Variable sequences outside the SAM-binding core critically influence the conformational dynamics of the SAM-III/SMK box riboswitch.

ABSTRACT: The S(MK) box (SAM-III) translational riboswitches were identified in S-adenosyl-l-methionine (SAM) synthetase metK genes in members of Lactobacillales. This riboswitch switches between two alternative conformations in response to intracellular SAM concentration and controls metK expression at the level of translation initiation. We previously reported the crystal structure of the SAM-bound S(MK) box riboswitch. In this study, we combined selective 2'-hydroxyl acylation analyzed by primer extension chemical probing with mutagenesis to probe the ligand-induced conformational switching mechanism. We revealed that while the majority of the apo S(MK) box RNA molecules exist in an alternatively base-paired (ON) conformation, a subset of them pre-organize into a SAM-bound-like (READY) conformation, which, upon SAM exposure, is selectively stabilized into the SAM-bound (OFF) conformation through an induced-fit mechanism. Mutagenesis showed that the ON state is only slightly more stable than the READY state, as several single-nucleotide substitutions in a hypervariable region outside the SAM-binding core can alter the folding landscape to favor the READY state. Such S(MK) variants display a "constitutively OFF" behavior both in vitro and in vivo. Time-resolved and temperature-dependent selective 2'-hydroxyl acylation analyzed by primer extension analyses revealed adaptation of the S(MK) box RNA to its mesothermal working environment. The latter analysis revealed that the SAM-bound S(MK) box RNA follows a two-step folding/unfolding process.

SUBMITTER: Lu C 

PROVIDER: S-EPMC3479645 | biostudies-other | 2011 Jun

REPOSITORIES: biostudies-other