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.