Cobalamin riboswitches exhibit a broad range of ability to discriminate between methylcobalamin and adenosylcobalamin

Abstract

Riboswitches are a widely distributed class of regulatory RNAs in bacteria that modulate gene expression via small-molecule-induced conformational changes. Generally, these RNA elements are grouped into classes based upon conserved primary and secondary structure and their cognate effector molecule. Although this approach has been very successful in identifying new riboswitch families and defining their distributions, small sequence differences between structurally related RNAs can alter their ligand selectivity and regulatory behavior. Herein, we use a structure-based mutagenic approach to demonstrate that cobalamin riboswitches have a broad spectrum of preference for the two biological forms of cobalamin using isothermal titration calorimetry. This selectivity is primarily mediated by the interaction between a peripheral element of the RNA that forms a T-loop module and a subset of nucleotides in the cobalamin-binding pocket. Cell-based fluorescence reporter assays in revealed that mutations that switch effector preference lead to differential regulatory responses in a biological context. These data demonstrate that a more comprehensive analysis of representative sequences of both previously and newly discovered classes of riboswitches might reveal subgroups of RNAs that respond to different effectors. Furthermore, this study demonstrates a second distinct means by which tertiary structural interactions in cobalamin riboswitches dictate ligand selectivity.

Applications and limitations of regulatoryRNAelements in synthetic biology and biotechnology

“…Cobalamin comprises different chemical forms of vitamin B12 that are classified based on the upper axial ligand of the cobalt ion (Polaski et al . ). Those chemical forms (with their upper axial ligands) are cyanocobalamin, hydroxocobalamin, methylcobalamin and adenosylcobalamin (Polaski et al .…”

“…However, when a greater ligand specificity is required, it may be necessary to edit the sequence of the riboswitch to enable it to recognize subtle chemical differences, as demonstrated for the cobalamin riboswitch (Polaski et al . ).…”

“…These metabolites—for instance, cobalamin metabolites (Polaski et al . )—are also bound by certain proteins and used as cofactors (Romine et al . ).…”

“…The ligands of riboswitches are small molecules and metabolites, such as nucleotides and metal ions. These metabolites-for instance, cobalamin metabolites (Polaski et al 2017)-are also bound by certain proteins and used as cofactors (Romine et al 2017). Multiple riboswitches can be found in the same bacterium (Mandal et al 2003) and could hypothetically compete with one another, or with other cellular components, for ligandbinding in vivo.…”

“…For instance, the ligand cobalamin contains cobalt metal at the centre of its structure, which is linked to four pyrrole groups by nitrogen bonds. Cobalamin comprises different chemical forms of vitamin B12 that are classified based on the upper axial ligand of the cobalt ion (Polaski et al 2017). Those chemical forms (with their upper axial ligands) are cyanocobalamin, hydroxocobalamin, methylcobalamin and adenosylcobalamin (Polaski et al 2017).…”