Aminoacyl-tRNA synthetases as gatekeepers of the standard genetic code

Funding agency
Croatian Science Foundation - Official project page

Duration
01.03.2017. - 28.02.2021.

Project leader
dr. sc. Ita Gruic-Sovulj

Partnering institutions
University of Zagreb, Faculty of Science

Summary
The genetic code provides the basis for translation of genetic information into functional proteins. Codon assignments are established by aminoacyl-tRNA synthetases (aaRS), which couple amino acids to their cognate tRNAs. Mistakes in translation are generally kept low, in part by inherent aaRS hydrolytic editing. The extent to which mistranslation occurs presents a fundamental question in basic research with impact on medicine and biotechnology. AaRS quality control mechanisms also act as gatekeepers of the standard genetic code and prevent infiltration of natural but non-coded amino acids. Consequently, they present an obstacle for synthetic amino acid translation in rational protein design. Here we propose to investigate the synthetic and editing mechanisms of three aaRSs responsible for translation of amino acids that build the protein core: leucine, valine and isoleucine. We will compare the catalytic principles and gained specificity of the enzymes’ synthetic and editing sites against proteinogenic and non-coded amino acids. The cellular toxicity and stress responses of induced non-canonical mistranslation will be addressed. The goal is to improve our understanding of the basis for selection of the natural amino acid alphabet, and how violation of the coding principles influences cell physiology. Mechanistic insight will extend our ability to apply fluorinated amino acids in protein rational design. A possible link between altered translational fidelity and adaptation to environmental stress will be sought. This research couples biochemistry and chemical biology through the interest in reassignment of the genetic code. The assembled expertise through the international (Prof. Budiša, TU Berlin, Prof. Anderluh, NIC, Ljubljana, and Prof. Maček, Proteome Center Tübingen) and national (Dr. Vianello, RBI, Zagreb) collaborators will strongly enhance the capacity of our group, which is well-recognized in the field of protein translation, to conduct this research.

Non-canonical roles of aminoacyl-tRNA synthetases

Funding agency
Croatian Science Foundation

Duration
02.01.2013.- 01.04.2016.

Project leader
dr. sc. Ita Gruic-Sovulj

Partnering institutions
University of Zagreb, Faculty of Science
Institute Ruđer Bošković, Zagreb
University Hospital Centre, Zagreb
ETH Zürich, Institute of Molecular Biology and Biophysics 
The Ohio State University, Columbus

The origin of amino acid specificity in editing class I aminoacyl-tRNA synthetases and cellular requirements for proofreading

Funding agency
Unity Through Knowledge Fund

Duration
15.10.2013. - 14.10.2015.

Project leader
Dr. Ita Gruić Sovulj, associate professor, Faculty of Science, University of Zagreb

Project co-leader
Dr. Boris Lenhard, Imperial College London

Collaborators
Dr. Stephen Cusack, European Molecular Biology Laboratory, Grenoble
Dr. Mario Cindrić, Institute Ruđer Bošković, Zagreb

Partnering Institutions
Imperial College London
European Molecular Biology Laboratory, Grenoble
Institute Ruđer Bošković, Zagreb

Summary
Aminoacyl-tRNA synthetases (aaRSs) covalently pair tRNAs with their cognate amino acids for ribosomal protein synthesis. Some aaRSs are unable to discriminate against similar proteinogenic and non-proteinogenic amino acids at the synthetic reactions of aminoacylation with the accuracy above threshold levels of translational fidelity (1 in 10 000). Therefore, they employ a diverse set of proofreading (editing) reactions to hydrolyze incorrectly formed intermediate and/or product. It has been 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 editing reactions in leucyl-, isoleucyl- and valyl-tRNA synthetases from E. coli will be complemented with analysis of the cellular requirements for proofreading. 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, whose participation in protein synthesis is 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. Our further goal is to uncover cellular responses and defects introduced by attenuation of proofreading. We are particularly interested in correlating mistranslation with transcriptional response in the cell. This will establish a link between requirements for protein quality control, and cell physiology and signaling.