Conformational Flexibility and Dynamics of the Internal Loop and

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Conformational Flexibility and Dynamics of the Internal Loop and Helical Regions of the Kink-Turn Motif in the Glycine Riboswitch by Site-Directed Spin Labeling Jackie Marie Esquiaqui, Eileen M Sherman, Jingdong Ye, and Gail E. Fanucci Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00287 • Publication Date (Web): 18 Jul 2016 Downloaded from http://pubs.acs.org on July 23, 2016

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Title Page Title. Conformational Flexibility and Dynamics of the Internal Loop and Helical Regions of the KinkTurn Motif in the Glycine Riboswitch by Site-Directed Spin Labeling

Funding Source Statement. This work was supported by the National Science Foundation DGE-0802270 (J.M.E.), National Science Foundation MCB1329467 (GEF), start-up funds at the University of Central Florida (J.Y.) and National Institutes of Health CA175625 (J.Y.) and GM105409 (G.E.F.). Instrumentation utilized in this research was supported by National Institutes of Health S10RR031603 (G.E.F) and the National High Magnetic Field Laboratory – in House Research Program (G.E.F), Byline. Jackie M. Esquiaqui,1 Eileen M. Sherman,2 Jing-Dong Ye,2 Gail E. Fanucci,1* 1

Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611, United

States 2

Department of Chemistry, University of Central Florida, 4000 Central Florida Blvd, Orlando,

FL 32816, United States *Tel: 1-352-392-2345; fax: 1-352-392-0872; email: [email protected]

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Abbreviations and Textual Footnotes. Site-directed spin labeling (SDSL), continuous wave (CW), electron paramagnetic resonance (EPR), double electron-electron resonance (DEER), Vibrio cholerae (VC)


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Abstract.

Site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy provides a means for a solution state description of site-specific dynamics and flexibility of large RNAs, facilitating understandings of environmental conditions such as ligands and ions on RNA structure and dynamics. Here, the utility and capability of EPR line shape analysis and distance measurements to monitor and describe site-specific changes in the conformational dynamics of internal loop nucleobases as well as helix-helix interactions of the kink-turn motif in the Vibrio cholerae (VC) glycine riboswitch that occur upon sequential K+/Mg2+/glycine induced folding was explored. Spin-labels were incorporated into the 232 nt sequence via splinted ligation strategies. Thiouridine nucleobase labeling within the internal loop reveals unambiguous differential dynamics for two successive sites labeled, with varied rates of motion reflective of base flipping and base stacking. EPR based distance measurements for nitroxide spin-labels incorporated within the RNA backbone in the helical regions of the kink-turn are reflective of helical formation and tertiary interaction induced by ion stabilization. In both instances, results indicate that the structural formation of the kink-turn in the VC glycine riboswitch can be stabilized by 100 mM K+ where the conformational flexibility of the kink-turn is not further tightened by subsequent addition of divalent ions. Although glycine binding is likely to induce structural and dynamic changes in other regions, SDSL indicates no impact of glycine binding on the local dynamics or structure of the kink-turn as investigated here. Overall these results demonstrate the ability of SDSL to interrogate site specific base dynamics and packing of helices in large RNAs and demonstrate ion induced stability of the kink-turn fold of the VC riboswitch.


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Solution-state spectroscopic studies of RNA dynamics and conformational changes are indispensable for investigating the fundamental properties governing RNA folding pathways and are therefore invaluable for understanding the function of many RNAs. To probe RNA dynamics and folding, many well suited solution-state techniques exist and have been utilized including forester energy transfer (FRET), small angle X-ray scattering (SAXS), X-ray scattering interferometry (XSI), nuclear magnetic resonance (NMR) spectroscopy, and site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy.1-6 SDSL EPR spectroscopy has been of interest for studying many RNAs and offers several benefits including, no biomolecular size limitation, probing of discrete site-specific local dynamics, and evaluating conformational equilibria. In this work we show the feasibility, utility, and capability of SDSL EPR to monitor and describe site-specific changes during RNA folding in large RNAs, specifically, the kink-turn motif in the VC glycine riboswitch and we demonstrate the unique advantages SDSL EPR studies offer for probing site-specific dynamics within varying solution state environmental conditions. SDSL EPR methods require the incorporation of one or two unpaired electrons into the molecules of interest. For site-specific labeling of nucleic acids, numerous labeling strategies exist and in most cases solid phase synthesis is utilized to integrate nitroxide spin labels into the desired location(s) in RNA/DNA (for other methods see references 7-14).7-14 The continuous wave (CW) nitroxide EPR spectral line shape reports on mobility and changes in mobility within the 0.1-50 ns time regime. Additionally, the CW EPR line shape is also influenced by the experimental frequency/field utilized and studies are therefore benefited by a multi-frequency approach wherein varying frequencies provide sensitivity to varying time scales of motion sampled by the biological molecule.15-22 Mobility is defined as a combination of both motional rate and orientational fluctuations.16 Hence, for a singly incorporated spin label, CW SDSL EPR is a powerful approach to characterize the impact that the local environment has on biomolecular conformation and dynamics. One specific advantage of utilizing CW SDSL EPR to study RNA dynamics is the unique ability to spin label different regions within a given single nucleotide, including the ribose sugar, phosphate backbone, or nucleobases.8,

