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Cooperability
| Project leader |
Gruic-Sovulj Ita |
| Project co-leader: |
Dr. Boris Lenhard |
| Administering organization: |
Chemistry Department, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia
www.chem.pmf.hr
OIB:28163265527
Contact person: Antonio Šimunović, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, international@dekanat.pmf.hr
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| Partner Institution/Company: |
Imperial College London |
| Grant type: |
1B |
| Project title: |
The origin of amino acid specificity in editing class I aminoacyl-tRNA synthetases and cellular requirements for proofreading |
| Project summary: |
Expression of genetic information into pool of proteins represents biochemical foundation of life. Protein biosynthesis occurs at ribosomes where the information encoded as nucleic acids in mRNA is translated into amino acids that make up proteins. Aminoacyl-tRNA synthetases (aaRSs) are enzymes that catalyze ATP-dependent covalent pairing of tRNAs with their cognate amino acids. Formed aminoacyl-tRNAs bring the amino acids to the ribosomes where their incorporation into proteins is dictated by specific tRNA:mRNA interactions.
Tolerated error in translation is about one misincorporated amino acid per 104 translated codons. Yet, some aaRSs are unable to discriminate among structurally similar proteinogenic and non-proteinogenic amino acids with high accuracy in the synthetic reactions of aminoacylation. Therefore, they employ a diverse set of proofreading (editing) reactions to hydrolyze incorrectly formed intermediate and/or product. It was generally assumed that all editing reactions reside in the editing domain. However, we have recently shown that the synthetic site also has the capacity to edit aminoacyl-adenylate intermediate.
The aim of this project is to expand our understanding of aaRS proofreading; detailed mechanistic approach in studying synthetic site- and editing site-based proofreading reactions in leucyl-, isoleucyl- and valyl-tRNA synthetases from E. coli will be complemented with analysis of the cellular requirements for proofreading. It is evident now that the necessities for editing vary greatly depending on cellular physiology. E. coli is a facultative anaerobe that may experience oxygen deprivation in different ecological niches. Under these conditions, E. coli accumulates non-proteinogenic amino acid norvaline. Norvaline appears as a reasonably good substrate for the aforementioned aaRSs. Yet, its participation in protein synthesis seems to be largely prevented by aaRS editing activity. This sets an ideal system for the assessment of the requirements for proofreading under normal and mistranslation-prone (microaerobic) conditions. The proposed work further aims to uncover cellular responses and defects introduced by attenuation of proofreading. We are particularly interested in correlating norvaline mistranslation with transcriptional response in the cell. This will establish a link between requirements for protein quality control, and cell physiology and signaling.
This project will introduce bacterial genomics and transcriptomics methodology in Croatia and correlate it with the advanced biochemical studies. Proposed structural approach will strengthen mechanistic concepts of proofreading. Implementation of the novel methodologies and established collaborations with the top research groups in Europe will strengthen scientific competence of the Croatian group.
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| Hrvatski sažetak: |
Prijenos genetičke informacije iz DNA u molekule proteina predstavlja biokemijsku osnovu života. Biosinteza proteina događa se na ribosomu kada se informacija pohranjena u slijedu nukleotida mRNA prevodi u slijed aminokiselina koje grade protein. Aminoacil-tRNA-sintetaze (aaRS) su enzimi koji kataliziraju kovalentno sparivanje pripadnog para aminokiseline i tRNA uz utrošak ATP-a. Nastala aminoacilirana tRNA donosi aminokiselinu na ribosom, gdje njezinu ugradnju u protein diktiraju specifične interakcije koje se ostvaruju između tRNA i mRNA.
Prihvatljiva pogreška biosinteze proteina iznosi 1 u 10 000; dakle na 10 000 ugrađenih aminokiselina jedna je pogrešna. Neke aaRS ne mogu, s dovoljnim stupnjem točnosti, razlikovati pripadnu aminokiselinu od strukturno slične, ali nepripadne aminokiseline, u reakcijama aminoacilacije. Zbog toga su aminoacil-tRNA-sintetaze razvile specifične mehanizme popravka pogreške kojima hidroliziraju pogrešno stvoren međuprodukt i/ili finalni produkt. Dugo se smatralo da se sve hidrolitičke korektivne reakcije događaju u specijaliziranoj domeni za popravak. Ipak, naša nedavna istraživanja su pokazala da se hidroliza međuprodukta događa unutar mjesta gdje i nastaje, što je pokazalo da sintetsko aktivno mjesto ovih enzima također ima sposobnost hidrolitičke korekcije.
Cilj projekta je proširiti saznanja o mehanizmima popravka kojeg koriste aminoacil-tRNA-sintetaze. Mehanistički pristup s ciljem detaljne karakterizacije popravka u sintetskom mjestu, odnosno unutar domene za popravak, istraživat će se na primjeru tri enzima iz bakterije Escherichia coli: izoleucil-, valil- i leucil-tRNA-sintetaze. Paralelno s mehanističkim pristupom, istraživat će se i važnost mehanizama popravka u životu stanice. Danas je posve jasno da neophodnost popravka izuzetno ovisi o trenutnom fiziološkom statusu stanice. Bakterija E. coli je fakultativni anaerob, koji se tijekom života povremeno nalazi u okolini kojoj manjka kisika. U tim mikroaerobnim uvjetima dolazi do akumulacije neproteinogene aminokiseline, norvalina, u stanicama bakterije. Norvalin se pokazao kao relativno dobar supstrat aminoacil-tRNA-sintetaza, a njegova ugradnja u proteine izgleda da je spriječena hidrolitičkim popravkom navedenih enzima. Ovaj sustav predstavlja idealan sistem za proučavanje važnosti popravka u normalnim odnosno pogrešci sklonijim (mikroaerobnim) uvjetima. Cilj nam je spoznati stanične odgovore koji nastaju utišavanjem mehanizama popravka. Posebno nas zanimaju promjene u transkripcijskom odgovoru stanice izazvane pojačanom ugradnjom norvalina u proteine. Krajnji cilj ovog istraživanja je ustanoviti da li, i u kojoj mjeri, pogreške u biosintezi proteina utječu na staničnu fiziologiju i njezinu komunikaciju s okolinom.
Ovaj projekt omogućit će uvođenje bakterijske genomike i transkriptomike u hrvatsku znanstvenu zajednicu i njihovo povezivanje s naprednim biokemijskim istraživanjima. Metode strukturne biologije doprinijet će kvaliteti mehanističkih istraživanja popravka pogreške. Uspostavljanje novih metodologija, kao i predložene suradnje s vrhunskim znanstvenim grupama u Europi, značajno će doprinijeti jačanju znanstvene prepoznatljivosti hrvatskog tima s ovog projekta.
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| Amount requested from UKF: |
1.400.000,00 HRK |
| Amount of matching funding: |
350.000,00 HRK |
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