UKF 1B grant 8/13

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


Funding: Unity Through Knowledge Fund



15.10.2013. - 14.10.2015.


Project financial value

UKF funding: 1.400.000 kn
Total UKF + matching funding: 1.750.000 kn


Project leader

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


Project co-leader

Dr. Boris Lenhard, Imperial College London



Dr. Stephen Cusack, European Molecular Biology Laboratory, Grenoble

Dr. Mario Cindrić, Institute Ruđer Bošković, Zagreb


Team members

Nevena Cvetešić, Faculty of Science, University of Zagreb, Ph.D. student

Morana Dulić, Ph.D., Faculty of Science, University of Zagreb,postdoctoral fellow

Andres Palencia, Ph.D., European Molecular Biology Laboratory, Grenoble, postdoctoral fellow

UKF funded early-stage researcher:
Mirna Biluš, Faculty of Science, University of Zagreb, Ph.D. student


Partnering Institutions

Imperial College London

European Molecular Biology Laboratory, Grenoble

Institute Ruđer Bošković, Zagreb



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.



  • Cvetesic N., Dulic M., Sostaric N., Bilus M., Lenhard B., Gruic-Sovulj I.  Evolutionary Adaptability of the Synthetic Site Is Not Constrained by tRNA-Dependent Pre-Transfer Editing in Isoleucyl-tRNA Synthetase, submitted to J. Biol. Chem




Conferences and workshops

  • Nevena Cvetesic, Andres Palencia, Stephen Cusack, Ita Gruic-Sovulj: The prime function of Escherichia coli LeuRS CP1 domain is editing of norvaline not isoleucine
    Barcelona BioMed Conference Gene Translation: Fidelity and Quality Control, Barcelona, Spain 2.-4.12. 2013. Poster presentation.
  • Nevena Cvetesic, Ita Gruic-Sovulj: Escherichia coli LeuRS CP1 domain operates to exclude norvaline from the cellular proteome
    Workshop on Phosphopeptide enrichment, labeling strategies and proteomics data analysis, Split, Croatia 22-23.10. 2013. Poster presentation.
  • Nevena Cvetesic, Mirna Bilus,  Andres Palencia, Morana Dulic, Stephen Cusack, Ita Gruic-Sovulj: Class I aaRS quality control mechanisms preserve canonical translation in Escherichia coli
    25th tRNA Conference 2014, Kyllini, Greece 21.-25.09.2014. Plenary talk.
  • Nevena Cvetesic, Boumediene Soufi, Maja Semanjski, Boris Macek, Ita Gruic-Sovulj: Quantitative analysis of the Escherichia coli proteome in the absence of LeuRS proofreading
    25th tRNA Conference 2014, Kyllini, Greece 21.-25.09.2014. Poster presentation.
  • Morana Dulic, John Joseph Perona, Ita Gruic- Sovulj: A single synthetic site residue modulates partitioning of pre- and post- transfer editing pathways in overall editing by isoleucyl-tRNA synthetase from Escherichia coli 
    Congress of the Croatian Society of Biochemistry and Molecular Biology - HDBMB2014: "The Interplay of Biomolecules". Zadar, Croatia 24.-27.09.2014. Oral presentation.
  • Mirna Bilus, Ita Gruic-Sovulj: IleRS eliminates norvaline from Escherichia coli proteome via pre- and post-transfer editing pathways
    12th Greta Pifat Mrzljak International School of Biophysics: Biomolecular complexes and assemblies, Primosten, Croatia, 27.09.-06.10.2014. Poster presentation. 
  • Nevena Cvetesic, Andres Palencia, Stephen Cusack, Ita Gruic-Sovulj: Reassessment of LeuRS discriminatory power unveils norvaline as a prime quality control target
    12th Greta Pifat Mrzljak International School of Biophysics: Biomolecular complexes and assemblies, Primosten, Croatia, 27.09.-06.10.2014. Oral and poster presentation.
  • Nevena Cvetesic, Andres Palencia, Stephen Cusack, Ita Gruić-Sovulj: The prime function of leucyl-tRNA synthetase proofreading is prevention of the non-canonical mistranslation in Escherichia coli
    XXIV Croatian meeting of chemists and chemical engineers, Zagreb, 21.04.-24.04.2015. Oral presentation.
  • Ita Gruic-Sovulj: Translational quality control mechanisms that eliminate the non-canonical norvaline from the genetic code in Escherichia coli.
    Bacterial networks (BACNET 15), Sant Feliu de Guixols, 09.05.-14.05.2015. Oral presentation.
  • Morana Dulic : Amino acid specifi city of the Escherichia coli leucyl-tRNA synthetase editing domain.
    FEBS Congress: The Biochemical Basis of Life, Berlin, 04.07.-09.07.2015. Poster presentation.
  • Ita Gruic-SovuljAminoacyl-tRNA synthetase editing preserves the canonical genetic code.
    FEBS3+Meeting, Portorož, 16.09.-19.09.2015. Invited lecture.
  • Morana Dulic, Andres Palencia, Nevena Cvetešić, Stephen Cusack, Ita Gruić-Sovulj: Specificity of leucyl-tRNA synthetase’s editing domain is determined by the chemical step of proofreading.
    FEBS3+Meeting, Portorož, 16.09.-19.09.2015. Oral presentation.