10, 23, 24

Structurally, these

regions are implicated in various important roles within RNAs, thus, a technique to study local dynamics for each is highly informative. Incorporation of a single spin label into numerous sites within a given region of RNA can also be used to probe secondary structures, such as duplex 4 ACS Paragon Plus Environment

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formation, as monitored by the changes in dynamics upon folding for varying environmental conditions. Furthermore, through spectral simulation of the EPR line shape, timescales of motion can be quantitatively assessed to ascribe, specifically, a measure of flexibility within a sitespecific region of RNA.25-27 In addition to monitoring site-specific local dynamics for a singly incorporated spin label through CW EPR methods, distance measurements between two incorporated labels can also be performed through the pulsed EPR technique commonly known as double electron-electron resonance (DEER) or pulsed electron double resonance (PEDLOR). EPR based distance measurements between two unpaired electrons have matured as methods for aiding in structural refinement and probing conformational changes in biomolecules.28-41 DEER measures throughspace electron spin dipolar coupling and thus requires incorporation of two unpaired spins wherein inter-nitroxide distances in the range of 20-80 Å can readily be determined. For protein systems, well established techniques exist for incorporation of two spin labels for DEER experiments, and the utility of such studies is reflected by a vast and rich literature.28, 30, 32, 33, 35, 36, 42, 43

DEER is also well suited for studying RNAs, and results can readily be applied to study

structure or interpreted to reflect tertiary interactions and conformational dynamics within RNA folding pathways that are important for understanding RNA function.37-41 Specifically, analysis of the resultant distance profiles allows for a description of the conformational equilibria and sampling flexibility under varying conditions, which has been done for protein systems to date.4447

The heterogeneity of RNA conformers present within an ensemble can be separated to provide

information regarding various populations of conformational states for a given environmental condition. Shifts in through-space tertiary interactions and the conformational landscape, which may be induced by ion-induced folding or ligand binding, can therefore be directly monitored. SDSL EPR has been used to a lesser extent in large (> 100 nt) nucleic acids6,

11, 48, 49

owing mostly to the challenge to site-specifically incorporate spin probes into large systems. Solid-phase RNA synthesis imposes a practical limit to the length of RNA that can be efficiently produced (typically up to 100 nt). Therefore, to site specifically spin label the 232 nt VC glycine riboswitch, we followed our previously demonstrated splinted ligation methodologies whereby a relatively short (20 nt), synthetically modified and spin labeled fragment is joined to a larger (212 nt) in vitro transcribed fragment.48, 49


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Riboswitches encompass a unique class of mRNA elements that function to regulate genetic expression through selective recognition and binding of a cognate ligand at threshold cellular concentrations.50,

51

Riboswitch sequences contain an aptamer domain(s) and a

downstream expression platform. Ligand binding to the aptamer induces changes in the conformational dynamics of both domains, governing the expression of downstream genes associated with metabolism of the bound ligand.51 The glycine riboswitch, studied in this work, is unique in that it contains two tandem aptamers connected by a short linker region of several nucleotides.52 Recent investigations have shown that the linker sequence forms a conserved (>90%) P0 duplex with nucleotides in the leader sequence, which is located 5′ to aptamer I (Figure 1A).53,

54

Furthermore, in approximately 50% of glycine riboswitch sequences, the P0

duplex, also termed the leader-linker interaction, engenders the formation of an RNA secondary structural element called a kink-turn motif (Figure 1A).53 Kink-turn motif function is diverse, and includes facilitating tertiary interactions in riboswitch folding and function.55 Specifically, for the glycine riboswitch, the impact of the kink-turn motif on folding, function, and ligand binding affinity has been investigated by biophysical and biochemical techniques including, in-line probing (ILP), electrophoretic mobility shift assays (EMSA), isothermal titration calorimetry (ITC), and small angle X-ray scattering (SAXS).3, 52, 53, 56 The presence of the kink-turn has been shown to promote interaptamer interactions and a globally more compact conformation, which results in an increased glycine binding affinity.3, 53 In this work, we demonstrate the utility of SDSL for probing solution state conformation and dynamics of the kink-turn region with emphasis on environment induced changes of site-specific internal loop nucleobase dynamics and helical structure and packing of the kink-turn motif. In general, SDSL EPR provides a spectroscopic method to interrogate site-specific dynamics and structure in various solution RNA folding states. Within, both continuous wave line shapes and distances from double electron-electron resonance are utilized and discussed. Furthermore, these experiments establish the capacity of SDSL EPR studies to characterize secondary structural elements, such as differentiating local RNA nucleobase dynamics, as well as, discrete RNA folding dynamics for large (>200 nt) RNAs. Materials and Methods. Synthetic RNAs. All chemically synthesized, 20 nt, RNA fragments were purchased from Dharmacon (Lafayette, CO) containing either a single 4-thiouridine modification incorporated at 6 ACS Paragon Plus Environment

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a site-specific nucleobase or two phosphorothioate modifications located within the RNA backbone (Figure 1C&E). Supporting

Information

Sequences for all fragments and modified sites are shown in Table

S1.

