title: Optimization of protein synthesis in isolated higher plant chloroplasts. Identification of paused translation intermediates.
abstract: 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.
mesh_terms: 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
chemical_list: D010940:Plant Proteins; D013462:Sulfur Radioisotopes; D000255:Adenosine Triphosphate; D003513:Cycloheximide; D008715:Methionine; D008274:Magnesium