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URMC / Labs / Wedekind Lab / Research

Research

Figure 1

Figure 1

Riboswitches are structured RNA molecules that regulate genes by binding small molecule effectors. Although present in all domains of life, they are typically located in the 5'-leader sequence of bacterial messenger RNAs where they control transcription or translation (Figure 1).

Due to a long-standing interest in RNA editing and modification, our lab began investigating translational regulation by bacterial riboswitches that bind the secondary metabolite prequeuosine1 (7-deaza-7-aminomethyl-guanine or preQ1), a precursor to the hypermodified tRNA-base queuosine (Q).

Figure 2

Figure 2

Q production is unique to bacteria and preQ1 riboswitches are of interest due to their potential as novel antibiotic targets.

When we entered the field, little was known about preQ1 recognition by riboswitches. As such, we determined the first crystal structure of a translational preQ1 (class I) riboswitch, and subsequently analyzed its ligand-binding kinetics and thermodynamics.

Figure 3

Figure 3

We subsequently investigated preQ1-binding and dynamics using single molecule (sm)FRET, which revealed an induced-fit ligand binding pathway (Figure 2). Subsequently, we determined the structure of a phylogenetically distinct, and more complex preQ1 riboswitch (class II) that buries its entire Shine-Dalgarno sequence (SDS) within an HLout pseudoknot core (Figure 3).

Together with chemical modification analysis, and computational modeling, our results suggested enhanced SDS docking and undocking with added preQ1, with regulation controlled by rapid dynamics (Figure 4).

Figure 4

Figure 4

Our work on riboswitches is ongoing, including the biochemical and biophysical analysis of poorly characterized riboswitches, and the role of tandem aptamers in gene regulation.  Most recently we determined the structure of the newly discovered preQ1-III riboswitch, revealing an unusual architecture wherein the 5´-aptamer domain is positioned remotely relative to the 3´-expression platform harboring the SDS. We leveraged the structure to analyze the ligand-dependent sequestration of the SDS by smFRET.

Recent Publications

Tails of three knotty switches: how preQ1 riboswitches control protein translation. Belashov I.A., Dutta D., Salim, M, and Wedekind, J.E. (2015) eLifeSciences (In press).

Molecular mechanism for preQ1-II riboswitch function revealed by molecular dynamics. Aytenfisu, A.H., Liberman, J.A., Wedekind J.E. and Mathews, D.H (2015) RNA (In press).

Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics. Liberman J.A., Suddala, K.C., Aytenfisu A.H., Chan D., Belashov I.A., Salim M., Mathews D.H., Spitale, R.C., Walter N.G. and Wedekind J.E. (2015) Proc. Natl. Acad. Sci. U.S.A. 112, E3485–E3494.

Single transcriptional and translational preQ1 riboswitches adopt similar pre-folded ensembles that follow distinct folding pathways into the same ligand-bound structure. Suddala K.C., Rinaldi A.J., Feng J., Mustoe A.M., Eichhorn C.D., Liberman J.A., Wedekind J.E., Al-Hashimi H.M., Brooks C.L., and Walter N.G. (2013) Nuc. Acids Res. 41, 10462-75.

Structure of a class II preQ1 riboswitch reveals ligand binding by a new fold. Liberman J.A., Salim M., Krucinska J. and Wedekind J.E. (2013) Nat. Chem. Biol. 9, 953-955.

Molecular mechanism of preQ1 riboswitch action: a molecular dynamics study. Banáš P., Sklenovský P., Wedekind J.E., Sponer J. and Otyepka M. (2012) J. Phys. Chem. B. 116, 12721-34.

Comparison of a preQ1 riboswitch aptamer in metabolite-bound and free states with implications for gene regulation. Jenkins J.L., Krucinska J., McCarty R.M., Bandarian V. and Wedekind J.E. (2011) J. Biol. Chem. 286, 24626-37.

The Structural Basis for Recognition of the PreQ0 Metabolite by an Unusually Small Riboswitch Aptamer Domain. Spitale R.C., Torelli A.T., Krucinska J., Bandarian V. and Wedekind J.E. (2009) J. Biol. Chem. 284, 11012-6.