Removal

of

the

protecting

group,

2’-O-bis(2-

acetoxyethoxy)methyl, also known as ACE, which is synthetically incorporated into each 2'OH position of the synthetic RNAs proceeded according to the vendor’s instructions. For R5 labeling, RNAs were dissolved in water57 whereas for IAP labeling, RNAs were dissolved in 100 mM sodium phosphate buffer (pH 8)58 (Fisher Scientific, Pittsburgh, PA). RNA concentration was determined using UV absorbance at 260 nm with a Cary WinUV 50 spectrophotometer (Varian Instruments, Walnut Creek, CA) in a quartz cuvette (Agilent Technologies, Santa Clara, CA). All RNA concentrations mentioned hereafter were determined similarly and OligoCalc59 was used to determine the extinction coefficients of each. RNA spin labeling. All deprotected synthetic RNA fragments were spin labeled prior to preparation of the full length RNA constructs via splinted ligation. The rationales for spin label choice were based upon availability of already published protocols, as well as, for the formation of the more stable carbon sulfur bond post spin labeling vs. formation of a reducible disulfide such as with other published spin labeling protocols. Spin-labeling schemes are depicted in Figure 1. For CW EPR experiments, synthetic RNA oligomers containing the 4-thiouridine modification were spin labeled with 3-(2-Iodoacetamido)-PROXYL (IAP), which was purchased from Sigma Aldrich (St. Louis, MO). Spin labeling procedures were followed in accordance with a previously published protocol58 with few modifications. The 4-thiouridine modified RNA oligomers were incubated at room temperature, in the dark with 200-fold molar excess of IAP for 24 hours, and with constant rotation. The RNAs were then PCA extracted, ethanol precipitated, and dissolved in 1X Tris-EDTA (TE) buffer (10 mM Tris-HCl and 1 mM EDTA, pH 7.5) (Fisher Scientific, Pittsburgh, PA) before concentration determination and ligation. For DEER studies, the doubly phosphorothioate modified RNA oligomer was spin labeled with 1-oxyl-2,2,5,5,-tetramehtylpyrroline (R5) utilizing a previously published protocol.57 Accordingly, the R5 spin label was freshly prepared using the R5 precursor, 1-oxyl2,2,5,5-tetramethyl-∆3-methane-sulfonyloxy-methylpyrroline that was purchased from Toronto Research Chemicals, Inc. (Toronto, Ontario). The RNA was incubated with 100 fold molar excess of R5 for 24 hours and the reaction was allowed to proceed in the dark, at room


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temperature, and with constant rotation. Subsequently, the RNA was PCA extracted, ethanol precipitated, and dissolved in TE buffer before concentration determination and ligation. For both CW and DEER RNA constructs, the percentage of spin labeled full-length RNA was estimated by calculating the ratio of spin label concentration to the concentration of RNA. Spin label concentration was determined through utilization of a 4-hydroxy TEMPOL standard curve, whereby spin concentration is obtained by double integration of the X-band (9.5 GHz) CW EPR spectra. Accordingly, double integration of EPR spectra for RNA samples was used to determine the spin concentration of each construct. The concentration of RNAs was calculated, as previously described, using UV absorbance at 260 nm and an extinction coefficient of 2,890,173 (2.89 x 106) M-1 cm-1. Spin labeling efficiency describes both the attachment of spin labels to the corresponding sites within the biomolecule and the resultant EPR active spin concentration. Given it does not distinguish between these two, we report the ratio of spin concentration to RNA concentration to determine the percentage of EPR active spin-labeled RNA for each prepared construct, which averaged approximately 41% for IAP labeled RNAs and 64% for the R5 labeled RNA. In vitro transcribed RNA. The VC glycine riboswitch consists of 232 nt. For EPR constructs, the first 20 nt are derived from a synthetic oligonucleotide sequence, whereas the remaining 212 nt sequence is obtained and purified using in vitro transcription with T7 RNA polymerase according to our previously published procedures.49 For splinted ligation using T4 DNA ligase, the synthetic RNA fragment is used as the 3ʹ OH acceptor, while the in vitro transcribed RNA serves as the 5ʹ monophosphate donor. Therefore, to prepare the triphosphorylated in vitro transcribed RNA for ligation, dephosphorylation using the alkaline phosphatase, FastAP (Thermo Scientific, Waltham, MA), followed by monophosphorylation with the kinase T4 polynucleotide kinase (New England Biolabs, Ipswich, MA), were performed according to our previously published optimized protocol.48 RNA splinted ligation with T4 DNA ligase. Ligation of the synthetic, 20 nt, spin-labeled, RNA oligomers to the 212 nt in vitro transcribed monophosphorylated RNA was achieved using T4 DNA ligase, following a previously published protocol.48 T4 DNA ligase is used in stoichiometric quantities, and it is therefore expressed and purified following previously published protocols.60,

