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The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2003 (English)In: EMBO Journal, ISSN 0261-4189, Vol. 22, no 20, 5593-5601 p.Article in journal (Refereed) Published
Abstract [en]

The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non-cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2–GDPNP binds strongly to post-initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase-deficient mutants of eIF5B and IF2.

Place, publisher, year, edition, pages
2003. Vol. 22, no 20, 5593-5601 p.
Keyword [en]
G protein, IF2, initiation of translation, protein synthesis, ribosome
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-93386DOI: 10.1093/emboj/cdg525OAI: oai:DiVA.org:uu-93386DiVA: diva2:166846
Available from: 2005-09-01 Created: 2005-09-01 Last updated: 2009-10-09Bibliographically 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 83
Keyword
Cell and molecular biology, Initiation, Protein Synthesis, Initiation Factors, Light Scattering, Ribosomes, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
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
Opponent
Supervisors
Available from: 2005-09-01 Created: 2005-09-01Bibliographically approved

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