The aim of this work was to understand the molecular mechanism of translation and the mechanism of translation termination, in particular. Cleavage of peptidyl-tRNA and peptide release terminates translation of mRNA on the ribosome. In prokaryotes, three release factors (RFs) are involved in this process. RF1 and RF2 recognise the three stop codons on mRNA and induce hydrolysis of the ester bond in peptidyl-tRNA. RF3 accelerates the rate of RF1 and RF2 recycling between ribosome in a GTP-dependent manner. We have clarified the mechanism of action of peptide release factor RF3. In the cell, free RF3 is in the GDP conformation. When RF3∙GDP binds to ribosome in complex with RF1 or RF2, these ribosome complexes act as guanine exchange factors for RF3 by inducing rapid dissociation of GDP. If, and only if, the peptide has been removed from tRNA, GDP is quickly replaced by GTP. Binding of GTP to RF3 induces a conformation of the factor with high affinity for the ribosome, which forces RF1 or RF2 to rapidly dissociate. Subsequent hydrolysis of GTP on RF3 induces a factor conformation with low affinity for the ribosome and rapid release of RF3∙GDP. It was further shown how the position of peptidyl-tRNA on the ribosome and the presence or absence of its peptide regulates the binding and GTPase activity of translation factors IF2, EF-G and EF-Tu. The result explains how idling GTPase hydrolysis and negative interference between different translation factors are minimized in living cells. The present biochemical observations, in conjunction with cryo-EM results, lead to new proposals for the role of hybrid sites in translocation of tRNAs, recycling of RF1 and RF2 by RF3 and recycling of post-termination ribosomes back to a new round of initiation.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-4569 |
| Date | January 2004 |
| Creators | Zavialov, Andrey |
| Publisher | Uppsala universitet, Institutionen för cell- och molekylärbiologi, Uppsala : Acta Universitatis Upsaliensis |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1104-232X ; 1017 |
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