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-Ihl Chang

Research output: Contribution to journalArticle

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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Peptide Initiation Factors
Proteins
Centrifugation
Ammonium Sulfate
Fractionation
Chromatography
Escherichia coli
Ribosome Subunits
Plasmids
Peptide Hydrolases
Gels
Genes
Growth
fMet-tRNA(fMet)
formylmethionine-tRNA
Transfer RNA
phosphocellulose
Mono-S
Sulfates

Keywords

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

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Microbiology

Cite this

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title = "Translation initiation factor IF1-dependent stimulation of 30 S preinitiation complex formation: Rapid isolation an fMet-tRNA binding activity of IF1",
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.",
keywords = "fMet-tRNA binding, IF1, infA, NEPHGE/SDS 2-D gel, Ribosome",
author = "Sang-Yun Choi and Kim, {Hyun Jung} and Yang, {Jung Ik} and Hyo-Ihl Chang",
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T2 - Rapid isolation an fMet-tRNA binding activity of IF1

AU - Choi, Sang-Yun

AU - Kim, Hyun Jung

AU - Yang, Jung Ik

AU - Chang, Hyo-Ihl

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N2 - 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.

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