61

For the two IAP spin labeled constructs, procedures were followed

exactly in accordance with our previously published protocol.48 For the R5 spin labeled RNA, the 8 ACS Paragon Plus Environment

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scale of the ligation reaction was increased 4-fold to ensure sufficient sample quantity for DEER experiments. Purification was performed via a 6% denaturing PAGE allowing for the 232 nucleotide ligated product to be separated from the un-ligated RNA. The ligated RNA was excised from the gel and electroeluted overnight at 4°C in TE buffer followed by PCA extraction and ethanol precipitation. The average percent yield of purified ligated RNA was 46%. Continuous Wave (CW) EPR. CW EPR spectra were obtained with a Bruker E500 spectrometer X-Band (9.5 GHz) outfitted with a loop gap resonator (Medical Advances, Milwaukee, WI). Spectra are reported as the average of 20 scans using the following spectral parameters: 100 Gauss (G) sweep width, 0.6 modulation amplitude, 70 ms conversion time, 100 kHz field modulation amplitude and with 2 mW incident microwave power. Sample temperature was monitored by an Omega temperature sensor and probe (OMEGA Engineering, INC, Stamford, CT) and was regulated at 25°C ± 0.3 by passing air through a copper coil submerged in a recirculating water bath at approximately 50-55 °C (Thermo Scientific, Waltham, MA). To help maintain stable sample temperature, a quartz Dewar (Wilmad-Labglass, Vineland, NJ) was utilized that surrounded the loop gap resonator. Four EPR samples for each riboswitch construct were prepared at approximately 100 µM RNA, with spin concentration in the range of 21 µM (U3) - 58 µM (U2), in the following varying conditions: RNA in water only, RNA with 100 mM KCl, RNA with 100 mM KCl and 5 mM MgCl2, and lastly, RNA with 100 mM KCl, 5 mM MgCl2, and 5 mM glycine. The rationale for sequential ion/ligand addition was based upon monitoring changes in RNA dynamics specifically induced by monovalent vs. divalent vs. ligand binding folding. Each sample contained 3-5 µL and was loaded into a flame sealed glass capillary tube with dimensions of 0.6mm I.D. x 0.85mm O.D. (Fiber Optic Center, New Bedford, MA). Spectra were baseline corrected and normalized using Labview software provided by Dr. Christian Altenbach and Dr. Wayne Hubbell (https://sites.google.com/site/altenbach/labviewprograms) and are reported here as double integral area normalized with scaled intensities, accordingly. Data for h(0) and h(+1) intensities were also obtained using these programs and the error bars described for the empirical plot represent the standard deviation based upon triplicate CW EPR measurements for each sample. Triplicate measurements were made on different days to account for fluctuations in temperature stability. DEER spectroscopy. For the doubly R5 spin labeled riboswitch construct, four samples were prepared for DEER distance measurements with the following varying conditions: RNA in 9 ACS Paragon Plus Environment

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D2O only, RNA with 100 mM KCl, RNA with 100 mM KCl and 5 mM MgCl2, and lastly, RNA with 100 mM KCl, 5 mM MgCl2, and 5 mM glycine. Each prepared sample contained approximately 109 µM RNA with (~140 µM total spin-concentration, 64% efficiency) dissolved in D2O with 30% glycerol-d8 (Cambridge Isotope Laboratories, Tewksbury, MA). Each added reagent (salt or glycine) was also prepared using D2O and, after addition of each, the samples were incubated at room temperature for approximately 30 minutes to allow for riboswitch folding. The 40-45 µL samples were loaded into flame sealed, quartz capillary tubes (2 mm i.d., 3 mm o.d., Technical Glass Products, Painesville Twp., PA) and were flash frozen in liquid nitrogen. All DEER measurements were performed at X-Band (9.5 GHz) using a Bruker ELEXSYS E580 spectrometer equipped with the EN 4118X-MD4 resonator at 65 K. A fourpulse DEER sequence was used28 with pulse lengths for π/2 of 16 ns and for π of 32 ns. Spinecho decay experiments were performed for each construct to evaluate the phase memory time and, subsequently, an interpulse delay time of 3 µs was used. For each sample, the observer frequency was set to the maximum of the low field and the pump frequency was set to the center field line of the absorption spectrum, approximately 70 MHz or more apart. Signal averaging was performed for 24 to 48 hours. To determine interspin distances from the resultant dipolar evolution data, Tikhonov regularization (TKR) was performed using DeerAnalysis2013.2 software (http://www.epr.ethz.ch/software). The dipolar modulation curves were long-pass filtered and background corrected with the correct zero time, which was determined by fitting with a Gaussian function.36, 47 The TKR distance profiles were then obtained by selection of the optimal regularization parameter.46 In-line probing. To show that spin label attachment does not alter the function of the glycine riboswitch, in-line probing was performed (details provided in Supporting Information Figure S1). The obtained glycine binding affinities (Kd) are similar to those reported for the wildtype VC glycine riboswitch (5.0 ± 4.0 µM).53 The Kd is 6.3 ± 5.0 µM for site U2 ; the Kd is 4.3 ± 4.1 µM for site U3. In-line probing assays were carried out similar to previously reported procedures.62, 63 The reported binding affinities were calculated as average Kd values obtained from the individual glycine-perturbed regions in two independent trials with curve coefficients better than 0.97.

