Biophysical Characterization of Interaction between E. coli Alanyl-tRNA Synethase with its Promoter DNA

Author(s): Baisakhi Banerjee, Sayak Ganguli, Rajat Banerjee*

Journal Name: Protein & Peptide Letters

Volume 27 , Issue 7 , 2020

Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Background: Aminoacyl-tRNA Synthetases (aaRSs) are well known for their role in the translation process. Lately investigators have discovered that this family of enzymes are also capable of executing a broad repertoire of functions that not only impact protein synthesis, but extend to a number of other activities. Till date, transcriptional regulation has so far only been described in E. coli Alanyl-tRNA synthetase and it was demonstrated that alaRS binds specifically to the palindromic DNA sequence flanking the gene’s transcriptional start site and thereby regulating its own transcription.

Objective: In the present study, we have characterized some of the features of the alaRS-DNA binding using various biophysical techniques.

Methods: To understand the role of full length protein and oligomerization of alaRS in promoter DNA binding, two mutants were constructed, namely, N700 (a monomer, containing the N-terminal aminoacylation domain but without the C-terminal part) and G674D (previously demonstrated to form full-length monomer). Protein-DNA binding study using fluorescence spectroscopy, analytical ultracentrifugation, Isothermal Titration Calorimetry was conducted.

Results: Sedimentation equilibrium studies clearly demonstrated that monomeric variants were unable to bind promoter DNA. Isothermal Calorimetry (ITC) experiment was employed for further characterization of wild type alaRS-DNA interaction. It was observed that full length E. coli Alanyl-tRNA synthetase binds specifically with its promoter DNA and forms a dimer of dimers. On the other hand the two mutant variants were unable to bind with the DNA.

Conclusion: In this study it was concluded that full length E. coli Alanyl-tRNA synthetase undergoes a conformational change in presence of its promoter DNA leading to formation of higher order structures. However, the exact mechanism behind this binding is currently unknown and beyond the scope of this study.

Keywords: Alanyl-tRNA synthetase, transcriptional regulation, analytical ultracentrifuge, isothermal calorimetry, Protein- DNA interaction, biophysical techniques.

