title: Development of a chaperone-deficient system by fractionation of a prokaryotic coupled transcription/translation system.
abstract: A coupled transcription/translation system from Escherichia coli has been developed that is very active for protein synthesis but deficient in chaperone proteins. The chaperones GroEL and DnaK distribute during the first ultracentrifugation of the E. coli extract partially with the ribosomes and partially in a liquid, viscous fraction above the ribosomes. Gel filtration chromatography of this latter fraction separates GroEL and DnaK as high-molecular-weight components from the peak of activity of the factors required for protein synthesis. Thus, a chaperone-deficient transcription/translation system can be reconstituted with salt-washed ribosomes. This chaperone-deficient system was used to study synthesis and folding of bacterial dihydrofolate reductase and of rhodanese, a eukaryotic mitochondrial enzyme. Both enzymes were synthesized from nonlinearized plasmids that had the respective coding sequence under the SP6 promoter. Both enzymes were synthesized in active form and with high specific activity in the chaperone-deficient system. A high proportion, about 20% of newly synthesized dihydrofolate reductase and about 50% of rhodanese, stayed with the ribosomes after coupled transcription/translation. No enzymatic activity was detected in this fraction. Addition of the chaperones GroEL/ES and DnaK resulted in a shift of rhodanese molecules from the ribosomes into the supernatant fraction. Nearly all molecules in the supernatant were enzymatically active.
authors.affiliation: Department of Chemistry & Biochemistry, University of Texas at Austin 78712.
authors.forename: W
authors.initials: W
authors.lastname: Kudlicki
authors.forename: M
authors.initials: M
authors.lastname: Mouat
authors.forename: J P
authors.initials: JP
authors.lastname: Walterscheid
authors.forename: G
authors.initials: G
authors.lastname: Kramer
authors.forename: B
authors.initials: B
authors.lastname: Hardesty
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000502:physiology*; D002458:Cell Fractionation; D018834:Chaperonin 60; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000032:analysis / Q000172:deficiency* / Q000502:physiology*; D014176:Protein Biosynthesis; D017510:Protein Folding; D012270:Ribosomes / Q000502:physiology; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D013762:Tetrahydrofolate Dehydrogenase; D013884:Thiosulfate Sulfurtransferase; C063842:dnaK protein, E coli