Results. 10 ACS Paragon Plus Environment

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Differential Nucleobase Dynamics of the Internal Loop Numerous

crystallographic

models

of

kink-turn

(http://www.lifesci.dundee.ac.uk/groups/nasg/kturn/kturns_known.php),64 as

exemplified

Figures 2A-2F for the 23S rRNA of Haloarcula marismortui (PDB:3CC2) and riboswitch (PDB:2GIS), place the nucleotide at position L2

motifs in

the SAM

in a syn conformation that

participates in stacking interactions with nucleotides of the P1 non-canonical helix (position 1n or 1b).65,

66

In contrast, the nucleotide at L3 takes on a solvent exposed conformation in the

folded kink-turn structure. Accordingly, disparate nucleobase dynamics at these analogous sites in the VC glycine riboswitch upon folding of the kink-turn are expected. To probe if these disparate base conformations are present within the kink-turn of the VC glycine riboswitch and to determine which environmental conditions are responsible for the folded structure, we chose to singly label two specific modified nucleobases within the internal loop region of the kink-turn, one containing a spin-labeled thiouridine at L2 and the other at L3. These constructs, which are shown in Figure 1B, are hereafter referred to as U2 and U3, where U2 corresponds to position L2 and U3 to position L3 as defined by the kink-turn nomenclature described by Lilley and colleagues.64, 66, 67 For each construct, X-Band CW EPR spectra were collected under various environmental conditions, such as water only, addition of salts, KCl (K+) and MgCl2 (Mg2+), as well as, glycine ligand. Samples are referred to within as R, RK, RKM and RKMG; respectively. The resultant EPR spectra for U2 and U3 under these varying conditions are shown in Figure 3A (and spectral simulations of each in Supporting Information Figure S2 and Table S2).22, 68 Analysis of the resultant nitroxide line shapes provides a description of the local mobility and changes in dynamics upon environmentally stimulated folding of the kink-turn motif. Nitroxide line shapes are reflective of motional averaging; where narrower and more intense lines are indicative of higher mobility and where broadening is indicative of a more restricted mobility.69 Although the normalized central intensity is a common method for analyzing line shape changes related to local dynamics, because baseline correction in noisy spectra can introduce relatively large errors in area integration; a more robust way to characterize changes in mobility based upon line shapes is to utilize parameters that are not dependent upon area normalization, such as the scaled mobility,70,

71

or the ratio of the intensities of the low field to central field

transitions, h(+1)/h(0),15 or spectral simulation.72, 73 Therefore, we have chosen to plot spectra in 11 ACS Paragon Plus Environment

