Cloning and sequencing of riboswitches of Alishewanella tabrizica strain RCRI4 and comparative studies on similar riboswitches in other bacteria

Abstract

Introduction: Riboswitches, as cis acting non-coding RNA elements, regulate gene expression via specific binding of various small molecules. Their conserved structures are the key characteristics for binding specific molecules and gene regulation. Also, the contribution of riboswitches in antibiotic targeting is possible. In this study, we aimed first to find out the structural and functional level of similarity among riboswitches and rRNA structures including binding of aminoglycosides to riboswitches through computational tools. On the other side, riboswitches are rarely characterized in environmental bacteria. In this study, TPP riboswitch sourced from novel characterized specie Alishewanella tabrizica, was identified. Methods: After PDB structures’ selection, multiple sequence and structural alignment were carried out. Highly similar rRNA motifs with riboswitches (including “A site”) were sorted out. Subsequently, the probable interaction of riboswitches with aminoglycosides ere studied using docking studies. For identifying of TPP riboswitch of A. tabrizica, kinetic and affinity analysis of TPP binding to different TPP riboswitch aptamer domains sourced from A. tabrizica, Alishewanella aestuarii, E. coli, B. subtilis were studied and compared using In-line probing and Surface Plasmon Resonance (SPR) methods. Results: Docking analysis showed significant high binding energies between riboswitches-aminoglycosides. Accordingly, lysine, glycine and SAM-I riboswitches were recognized as the best RNA targets for all of the aminoglycosides. Docking results were also validated through rDock program and MD simulation. According to the KD values from experimental analysis, the affinity of TPP binding was the highest in A. tabrizica. In addition, the order of TPP binding affinity was TPP aptamer domains sourced from A. tabrizica > A. aestuarii > E. coli > B. subtilis. Conclusion: Taking together, it can be proposed that riboswitches have this potential to be the target of aminoglycosides. The observed variation TPP riboswitches affinity could be referred to the studied bacteria adaptation to diverse environmental conditions.