How To Turn On A Riboswitch: Structural And Mechanistic Lessons Learned From S-Adenosyl-L-Methionine- And Manganese-Sensing Rnas

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Riboswitches are widespread structured RNA elements that usually occur in 5'-untranslated regions (UTRs) of bacterial mRNAs. They directly regulate the expression level of the host gene, generally at the level of translation or transcription, in response to cellular factors such as metabolites, ions, or signaling molecules. The ability of these cis-regulatory elements to sense ligand stringently and to undergo drastic conformational changes is intriguing. Here I present two of my projects dissecting two riboswitches that sense S-adenosylmethionine (SAM) and Manganese. I first present our findings on the conformational switching mechanism of the SAM-III riboswitch. Though the Ke lab has already described the SAM-binding site of this RNA [1], the mechanism by which it switches conformations back and forth between expression "on" and "off" states is not understood, and is the goal of my first project. Here we present several structural snapshots SAMfree intermediate states, including a previously unanticipated structure. Interconversion between these conformations was confirmed in solution by collaborators using single-molecule (sm)FRET. I present a model for SAM-III's conformational switching in which it takes on multiple conformations in the absence of SAM and undergoes two distinct levels of conformation selection to converge to a single conformation in the presence of SAM. These findings exemplify RNA's built-in conformational flexibility in its function. Many putative riboswitches have been identified bioinformatically, but have unknown ligands. Study of these riboswitches has been historically challenging, but offers the greatest reward. In the second project presented, I show that one such "orphan", the widespread yybP-ykoY [2], is a Mn2+responsive riboswitch class. This finding suggests roles for its mostly-uncharacterized regulated genes in manganese homeostasis or oxidative stress. Additionally, I present the crystal structure of a transcriptional yybP-ykoy riboswitch and describe its mechanism of discriminating Mn2+ from other metals by both geometry and chemical "hardness". We also find that the structure of an E. coli yybPykoY riboswitch in the absence of Mn2+ displays a destabilized Mn2+ binding region. We further show, via the structure of a binding site mutant, that the riboswitch's metal specificity can be altered. Through these and new structures and an ongoing collaboration using single-molecule (sm)FRET, we suggest a scheme for the conformation dynamics of this riboswitch. Using the information gleaned from these structures, we are also designing fluorescent Mn2+ sensors, based on the Spinach/Broccoli fluorophorebinding RNA platform [3].

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riboswitch; RNA structure; manganese
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Lis,John T
Pleiss,Jeffrey A.
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Ph. D., Biochemistry
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Doctor of Philosophy
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dissertation or thesis
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