PMID:12061803
| Citation |
Kramer, G, Ramachandiran, V, Horowitz, PM and Hardesty, B (2002) The molecular chaperone DnaK is not recruited to translating ribosomes that lack trigger factor. Arch. Biochem. Biophys. 403:63-70 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1016/S0003-9861(02)00213-8 |
| Keywords |
Arginine/metabolism; Blotting, Western; Cell-Free System; Electrophoresis, Polyacrylamide Gel; Escherichia coli/metabolism; Escherichia coli Proteins; HSP70 Heat-Shock Proteins/metabolism; Peptide Initiation Factors/metabolism; Peptides/chemistry; Plasmids/metabolism; Prokaryotic Initiation Factor-3; Protein Biosynthesis; Protein Structure, Tertiary; Ribosomes/metabolism; Streptavidin/pharmacology; Thiosulfate Sulfurtransferase/metabolism; Transcription, Genetic |
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