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Fusion with Anticodon Binding Domain of GluRS is Not Sufficient to Alter the Substrate Specificity of a Chimeric Glu-Q-RS

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Abstract

Glutamyl-queuosine-tRNAAsp synthetase (Glu-Q-RS) is a paralog of glutamyl-tRNA synthetase (GluRS) and is found in more than forty species of proteobacteria, cyanobacteria, and actinobacteria. Glu-Q-RS shows striking structural similarity with N-terminal catalytic domain of GluRS (NGluRS) but it lacks the C-terminal anticodon binding domain (CGluRS). In spite of structural similarities, Glu-Q-RS and NGluRS differ in their functional properties. Glu-Q-RS glutamylates the Q34 nucleotide of the anticodon of tRNAAsp whereas NGluRS constitutes the catalytic domain of GluRS catalyzing the transfer of Glu on the acceptor end of tRNAGlu. Since NGluRS is able to catalyze aminoacylation of only tRNAGlu the glutamylation capacity of tRNAAsp by Glu-Q-RS is surprising. To understand the substrate specificity of Glu-Q-RS we undertook a systemic approach by investigating the biophysical and biochemical properties of the NGluRS (1–301), CGluRS (314–471) and Glu-Q-RS-CGluRS, (1–298 of Glu-Q-RS fused to 314–471 from GluRS). Circular dichroism, fluorescence spectroscopy and differential scanning calorimetry analyses revealed absence of N-terminal domain (1–298 of Glu-Q-RS) and C-terminal domain (314–471 from GluRS) communication in chimera, in contrast to the native full length GluRS. The chimeric Glu-Q-RS is still able to aminoacylate tRNAAsp but has also the capacity to bind tRNAGlu. However the chimeric protein is unable to aminoacylate tRNAGlu probably as a consequence of the lack of domain–domain communication.

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Abbreviations

Glu-Q-RS:

Glutamyl-queuosine-tRNAAsp Synthetase

GluRS:

Glutamyl-tRNA synthetase

NGluRS:

N-terminal catalytic domain of GluRS

CGluRS:

C-terminal anticodon binding domain

aaRS:

Aminoacyl tRNA synthetase

Ec :

Escherichia coli

Tth :

Thermus thermophilus

CP:

Connective peptide

l-Glu:

l-Glutamic acid

IPTG:

Isopropyl β-D-1-thiogalactopyranoside

PMSF:

Phenylmethanesulfonyl fluoride

CD:

Circular dichroism

UV:

Ultra violet

MRE:

Mean residue ellipticity

θ :

Observed ellipticity

GdnHCl:

Guanidine chloride

f D :

Fraction unfolded

∆G:

Free energy change

m :

Slope of the transition

DSC:

Differential scanning calorimetry

Tm :

Midpoint of transition

Kd :

Dissociation constant

GoA:

Glutamol-AMP

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Acknowledgments

We are grateful to Professor Gautam Basu, Department of Biophysics, Bose Institute for allowing us to use the CD instrument. Dr. Saumya Dasgupta is acknowledged for his assistance during CD measurements. Ec His-tagged GluRS overproducing strain, pKR15 plasmid expressing Ec tRNAGlu and JP1449 (DE3) were kindly gifted by Professor Jacques Lapointe (Department of Biochemistry and Microbiology, Université Laval, Québec, Canada). We also like to thank Professor Gabor Igloi, University of Freiburg, Germany for critical reading of the manuscript and for providing in vitro transcribed jack bean tRNAArg. Molecular Mechanism of Disease and Drug Action (MMDDA) project (Grant 11-R&D-SIN-5.04) Department of Atomic Energy, Govt. of India is kindly acknowledged for providing the fund for purchasing differential scanning calorimetry (DSC) instrument. S.R. thanks to University of Calcutta for providing fellowship.

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Authors and Affiliations

  1. Department of Biotechnology and Dr. B C Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700 019, India

    Sutapa Ray, Bappaditya Roy & Rajat Banerjee

  2. Department of Molecular Biology and Genetics, CARB Centre, Aarhus University, Gustav Wieds Vej 10c, 8000, Aarhus, Denmark

    Mickael Blaise

  3. Biophysics Division, Saha Institute of Nuclear Physics, Sector-1, Block-AF, Bidhannagar, Kolkata, 700064, India

    Saptaparni Ghosh

  4. UPR Architecture et Réactivité de l’ARN, CNRS, Institut de Biologie Moléculaire et Cellulaire, Université Louis Pasteur de Strasbourg, 15, Rue René Descartes, 67084, Strasbourg Cédex, France

    Daniel Kern

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  1. Sutapa Ray
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  2. Mickael Blaise
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Correspondence to Rajat Banerjee.

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Ray, S., Blaise, M., Roy, B. et al. Fusion with Anticodon Binding Domain of GluRS is Not Sufficient to Alter the Substrate Specificity of a Chimeric Glu-Q-RS. Protein J 33, 48–60 (2014). https://doi.org/10.1007/s10930-013-9537-7

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