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Figure 3A with scaled intensity of the central linewidth so that change in the intensities of the h(+1) can be more readily observed and to report values of h(+1)/h(0) in Fig. 3B. Inspection of the spectra in Figure 3A leads to the following conclusions. Firstly, spectra for both U2 and U3 in water only are strikingly similar with line shapes reflective of fast isotropic mobility. Spectral simulations reveal “fast limit” motion with correlation times of 0.4 ns (simulation details given in Supporting Information and Figure S2).21, 74 This mobility is consistent with what would be expected for an unfolded/unstructured state in water. Secondly, the addition of K+ alters the line shapes of both sites, but the change in mobility is more dramatically decreased for U2, while having lesser of an impact on U3. Specifically, although the dynamics at site U3 decrease, this spectral line shape is still indicative of nearly isotropic motion with correlation time of 0.9 ns. In contrast, the line shape for U2 has more dramatic broadening of the high and low field transitions, with a simulated correlation time near 1.3 ns. Additionally, the values of the h(+1)/h(0) ratio of intensities exemplifies the differential mobility at these two sites when folding of the kink-turn is induced by K+; where a higher value of the h(+1)/h(0) indicates higher mobility. These spectral changes nicely reflect site specific dynamics that agree with folding-induced conformational changes whereby base stacking occurs at U2 vs solvent exposure of the nucleobase at U3. Thirdly, for both sites, neither the further addition of Mg2+, nor, glycine ligand induce significant changes in spin label mobility (statistical analysis of data in Figure 3B provided in the Supporting Information, Figure S3); suggesting that conformation of the internal loop structure of the kink-turn is likely firmly established upon addition of monovalent potassium ions. Although SAXS data demonstrate the presence of physiologically relevant concentrations of Mg2+ induces a more globally compact structure of the VC glycine riboswitch in both the glycine free and bound states,3 the EPR data show that this compaction does not alter the base dynamics of the kink-turn internal loop. Note, this result is not inconsistent with SAXS findings as the EPR results report on site-specific conformational changes and dynamics. The EPR data also indicate that in the presence of high potassium ion concentration, further addition of Mg2+ (which has been shown to induced compaction) nor binding of glycine to the aptamer domains transmit a change to the nucleobase dynamics of the kink-turn region of the VC glycine riboswitch. Modeling of Spin-Labels into Kink-Turn Structures


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Currently, a crystal structure for the full length VC glycine riboswitch does not exist and the solved structure for the Fusobacterium nucleatum (FN) glycine riboswitch does not include the kink-turn motif. Therefore, multiscale modeling of macromolecular systems (MMM) was utilized to model attachment of IAP to a stacked vs. solvent exposed 4-thiouracil nucleobase (details regarding spin label modeling are provided in Supporting Information and Figure S4); In the absence of an exact structural model, two alternative kink-turn motif crystal structures were selected for modeling including the 23S rRNA from Haloarcula marismortui (PDB: 3CC2) (kink-turn 42) and the SAM riboswitch (PDB: 2GIS). These two kink-turns were chosen based upon the sequences containing uridine nucleotides located at positions L2 and L3, analogous to the labeled sites U2 and U3 in the VC glycine riboswitch which is required for MMM modeling of IAP attachment to uracil nucleobases. We identified these utilizing the structural database for kink-turn

motifs,

provided

by

Lilley

and

colleagues64

(http://www.lifesci.dundee.ac.uk/groups/nasg/kturn/). The most probable IAP rotamers, as shown attached in Figure 2E and F, as well as in Figure S4, nicely demonstrate the disparate local environments of each attached spin label in agreement with the observed different nitroxide line shapes in Figure 3A. DEER Studies of Kink-turn Motif Conformational Shifts. The effects of sequential addition of KCl (K+), MgCl2 (Mg2+), and glycine ligand on the conformational dynamics of the formation of the P0 helix and its relative structural relationship to the P1 helix in the VC glycine riboswitch were monitored by DEER distance measurements. DEER distance profiles provide information about conformational changes and flexibility that can be obtained from both the most probable distance and span of distances; as these parameters are reflective of conformational flexibility and structural changes, which can be monitored for various environmental conditions.36, 47 For these experiments, two R5 spin labels, as shown in Figure 1D&E, were incorporated into backbone phosphorothioate sites.48, 57 The two sites chosen for labeling with R5 are shown in Figure 1D, and are located immediately 5ʹ to position -3b in the P0 duplex and immediately 3ʹ to position 3b in the P1 duplex of the kink-turn. The rationale for location choice is as follows. We desired one spin label to be located within the P1 helix, where we expected, based upon previous ILP studies,53 little to no structural variations to occur under the differing environmental


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conditions.56 The other site was chosen based upon both previous ILP and R5 spin labeling studies where we knew the P0 helix would form upon addition of K+.49, 53 With no crystal structure for the VC glycine riboswitch available and with the solved structure for the Fusobacterium nucleatum (FN) riboswitch lacking the leader sequence, we chose the kink-turn motif in the crystal structure of the SAM riboswitch (PDB: 2GIS), based upon sequence similarity, to estimate anticipated distances between SDSL sites by MMM analysis. Optimal distances for measurement by DEER range between 20-80 Å; accordingly, spin labeled sites are judiciously selected to reside within this range. Modeling with MMM, as described in previous sections, was again employed to attach R5 spin labels within the kink-turn motif of the SAM riboswitch crystal structure. It is known that the R5 labeling of phosphorothioate sites generates R and S diastereomers, and we evaluated the effects of the diastereomers on the expected distance profile. (For more information see Supporting Information Figure S5). From this model, distances in the range of 20 and 35 Å are expected; albeit structural variations may exist given this modeling was performed on the SAM riboswitch. DEER results are given in Figure 4 and Table 1. DEER experiments were performed for the same four varying environmental conditions described for CW EPR experiments (R, RK, RKM, RKMG) and interspin distances were determined for each sample. The backgroundcorrected dipolar modulation curves are shown in Figure 4A, and Figure 4B shows the corresponding distance profiles for the four varying environmental conditions. Completed details of DEER data analysis can be found in Supporting Information Figures S6-11. Table 1 summarizes the most probable distances and breadths of the profiles for each. Based upon the work of Lilley and colleagues, it is expected that in the unfolded state, the kink-turn exists in an extended conformation,66,

