne the inherent stability of an mRNA. To test the function of the inve rted repeat structures in chloroplast mRNA stability mutants were cons tructed in the psaB gene that eliminated the 3' flanking sequences of psaB or extended the open reading frame into the 3' inverted repeat. T he mutant psaB genes were introduced into the chloroplast genome of Ch lamydomonas reinhardtii. Mutants lacking the 3' stem-loop exhibited a 75% reduction in the level of psaB mRNA. The accumulation of photosyst em I complexes was also decreased by a corresponding amount indicating that the mRNA level is limiting to PsaB protein synthesis. Pulse-chas e labeling of the mRNA showed that the decay rate of the psaB mRNA was significantly increased demonstrating that the stem-loop structure is required for psaB mRNA stability. When the translation reading frame was extended into the 3' inverted repeat the mRNA level was reduced to only 2% of wild-type indicating that ribosome interaction with stem-l oop structures destabilizes chloroplast mRNAs. The non-photosynthetic phenotype of the mutant with an extended reading frame allowed us to t est whether infrequently used stop codons (UAG and UGA) can terminate translation in vivo. Both UAG and UGA are able to effectively terminat e PsaB synthesis although UGA is never used in any of the Chlamydomona s chloroplast genes that have been sequenced.