No abstract

Summary Riboswitches are highly structured elements residing in the 5' untranslated region of messenger RNAs that specifically bind cellular metabolites to alter gene expression. While there are many structures of ligand bound riboswitches that reveal details of bimolecular recognition, their unliganded structures remain poorly characterized. Characterizing the molecular details of the unliganded state is crucial for understanding the riboswitch's mechanism of action because it is this state that actively interrogates the cellular environment and helps direct the regulatory outcome. To develop a detailed description of the ligand free form of an S-adenosylmethionine binding riboswitch at the local and global levels, we have employed a series of biochemical, biophysical, and computational methods. Our data reveals that the ligand binding domain adopts an ensemble of states that minimizes the energy barrier between the free and bound states to establish an efficient decision making brachpoint in the regulatory process.

Riboswitches are highly structured cis-acting elements located in the 59-untranslated region of messenger RNAs that directly bind small molecule metabolites to regulate gene expression. Structural and biochemical studies have revealed riboswitches experience significant ligand-dependent conformational changes that are coupled to regulation. To monitor the coupling of ligand binding and RNA folding within the aptamer domain of the purine riboswitch, we have chemically probed the RNA with N-methylisatoic anhydride (NMIA) over a broad temperature range. Analysis of the temperature-dependent reactivity of the RNA in the presence and absence of hypoxanthine reveals that a limited set of nucleotides within the binding pocket change their conformation in response to ligand binding. Our data demonstrate that a distal loop-loop interaction serves to restrict the conformational freedom of a significant portion of the three-way junction, thereby promoting ligand binding under physiological conditions.

The GAAA tetraloop-receptor is a commonly occurring tertiary interaction motif in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction makes to the folding properties of an RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA tetraloop domain and receptor domain were connected by a singlestranded A 7 linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking ([Co(NH 3 . Analysis of metal ion cooperativity yielded Hill coefficients of ≈ 2 for Na + -or K + -dependent docking versus ≈ 1 for the divalent ions and Co(NH 3 ) 6 3+ . Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloopreceptor docking. RNA constructs with U 7 and A 14 single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.RNA is an essential biological molecule that functions in numerous cellular processes, including catalyzing such critical reactions as protein synthesis and RNA splicing (1-3). To achieve its various functions, RNAs must adopt complex, well-defined three-dimensional structures, and determining how these RNA structures are formed and stabilized is critical to understanding their biological function. The process by which RNAs fold to these threedimensional structures is complex. Unlike most protein folding, RNA folding often proceeds by a hierarchical pathway, where the secondary structure forms prior to and independently of the tertiary structure, and the tertiary structure is stabilized by interactions between the secondary structural elements (4). † This work was supported in part by grants from: NIH (AI 33098), NSF, NIST and the W. M. Keck Foundation initiative in RNA science at the University of Colorado, Boulder. CDD was also supported in part by Biophysics Training Grant NIH (GM 65103).*To whom correspondence should be addressed. E-mail: arthur.pardi@colorado.edu, Department of Chemistry and Biochemistry, 215 UCB, University of Colorado, Boulder, CO 80309. Phone (303) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptA variety of RNA tertiary interaction motifs have been identified, including loop-loop interactions between hairpin or internal loops, A-minor interactions, and pseudoknots (5-10).One...