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Ribosomal recycling by the interplay of RRF and three conformations of EFG
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
Manuscript (Other academic)
URN: urn:nbn:se:uu:diva-93393OAI: oai:DiVA.org:uu-93393DiVA: diva2:166853
Available from: 2005-09-01 Created: 2005-09-01 Last updated: 2010-01-13Bibliographically approved
In thesis
1. Mechanism and Regulation of Initiation of Protein Synthesis in Eubacteria
Open this publication in new window or tab >>Mechanism and Regulation of Initiation of Protein Synthesis in Eubacteria
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Regleringen av proteinsyntesens initiering i Eubacteria och dess mekanistiska förklaring
Abstract [en]

Initiation of protein synthesis in E.coli 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.

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-tRNAfMet. 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.

The interplay between initiation factors in the rate and accuracy of protein synthesis was thoroughly studied. Using fMet-tRNAfMet (initiator tRNA), Met-tRNAfMet (non-formylated initiator tRNA) and Phe-tRNAPhe (elongator tRNA), we showed that the major player in the accuracy is IF2 through recognizing the formyl group on fMet-tRNAfMet, while IF3 acts by increasing both the on- and off-rate of tRNA from 30S pre-initiation complexes.

Collectively, these novel results describe a comprehensive model of initiation of protein synthesis. In this model, initiation factors increase the rate of fMet-tRNAfMet binding to 30S subunits, subsequently; the stabilization of fMet-tRNAfMet 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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 71 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 83
Cell and molecular biology, Initiation, Protein Synthesis, Initiation Factors, Light Scattering, Ribosomes, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-5907 (URN)91-554-6320-7 (ISBN)
Public defence
2005-09-30, B 42, BMC, Husargatan 3, UPPSALA, 10:00
Available from: 2005-09-01 Created: 2005-09-01Bibliographically approved

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