Optimization of protein synthesis in isolated higher plant chloroplasts. Identification of paused translation intermediates.
Protein synthesis in isolated, intact pea chloroplasts was optimized and compared to translation within chloroplasts in vivo. Many polypeptides labeled with [35S]methionine in isolated intact chloroplasts did not comigrate with polypeptides which were labeled within chloroplasts in vivo. Antibodies to the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) immunoprecipitated [35S]-labeled large subunit plus several lower-molecular-mass translation products of isolated chloroplasts. The lower-molecular-mass soluble translation products synthesized in pulse-labeled chloroplasts were converted into full-length large-subunit polypeptides during a subsequent chase period. This result suggests that many of the polypeptides observed in pulse-labeled chloroplasts are incomplete translation products which are the result of ribosome pausing at discrete points along chloroplast mRNAs. The pulse-chase technique was used to follow synthesis of the 34.5-kDa precursor of the psb A gene product and its processing to the mature 32-kDa polypeptide in isolated chloroplasts. Chloroplast translation profiles obtained using the pulse-chase assay were very similar to translation profiles obtained in vivo thus extending the utility of protein synthesis in isolated chloroplasts.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002736:Chloroplasts / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010940:Plant Proteins / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D013462:Sulfur Radioisotopes
D010940:Plant Proteins; D013462:Sulfur Radioisotopes; D000255:Adenosine Triphosphate; D003513:Cycloheximide; D008715:Methionine; D008274:Magnesium