  • Ita Gruic Sovulj: Synthetic and proofreading mechanisms of class I aminoacyl-tRNA synthetases
    Technische Universität Berlin, Berlin, Germany 26.06.2015.
  • Ita Gruic-Sovulj: Quality control in aminoacyl-tRNA synthesis preserves the canonical translation in Escherichia coli
    Preoteome Center Tübingen, University of Tübingen, Germany 25.03.2014.
  • Boris Lenhard: Promoter classes and promoter grammars in vertebrate genomes
    Department of chemistry, Faculty of science, University of Zagreb, Croatia 09.10.2014.


Milestones reached

We have characterized the origin of LeuRS specificity against isoleucine and showed that high level of specificity is established via both weak ground-state binding and the decreased rates of the chemical steps. We clearly demonstrate that previous work by other groups was in error because of the unrecognized contamination of the commercial isoleucine samples with cognate leucine. The major conclusion is that editing of isoleucine is not required during Escherichia coli protein synthesis. This finding has important implications for the development of resistance to antimicrobial agents targeting the LeuRS editing site.

Norvaline, a non-canonical amino acid that accumulates in Escherichia coli during downshift of oxygen, was recognized as a prime biological target for LeuRS editing. Our in vitro and in vivo experiments connected a requirement for editing with E. coli capacity to adapt to rapidly changing oxygen environment. Our work shows, for the first time, that aminoacyl-tRNA synthetase translational quality control mechanisms participate in bacterial adaptive response. This unveils an important biological role for LeuRS editing.

Norvaline was also recognized as a good substrate for IleRS synthetic and editing machinery in vitro. Detailed steady-state and pre-steady state kinetic analyses were performed to extract quantitative data describing origin of specificity against norvaline by IleRS.



  • Ita Gruic-Sovulj was awarded with the National award for scientific achievement for the year 2014
  • Nevena Cvetesic is analysing RNA-seq data in Boris Lenhard's group
  • Nevena Cvetesic was awarded by The Croatian Society of Biochemistry and Molecular Biology (HDBMB) with the Annual Award to young scientists  
  • Nevena Cvetesic was awarded with the L'Oreal-Unesco award for women in science
  • Morana Dulic performed structural and binding studies on Escherichia coli LeuRS at the EMBL laboratory in the group of Stephen Cusack.
  • Purchase of Sartorius Biostat A fermentor/bioreactor
  • Purchase of Eppendorf ThermoMixer C
  • Purchase of GE Healthcare Life Sciences Ultrospec 10 Cell Density Meter

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