Effect of Sequence on the Interactions of Divalent Cations with M-Box Riboswitches from Mycobacterium tuberculosis...

RNA is highly negatively charged and often acquires complex structures that require the presence of divalent cations. Subtle changes in conformation resulting from changes in sequence can affect the way ions associate with RNA. Riboswitches are RNA molecules that are involved in the control of gene expression in bacteria and are excellent systems for testing the effects of sequence variations on the conformation of RNA because they contain a highly conserved binding pocket but present sequence variability among different organisms. In this work, we have compared the aptamer domain of a proposed M-box riboswitch from Mycobacterium tuberculosis with the aptamer domain of a validated M-box riboswitch from Bacillus subtilis. We have in vitro transcribed and purified wild-type (WT) M-box riboswitches from M. tuberculosis and B. subtilis as well as a variety of mutated aptamers in which regions from one riboswitch have been replaced with regions from the other riboswitch. We have used ultraviolet unfolding experiments and circular dichroism to characterize the interactions of WT and related M-box riboswitches with divalent cations. Our results show that M-box from M. tuberculosis associates with Mg²⁺ and Sr²⁺ in a similar fashion while M-box from B. subtilis discriminates between these two ions and appears to associate better with Mg²⁺. Our overall results show that M-box from M. tuberculosis interacts differently with cations than M-box from B. subtilis and suggest conformational differences between these two riboswitches.