Martinis, S.A.; Plateau, P.; Cavarelli, J.; Florentz, C. AminoacyltRNA synthetases: a family of expanding functions. Mittelwihr, France, October 10-15, 1999. EMBO J., 1999, 18(17), 4591-4596.
[] [PMID: 10469639]
Rho, S.B.; Lincecum, T.L., Jr.; Martinis, S.A. An inserted region of leucyl-tRNA synthetase plays a critical role in group I intron splicing. EMBO J., 2002, 21(24), 6874-6881.
[] [PMID: 12486008]
Kwon, N.H.; Kang, T.; Lee, J.Y.; Kim, H.H.; Kim, H.R.; Hong, J.; Oh, Y.S.; Han, J.M.; Ku, M.J.; Lee, S.Y.; Kim, S. Dual role of methionyl-tRNA synthetase in the regulation of translation and tumor suppressor activity of aminoacyl-tRNA synthetaseinteracting multifunctional protein-3. Proc. Natl. Acad. Sci. USA, 2011, 108(49), 19635-19640.
[] [PMID: 22106287]
Sun, L.; Song, Y.; Blocquel, D.; Yang, X.-L.; Schimmel, P. Two crystal structures reveal design for repurposing the C-Ala domain of human AlaRS. Proc. Natl. Acad. Sci. USA, 2016, 113(50), 14300-14305.
[] [PMID: 27911835]
Putney, S.D.; Schimmel, P. An aminoacyl tRNA synthetase binds to a specific DNA sequence and regulates its gene transcription. Nature, 1981, 291(5817), 632-635.
[] [PMID: 6264314]
Moncrieffe, M.C.; Grossmann, J.G.; Gay, N.J. Assembly of oligomeric death domain complexes during Toll receptor signaling. J. Biol. Chem., 2008, 283(48), 33447-33454.
[] [PMID: 18829464]
Kar, S.R.; Lebowitz, J.; Blume, S.; Taylor, K.B.; Hall, L.M. SmtBDNA and protein-protein interactions in the formation of the cyanobacterial metallothionein repression complex: Zn2+ does not dissociate the protein-DNA complex in vitro. Biochemistry, 2001, 40(44), 13378-13389.
[] [PMID: 11683648]
Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J., 2000, 78(3), 1606-1619.
[] [PMID: 10692345]
Vistica, J.; Dam, J.; Balbo, A.; Yikilmaz, E.; Mariuzza, R.A.; Rouault, T.A.; Schuck, P. Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition. Anal. Biochem., 2004, 326(2), 234-256.
[] [PMID: 15003564]
Farris, F.J.; Weber, G.; Chiang, C.C.; Paul, I.C. Preparation, crystalline structure, and spectral properties of the fluorescent probe 4,4′-bis-1-phenylamino-8-naphthalenesulfonate. J. Am. Chem. Soc., 1978, 100(14), 4469-4474.
Munteanu, M.G.; Vlahovicek, K.; Parthasarathy, S.; Simon, I.; Pongor, S. Rod models of DNA: sequence-dependent anisotropic elastic modelling of local bending phenomena. Trends Biochem. Sci., 1998, 23(9), 341-347.
[] [PMID: 9787640]
Ulyanov, N.B.; James, T.L. Statistical analysis of DNA duplex structural features. Methods Enzymol., 1995, 261, 90-120.
[] [PMID: 8569515]
Babcock, M.S.; Pednault, E.P.D.; Olson, W.K. Nucleic acid structure analysis. Mathematics for local Cartesian and helical structure parameters that are truly comparable between structures. J. Mol. Biol., 1994, 237(1), 125-156.
[] [PMID: 8133513]
Tuszynska, I.; Magnus, M.; Jonak, K.; Dawson, W.; Bujnicki, J.M. NPDock: a web server for protein-nucleic acid docking. Nucleic Acids Res., 2015, 43(W1) W425-430.
[] [PMID: 25977296]
Narasimhulu, S. Quenching of tryptophanyl fluorescence of bovine adrenal P-450C-21 and inhibition of substrate binding by acrylamide. Biochemistry, 1988, 27(4), 1147-1153.
[] [PMID: 3259146]
Eftink, M.R.; Ghiron, C.A. Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. Biochemistry, 1976, 15(3), 672-680.
[] [PMID: 1252418]
Saha, R.; Banik, U.; Bandopadhyay, S.; Mandal, N.C.; Bhattacharyya, B.; Roy, S. An operator-induced conformational change in the C-terminal domain of the lambda repressor. J. Biol. Chem., 1992, 267(9), 5862-5867.
[PMID: 1532575]
Banerjee, B.; Banerjee, R. Urea unfolding study of E. coli alanyltRNA synthetase and its monomeric variants proves the role of Cterminal domain in stability. J. Amino Acids, 2015, 2015 805681.
Das, S.; Banerjee, B.; Hossain, M.; Thangamuniyandi, M.; Dasgupta, S.; Chongdar, N.; Kumar, G.S.; Basu, G. Characterization of DNA binding property of the HIV-1 host factor and tumor suppressor protein Integrase Interactor 1 (INI1/hSNF5). PLoS One, 2013, 8(7) e66581.
[] [PMID: 23861745]
Deb, S.; Bandyopadhyay, S.; Roy, S. DNA sequence dependent and independent conformational changes in multipartite operator recognition by λ-repressor. Biochemistry, 2000, 39(12), 3377-3383.
[] [PMID: 10727231]
Zandarashvili, L.; Nguyen, D.; Anderson, K.M.; White, M.A.; Gorenstein, D.G.; Iwahara, J. Entropic enhancement of Protein-DNA affinity by oxygen-to-sulfur substitution in DNA phosphate. Biophys. J., 2015, 109(5), 1026-1037.
[] [PMID: 26331260]
Morgunova, E.; Yin, Y.; Jolma, A.; Dave, K.; Schmierer, B.; Popov, A.; Eremina, N.; Nilsson, L.; Taipale, J. Structural insights into the DNA-binding specificity of E2F family transcription factors. Nat. Commun., 2015, 6, 10050.
[] [PMID: 26632596]
Bandyopadhyay, S.; Banik, U.; Bhattacharyya, B.; Mandal, N.C.; Roy, S. Role of the C-terminal tail region in the self-assembly of lambda-repressor. Biochemistry, 1995, 34(15), 5090-5097.
[] [PMID: 7711028]
Schlax, P.J.; Capp, M.W.; Record, M.T., Jr. Inhibition of transcription initiation by lac repressor. J. Mol. Biol., 1995, 245(4), 331-350.
[] [PMID: 7837267]
Frank, D.E.; Saecker, R.M.; Bond, J.P.; Capp, M.W.; Tsodikov, O.V.; Melcher, S.E. thermodynamics of the interactions of lac repressor with variants of the symmetric lac operator: effects of converting a consensus site to a non-specific site11edited by p. e. wright. j. mol. biol. 1997, 267(5), 1186-1205.
Barkley, M.D.; Riggs, A.D.; Jobe, A.; Burgeois, S. Interaction of effecting ligands with lac repressor and repressor-operator complex. Biochemistry, 1975, 14(8), 1700-1712.
[] [PMID: 235964]
Dou, S-X.; Wang, P-Y.; Xu, H.Q.; Xi, X.G. The DNA binding properties of the Escherichia coli RecQ helicase. J. Biol. Chem., 2004, 279(8), 6354-6363.
[] [PMID: 14665634]
Barranco-Medina, S.; Galletto, R. DNA binding induces dimerization of Saccharomyces cerevisiae Pif1. Biochemistry, 2010, 49(39), 8445-8454.
[] [PMID: 20795654]

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2020
Published on: 13 August, 2020
Page: [635 - 648]
Pages: 14
DOI: 10.2174/0929866526666191104123229
Price: $65

Article Metrics

PDF: 39