67

and that stabilization of the kinked

conformation can be induced by 1) binding of L7Ae and related proteins, 2) tertiary interactions, and 3) addition of either divalent or monovalent ions at µM or mM concentrations; respectively.55, 66, 75 The latter observation is consistent with our previous CW EPR backbone dynamic investigations utilizing single R5 labels within the kink-turn,49 suggesting that the kinkturn is unfolded and extended in the absence of potassium and magnesium ions. In the absence of added salts and ligand, DEER results are consistent with an unfolded P0 helix, where, a broad inter-nitroxide distance of 42 Å is observed and spans the range of 15 to 53 Å. Upon addition of K+ (RK sample), the inter-nitroxide distance shifts by 4-5 Å to 38 Å and the distance profile 14 ACS Paragon Plus Environment

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narrows, reflective of folding to a conformer with less conformational heterogeneity/flexibility. The trends of these DEER data for the folding of the P0 helix are similar to DEER results for monitoring a folding/unfolding transition in an outer membrane transporter.76 In those studies, for the unfolded state, broader DEER profiles are obtained with evidence for a distribution of conformational states. Here, however, because of the distribution of distances expected for the mixture of R/S diastereomers, we cannot make any conclusions regarding equilibrium distribution of conformations. Although the presence of R/S diastereomers did not impact analysis of distances in model CS DNA,77 MMM modeling of the SAM riboswitch predicts that alternate R/S diastereomers can generate different distances that cluster in populations centered nearly 10-15 Å apart (Figure S5). As expected, no further change in the distance was observed upon subsequent addition of Mg2+ (RKM sample) and glycine (RKMG sample). However, a slight narrowing of the breadth of the profile is observed. These results are in agreement with the expectation of a conformational shift from an extended to closed/folded kink-turn structure upon ion-induced stabilization. Furthermore, the indistinguishable change in distance from the RK to RKM to RKMG conditions but with a slight decrease in the overall breadth of the distance profile again suggests that this stabilized form of the kink-turn structure is well established by K+ alone and any further compaction of the riboswitch by Mg2+ or glycine induced conformational changes within the aptamer domains are only minimally transmitted to the P0/P1 helix interactions. As a control experiment, a mutant construct was generated to disrupt the P0 duplex/leader-linker interaction, and DEER experiments were performed accordingly as with the wild-type VC glycine riboswitch (full details of construct and data analysis are given in Supporting Information Figures S10-11, and results are included in Table 1). Results for the mutant construct in conditions commensurate with the fully folded WT (RKMG), illustrate an open-like conformation wherein the most probable distance is 3 Å greater than that of WTRKMG and with a FWHM value nearly double that for WT in the absence of ions or ligand (R condition; FWHM of 26.9 for the mutant vs. 13.8 for wild-type). Discussion. SDSL CW EPR and DEER studies provide a powerful approach for studying the role of site specific interactions in the context of local dynamics, as well as, environmental conditions and therefore allow for connections between structure, dynamics, and biomolecular folding to be 15 ACS Paragon Plus Environment

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established. In this work, SDSL EPR, in conjunction with T4 DNA ligase mediated splinted ligation, was used to investigate site-specific dynamics and conformational flexibility of the kink-turn motif found in the 232 nt VC glycine riboswitch. The expected disparate local environments and nucleobase interactions at positions L2 and L3 within the internal loop were monitored for varying RNA environmental conditions of the kink-turn, as well as, the conformational flexibility of the kink-turn helices, as probed through distance measurements obtained from DEER. Overall, our results from both CW and DEER experiments demonstrate that the structural formation of the VC glycine riboswitch kink-turn occurs in the presence of high concentration K+ at 25°C. Kink-turn folding has been studied extensively including work showing

how

kink-turn

binding

proteins,

tertiary

interactions,

and

metal

ion

species/concentration impact fold. Our findings are discussed below within the context of this work of others. A rich literature of kink-turn motif studies exists wherein probing of both structure and folding under varying environmental conditions have been tested.1, 5, 55, 65-67, 75, 78-81 In the work of Lilley and colleagues, stabilization of the kink conformation has been shown to be influenced by the addition of alkali and alkaline earth ions, binding of L7Ae and related proteins, and tertiary structural elements within larger RNAs.55,

