Objetivo Termination of translation in eukaryotes has for a long time been less well understood than the initiation and elongation steps of protein synthesis. However, the termination step is critically important for formation of normal-sized proteins and to prevent premature termination or elongation beyond the stop codon of mRNA. Hydrolysis of peptidyl-tRNA takes place within the ribosome particle when the A site is occupied by the stop codon and polypeptide chain release factor of class 1 (eRF1 in eukaryotes, aRF1 in Archaea). The mechanism of translational termination consists of recognition of stop codon or stop-codon-rRNA complex by eRF1 followed by transfer of a signal to a peptidyl transferase centre to induce release of the nascent polypeptide from peptidyl-tRNA. The mechanism of both of these steps remains unclear in eukaryotic ribosomes.Here, translational termination will be studied by the currently available strategies, namely(i) crosslinking of eRF1, mRNA and rRNA(s) within the ribosome in the presence of tRNA and synthetic mRNA; two types of crosslinkers will be used: 4-thioU-substituted mRNAs and mRNAs with photoreactive groups attached to one of heterocyclic bases of a stop codon;(ii) after crosslinking the appropriate groups in eRF1 and rRNA(s) will be identified to reveal the chemical nature of contact area between eRF1, mRNA and rRNA(s);(iii) X-ray crystallography of crosslinked eRF1-stop codon complexes isolated from ribosomes in non-denaturing conditions (recently eRF1 has been successfully crystallised by CO); the aim of this part of the work is to visualise the chemical groups of eRF1 recognizing stop codons and to understand the stereospecific basis of this interaction;(iv) in parallel with item (iii) which might be difficult to achieve due to non-crystallisability of the eRF1*mRNA complex or too low yield of the complex, attempt will be made to co-crystallise in solution without ribosomes eRF1 and/or aRF1 with mRNA and rRNA fragments known from item (ii) to be in contact with eRF1 in the ribosomes;(v) an independent approach will be used based by means of site-directed mutagenesis of the N-domain of eRF1 protein proposed to contain the stop-codon recognition site;(vi) the essential part of the project will be crystallisation of aRF1 known to resemble eRF1 to reveal common and distinct features of the 3D structures of eRF1 and aRF1 probably functionally essential. Collectively, all these approaches should result in understanding how three stop codons are decoded in eukaryotes, the last unresolved issue of the genetic code in general.CO is very well experienced in crystallisation of proteins and X-ray analysis, CR1 and CR2 are for many years successfully engaged in cross linking studies between nucleic acids, nucleic acids and proteins applying a wide set of photoreactive nucleic acid derivatives. CR3 is one of the leading groups in Europe in translation termination research possessing all modern biochemical and molecular-genetic strategies needed to achieve the goals of the project. CR4 has successfully crystallised and resolved many nucleic acid-protein complexes, and this experience will be widely used to reach the goals of the project. Due to mutual methodological complementarity the cooperation between the groups will be very productive and allow the project to succeed. It is noteworthy that all teams are internationally recognized and well experienced in international collaboration that might be helpful for productivity and proper management of the project. Programa(s) IC-INTAS - International Association for the promotion of cooperation with scientists from the independent states of the former Soviet Union (INTAS), 1993- Tema(s) 3 - Chemistry OPEN - OPEN Call Convocatoria de propuestas Data not available Régimen de financiación Data not available Coordinador Institute of Cancer Research Aportación de la UE Sin datos Dirección 237 Fulham Road SW3 6JB London Reino Unido Ver en el mapa Coste total Sin datos Participantes (4) Ordenar alfabéticamente Ordenar por aportación de la UE Ampliar todo Contraer todo CNRS, Universités Paris 6 et 7 Francia Aportación de la UE Sin datos Dirección Tour 43, 2 Place Jussieu 75251 Paris cedex 05 Ver en el mapa Coste total Sin datos National Academy of Sciences of Ukraine Ucrania Aportación de la UE Sin datos Dirección Zabolotny str., 150 252143 Kiev Ver en el mapa Coste total Sin datos Russian Academy of Sciences Rusia Aportación de la UE Sin datos Dirección Vavilova, 32 117984 Moscow Ver en el mapa Coste total Sin datos Siberian Branch of Russian Academy of Sciences Rusia Aportación de la UE Sin datos Dirección pr. Lavrentieva, 8 630090 Novosibirsk Ver en el mapa Coste total Sin datos
