title: The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch.
abstract: Riboswitch operation involves the complex interplay between the aptamer domain and the expression platform. During transcription, these two domains compete against each other for shared sequence. In this study, we explore the cooperative effects of ligand binding and Magnesium interactions in the SAM-I riboswitch in the context of aptamer collapse and anti-terminator formation. Overall, our studies show the apo-aptamer acts as (i) a pre-organized aptamer competent to bind ligand and undergo structural collapse and (ii) a conformation that is more accessible to anti-terminator formation. We show that both Mg(2+) ions and SAM are required for a collapse transition to occur. We then use competition between the aptamer and expression platform for shared sequence to characterize the stability of the collapsed aptamer. We find that SAM and Mg(2+) interactions in the aptamer are highly cooperative in maintaining switch polarity (i.e. aptamer 'off-state' versus anti-terminator 'on-state'). We further show that the aptamer off-state is preferentially stabilized by Mg(2+) and similar divalent ions. Furthermore, the functional switching assay was used to select for phosphorothioate interference, and identifies potential magnesium chelation sites while characterizing their coordinated role with SAM in aptamer stabilization. In addition, we find that Mg(2+) interactions with the apo-aptamer are required for the full formation of the anti-terminator structure, and that higher concentrations of Mg(2+) (>4 mM) shift the equilibrium toward the anti-terminator on-state even in the presence of SAM.
authors.affiliation: Theoretical Biology and Biophysics Group, Theoretical Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
authors.forename: Scott P
authors.initials: SP
authors.lastname: Hennelly
authors.forename: Irina V
authors.initials: IV
authors.lastname: Novikova
authors.forename: Karissa Y
authors.initials: KY
authors.lastname: Sanbonmatsu
mesh_terms: D002413:Cations, Divalent; D008024:Ligands; D008274:Magnesium / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D058928:Riboswitch; D012436:S-Adenosylmethionine / Q000378:metabolism*; D046968:Thermoanaerobacter / Q000235:genetics
chemical_list: D002413:Cations, Divalent; D008024:Ligands; D012329:RNA, Bacterial; D058928:Riboswitch; D012436:S-Adenosylmethionine; D008274:Magnesium