<p>Initiation of protein synthesis in <i>E.coli </i>involves several steps, which lead to the formation of the first peptide bond. This process requires three initiation factors: IF1, IF2 and IF3. Using a novel technique of combined light scattering and stopped-flow, we elucidated the importance of IF2•GTP conformation for the recruitment of 50S to 30S pre-initiation complex. Moreover, GTP hydrolysis is essential for IF2 release and later binding of ternary complex. Interestingly, a switch in IF2 affinity to G-nucleotides is induced during 30S pre-initiation complexes formation. </p><p>We found that IF1, previously with unknown functions in vitro, increases the rate of naked 70S dissociation by a factor 80 and acts as a fidelity factor in preventing 70S formation containing elongator tRNA instead of fMet-tRNA<sup>fMet</sup>. We showed that RRF/EFG/IF3 split both naked and post-termination complexes while IF1/IF3 split only naked ribosomes. The mechanisms of action of RRF/ EFG, the order of their binding to 70S, as well as, the three different conformation of EF-G on the ribosomes are emphasized. Interestingly, 70S formation rate is dependent on the concentration of IF3 and not linear with 50S subunits concentration. We demonstrated that the rate-limiting step in 70S formation is IF3 dissociation from 30S complexes.</p><p>The interplay between initiation factors in the rate and accuracy of protein synthesis was thoroughly studied. Using fMet-tRNA<sup>fMet</sup> (initiator tRNA), Met-tRNA<sup>fMet </sup>(non-formylated initiator tRNA) and Phe-tRNA<sup>Phe</sup> (elongator tRNA), we showed that the major player in the accuracy is IF2 through recognizing the formyl group on fMet-tRNA<sup>fMet</sup>, while IF3 acts by increasing both the on- and off-rate of tRNA from 30S pre-initiation complexes.</p><p>Collectively, these novel results describe a comprehensive model of initiation of protein synthesis. In this model, initiation factors increase the rate of fMet-tRNA<sup>fMet</sup> binding to 30S subunits, subsequently; the stabilization of fMet-tRNA<sup>fMet</sup> by IF2 increases the rate of IF3 dissociation. Later, IF2 in GTP conformation allows 50S docking to 30S pre-initiation complex free of IF3 followed by GTP hydrolysis allowing IF2 release for ternary complex to bind and start elongation of protein synthesis. </p>
| Identifer | oai:union.ndltd.org:UPSALLA/oai:DiVA.org:uu-5907 |
| Date | January 2005 |
| Creators | Antoun, Ayman |
| Publisher | Uppsala University, Department of Cell and Molecular Biology, Uppsala : Acta Universitatis Upsaliensis |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, text |
| Relation | Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1651-6214 ; 83 |
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