NMR Dissection of the Detailed Mechanism for Antibiotic Binding to Asite RNA

Abstract

A growing number of studies are suggesting the importance of non-coding RNAs that play a variety of roles by undergoing conformational changes in response to a specific cellular signal. It has been increasingly believed that this predisposition of RNA conformational changes in a specific manner is encoded by the flexibility that in turn is encoded in RNA sequences. Base flipping is a conformational transition that occurs ubiquitously across diverse RNA functional and structural contexts. A prominent example is Asite ribosomal RNA that contains two highly conserved internal loop adenines A1492 and A1493, which serve to decode the mRNA message by looping out and stabilizing a codon-anticodon mini-helix when it is formed between mRNA and its cognate aa-tRNA. Asite is also known to bind to many antibiotics where drug binds the internal loop, flips the two adenines out and the adenines are forced to bind the codon-anticodon minihelix irrespective of correctness of tRNA. In this study, we attempt to dissect the process of Asite drug binding further using NMR spin relaxation techniques. Site-specific measurement of fast dynamics (using transverse and longitudinal relaxation measurements) and slow dynamics (using R1rho dispersion measurements) on free and bound Asite RNA provide insights into the transition states of binding. Our results highlight the significance of various neighboring bases that dynamically encode the process of base flipping that is vital for an effective antibiotic binding.