The molecular chaperone DnaK is not recruited to translating ribosomes that lack trigger factor

Gisela Kramer, Vasanthi Ramachandiran, Paul M. Horowitz, Boyd Hardesty

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

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-tRNAf. 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.

Original languageEnglish (US)
Pages (from-to)63-70
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume403
Issue number1
DOIs
StatePublished - Jul 1 2002
Externally publishedYes

Keywords

  • Biotin-Met-tRNA
  • Coupled transcription/translation
  • DnaK
  • Fusidic acid
  • Nascent polypeptides
  • Rare codons
  • Translating ribosomes
  • Trigger factor

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Fingerprint

Dive into the research topics of 'The molecular chaperone DnaK is not recruited to translating ribosomes that lack trigger factor'. Together they form a unique fingerprint.

Cite this