• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.986-993
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• 2001

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.

• Journal of the Korean Magnetic Resonance Society
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• v.9 no.2
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• pp.110-121
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• 2005

E.coli methionyl tRNA synthetase consist of 676 amino acids and plays a key role in initiation of protein synthesis. The native form of this enzyme is a homodimer, but the monomeric enzyme truncated approximately C-terminal 120 amino acids retains the full enzymatic activities. X-ray crystal structure of the active monomeric enzyme shows that it has two domains. The N-terminal domain is thought to be a binding site for acceptor stem of tRNA, ATP, and methionine. The C-terminal domain is mainly a-helical and makes an interaction with the anticodon of $tRNA^{Met}$. Especially it is suggested that the region of helix-loop-helix including the tryptophan residue at the position 461 may be the essential for the interaction with anticodon of $tRNA^{Met}$. In this work the structure and function of E. coli methionyl-tRNA synthetase was studied by spectroscopic method (NMR, CD, Fluorescence). The importance of tryptophan residue at the position 461 was investigated by fluorescence spectroscopy. Tryptophan 461 is expected to be an essential site for the interaction between E. coli methionyl-tRNA synthetase and E. coli $tRNA^{Met}$. Proton and heteonuclear 2-dimensional NMR spectroscopy were also used to elucidate the protein-tRNA interaction.