Fig. 1. Protein-protein interactions between gp32 and gp59 on fDNA. (A) The fluorescence from individual molecules of fDNA with the proteins bound in the order as indicated at the side of each row. The gp32 protein is labeled with A488 (gp32 D ) and the gp59 protein is labeled with A555 (gp59 A ). The filter sets are described in Experimental Methods: F1 is for A488 emission, F2 for FRET between A488 and A555, and F3 for A555 emission. (B) Ensemble FRET studies of Oregon-green-488-maleimide-labeled gp59 titrated into a solution of 400 nM CPM-labeled gp32 and 100 nM fDNA. The fluorescence spectra of 400 nM CPM-gp32 alone (black line), the endpoint of the titration at 1 M Oregon-green-488-maleimide-gp59 (dark gray line), and several intermediate spectra (light gray lines) are shown. (C) Analysis of the donor quenching and acceptor sensitization plotted against the gp59 concentration determines the stoichiometry among gp32, gp59, and fDNA to be 1:1:1 with a calculated binding constant of Ϸ40 nM. The gp32 protein is labeled with A488 (gp32 D ), the gp59 protein is labeled with A555 (gp59 A ), and the gp41 protein is unlabeled. MgATP␥S (500 M) is present for the sample in row 2, and 500 M MgATP is present for the sample in row 3. (B) The gp32 protein is unlabeled, the gp59 protein is labeled with A555 (gp59 A ), and the gp41 is labeled with A488 (gp41 D ). MgATP␥S (500 M) is present for the sample in row 2, and 500 M MgATP is present for the sample in row 3 (5 min after addition of gp41 and nucleotide) and in row 4 (30 min after addition of gp41 and nucleotide). The filter sets are described in the legend to Fig. 1. RNA ͉ tertiary interactions ͉ heterogeneity ͉ time-correlated single photon counting T he discovery that RNAs can catalyze biological reactions has led to intensive effort aimed at identifying additional biological functions for RNA. More than 20 years later, we now know that RNAs play critical functional roles in metabolism, replication, regulation, and development in cells. Extensive biochemical and biophysical studies have led to a better understanding of the molecular mechanisms by which RNAs achieve their biological function, highlighting the important roles of both structure and dynamics. In this regard, single-molecule methods have recently emerged as particularly powerful tools. The folding dynamics of various functional RNAs have been investigated by single-molecule FRET experiments, which probe dynamics under equilibrium conditions via observation of the stochastic fluorescence trajectories as a function of time (1-6). These techniques offer a unique glimpse into subpopulations of a system and, in some cases, have identified conformational heterogeneity or the presence of intermediates that would otherwise be undetectable by ensemble methods (3-5, 7, 8).Unlike the highly cooperative, all-or-none, folding process observed for most protein domains, RNAs generally fold in a noncooperative manner, where the secondary structure forms independently of the tertiary structure. The thermodynamics for f...

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...

Proper assembly of RNA into catalytically active three-dimensional structures requires multiple tertiary binding interactions, individual characterization of which is crucial to a detailed understanding of global RNA folding. This work focuses on single-molecule fluorescence studies of freely diffusing RNA constructs that isolate the GAAA tetraloop-receptor tertiary interaction. Freely diffusing conformational dynamics are explored as a function of Mg(2+) and Na(+) concentration, both of which promote facile docking, but with 500-fold different affinities. Systematic shifts in mean fluorescence resonance energy transfer efficiency values and line widths with increasing [Na(+)] are observed for the undocked species and can be interpreted with a Debye model in terms of electrostatic relaxation and increased flexibility in the RNA. Furthermore, we identify a 34 +/- 2% fraction of freely diffusing RNA constructs remaining undocked even at saturating [Mg(2+)] levels, which agrees quantitatively with the 32 +/- 1% fraction previously reported for immobilized constructs. This verifies that the kinetic heterogeneity observed in the docking rates is not the result of surface tethering. Finally, the K(D) value and Hill coefficient for [Mg(2+)]-dependent docking decrease significantly for [Na(+)] = 25 mM vs. 125 mM, indicating Mg(2+) and Na(+) synergy in the RNA folding process.