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

Baisakhi Banerjee, Sayak Ganguli, Rajat Banerjee

Protein and peptide letters 2020

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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. In the present study, we have characterized some of the features of the alaRS-DNA binding using various biophysical techniques. 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. 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. 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. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.

Citation

Baisakhi Banerjee, Sayak Ganguli, Rajat Banerjee. Biophysical Characterization of Interaction between E. coli Alanyl-tRNA Synethase with its Promoter DNA. Protein and peptide letters. 2020;27(7):635-648