title: The molecular chaperone DnaK is not recruited to translating ribosomes that lack trigger factor.
abstract: The molecular chaperone DnaK and trigger factor (TF), a ribosome-associated protein with folding activity, have been implicated in assisting nascent polypeptides to acquire a three-dimensional structure on Escherichia coli ribosomes. We asked whether ribosomes that lack trigger factor would recruit DnaK for synthesis and folding of nascent peptides. For these analyses, translating ribosomes with a homogeneous population of nascent peptides were isolated. Truncated forms of rhodanese and E. coli translation initiation factor 3 (IF3) were generated with tandem rare arginine codons in the coding sequence. These codons cause strong translational pausing during coupled transcription/translation in E. coli extracts, generating nascent polypeptides on ribosomes. Protein synthesis in the TF(-) extract was initiated with biotin-Met-tRNA(f). Ribosomes with nascent polypeptides were isolated by interaction of the N-terminal biotin with streptavidin on magnetobeads. These translating ribosomes that lack TF contain the molecular chaperone DnaK in considerably less than stoichiometric amounts.
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712, USA. g.kramer@mail.utexas.edu
authors.forename: Gisela
authors.initials: G
authors.lastname: Kramer
authors.forename: Vasanthi
authors.initials: V
authors.lastname: Ramachandiran
authors.forename: Paul M
authors.initials: PM
authors.lastname: Horowitz
authors.forename: Boyd
authors.initials: B
authors.lastname: Hardesty
mesh_terms: D001120:Arginine / Q000378:metabolism; D015153:Blotting, Western; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D039665:Prokaryotic Initiation Factor-3; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000378:metabolism*; D019809:Streptavidin / Q000494:pharmacology; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D010448:Peptide Initiation Factors; D010455:Peptides; D039665:Prokaryotic Initiation Factor-3; D019809:Streptavidin; D001120:Arginine; D013884:Thiosulfate Sulfurtransferase; C063842:dnaK protein, E coli