Translation initiation factor IF1-dependent stimulation of 30 S preinitiation complex formation: Rapid isolation an fMet-tRNA binding activity of IF1

Sang Yun Choi, Hyun Jung Kim, Jung Ik Yang, Hyo Il Chang

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Abstract

Translation initiation in prokaryotes involves the formation of a 30 S preinitiation complex, in which translation initiation factors play a role in the stimulation of fMet-tRNA (fMet) binding. However, the specific function and precise mechanism of initiation factor IF1 are still unclear. One reason hindering the study of IF1 is the difficulty in obtaining a functionally active factor with a high purity. In the present study, a large quantity of active IF1 was rapidly purified, obtained by the overexpression of the infA gene, and then used for a functional study. The induction of infA did not appreciably affect the growth rate of the protease-deficient strain E. coli AR68 harboring the IF1 overproducing plasmid. The level of IF1 obtained was approximately 1-2% of the total cell protein, which enabled the yield of highly purified IF1 (>98% pure) to be increased to 0.15 mg of IF1/g of cells. The IF1 was isolated within one day by the centrifugation of the ribosomal washed fraction, by ammonium sulfate fractionation, chromatography on batch of phosphocellulose, and FPLC Mono S. The overexpressed IF1 was found to be comparable to the factor isolated from normal cells, as determined by migration in NEPHGE/SDS 2-D gels. For binding of fMet-tRNA(fMet) to the 30 S ribosomal subunits, relatively high levels of binding were obtained when IF2 was present. The addition of IF1 up to 110 pmol proportionally stimulated the binding to a variable extent. This IF1-dependent stimulation of the 30 S preinitiation complex formation demonstrated that IF1 would appear to be exclusively essential for promoting the initiation phase of protein synthesis.

Original languageEnglish
Pages (from-to)986-993
Number of pages8
JournalJournal of microbiology and biotechnology
Volume11
Issue number6
Publication statusPublished - 2001 Dec 1

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Keywords

  • IF1
  • NEPHGE/SDS 2-D gel
  • Ribosome
  • fMet-tRNA binding
  • infA

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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