Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Translation Initiation Factor IF1-Dependent Stimulation of 30 S Preinitiation Complex Formation: Rapid Isolation and fMEt-tRNA Binging Activity of IF1

  • CHOIK, SANG-YUN (Graduate School of Biotechnology, Korea University) ;
  • HYUN-JUNG KIM (Graduate School of Biotechnology, Korea University) ;
  • JUNG-IK YANG (Graduate School of Biotechnology, Korea University) ;
  • HYO-IL CHANG (Graduate School of Biotechnology, Korea University)
  • Published : 2001.12.01
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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 a functionally active factor with a high purity. In the present study a large quantity of active IF 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 centrifugatioin 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 subunitis, 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.

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References

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