title: In vitro synthesis of the tryptophan operon leader peptides of Escherichia coli, Serratia marcescens, and Salmonella typhimurium.
abstract: We used an in vitro DNA-dependent protein-synthesizing system to demonstrate de novo synthesis of the leader peptide specified by the tryptophan (trp) operons of several bacterial species. Peptide synthesis was directed by self-ligated short restriction fragments containing the trp promoter and leader regions. Synthesis of leader peptides was established by demonstrating that they were labeled in vitro only by those amino acids predicted to be present in the peptides. Leader peptide synthesis was abolished by the addition of the Escherichia coli trp repressor. The E. coli trp leader peptide was found to be extremely labile in vitro; it had a half-life of 3-4 min. In a highly purified DNA-dependent peptide-synthesizing system, synthesis of the di- and tripeptides predicted from the Salmonella typhimurium trp operon leader sequence, fMet-Ala and fMet-Ala-Ala, also was observed. Using this dipeptide synthesis system, we demonstrated that translation initiation at the ribosome binding site used for trp leader peptide synthesis was reduced 10-fold when the transcript contained a segment complementary to the ribosome binding site.
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001665:Binding Sites; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D009690:Nucleic Acid Conformation; D009876:Operon; D010455:Peptides / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics*; D012706:Serratia marcescens / Q000235:genetics*; D014364:Tryptophan / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D014364:Tryptophan