75, 79

Although, these investigations of ion-induced

folding of the kink-turn have included both monovalent and divalent ions, most of these studies focused on either the effects of Na+ or Mg2+. Their 2004 FRET studies showed that either ions can induce the kinked conformation of kink-turns; however, folding with monovalent ions (Na+) is obtained only at higher concentrations (~ 90 µM for divalent Mg2+ and ~30 mM for monovalent Na+).75 Furthermore, in their 2014 study, it was found that the sequence at the 3b•3n position can behave as a discriminator for kink-turn ion-dependent folding.81 Likewise, it was proposed that kink-turn sequences containing 3b•3n = A•G are predisposed to ion-induced folding, with the postulated rationale based upon an interaction between 3n = G and hydrated metal ions (either divalent or monovalent) as seen in a solved crystal structure. Our CW EPR and DEER results nicely agree with these previous ion-induced kink-turn folding observations and indeed our kink-turn sequence follows the 3b•3n = A•G. For our SDSL EPR investigations, high concentrations of K+ (100 mM) were utilized, given this concentration mimics cytoplasmic potassium ion concentrations, to test stabilization of the kink-turn under monovalent metal ion


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conditions and to observe if the further addition of divalent Mg2+ (5 mM) would influence additional folding of the kink-turn in VC glycine riboswitch. Work by Ferre-D’Amare and colleagues showed that for the kink-turn containing VC glycine riboswitch, addition of Mg2+ leads to further compaction (relative to constructs lacking the kink-turn) as observed by SAXS. Their SAXS work also showed that the kink-turn binding protein, YbxF, when bound to the Bacillus subtilis (BS) glycine riboswitch also induces a more compact conformation. Our findings are not inconsistent with these previously published data. SAXS provides information regarding the global structure of the riboswitch; whereas, SDSL results, as the name implies, provides site specific information for regions that contain the spinlabels. The SDSL findings reported within are specific only for the kink-turn and do not directly report on overall compaction. Importantly, our work adds to the understanding of conformational changes in the VC glycine riboswitch. The ion-induced shift observed for our DEER study reports changes within/between the kink-turn helices and shows that the global Mg2+ induced compaction observed by Ferre-D’Amare, as well as, ligand binding-induced folding by the aptamers, is not transduced to the kink-turn region. Future SDSL efforts are centered on introducing spin labels into the glycine binding loops such that site-specific information can also be obtained. By judicious choice of spin-labels throughout the riboswitch sequence, compaction and overall structural constraints may also be forthcoming; as these methods are oftentimes applied to protein systems. 82-84 Advantages of the site-specificity afforded by SDSL EPR can be seen when comparing the results from thiouridine nucleobase labeling, as reported here, to kink-turn internal loop backbone labeling, as reported earlier by our group. Via thiouridine base labeling, the ion induced folding is readily observed for both sites and the dynamics decreases compared to the sample prepared in water only (ie kink-turn unfolded). Additionally, it is seen that there are distinct differences in base dynamics between two consecutive nucleotides U2 and U3 within the loop of the kink-turn. The higher dynamics of the U3 is consistent with X-ray structures of kinkturns where the nucleobase at U3 is solvent exposed and the slower dynamics of the spin-label at U2 is consistent with base stacking seen for this site. Interestingly, in our previous work, R5 backbone labeling between these two sites (at the phosphate between U2 and U3) shows that the dynamics does not change from the unfolded water only structure to the ion-induced folded structure. These results are not conflicting, but simply indicate that alterations in base dynamics 17 ACS Paragon Plus Environment

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can occur with no observable change seen for the backbone dynamics. We believe this is an exemplary case that highlights the unique advantages of utilizing SDSL EPR: the ability to probe and distinguish local dynamics between the phosphate backbone, ribose sugar, or nucleobase of a given nucleotide. It is noteworthy to point out that the R5 spin-label used for our previous backbone labeling studies is quite dynamic and other specialized probes have been synthesized where the spin-label motion is minimized so that the more subtle changes in backbone dynamics may be interrogated.26 Perhaps differences in the backbone dynamics at the site between U2 and U3 can be observed if different labels are utilized or higher frequency investigations are undertaken.21, 22 The work described within emphasizes the capacity of CW EPR and DEER studies to monitor site-specific secondary and tertiary folding of large dynamic RNAs, as modulated by varying solution conditions, which is important for furthering an understanding of the relationship between structure, conformational flexibility, and function. The ligation strategy allows for the characterization of the sequential effects of K+, Mg2+, and ligand induced RNA folding dynamics for the large VC glycine riboswitch, as opposed to an in-trans assembly approach for spin-labeling where sequential addition of salts to monitor pre-ligand induced folding may not be feasible.6 This is of particular importance as ligand-induced conformational changes in riboswitches are directly responsible for mediating genetic regulatory function; thus, elucidation of both pre-ligand induced vs. ligand-induced dynamics and conformations is required for a detailed mechanistic evaluation that interfaces an understanding of riboswitch structure, dynamics, and function. As demonstrated from the studies presented here, we learn that, for the glycine riboswitch, pre-ligand induced formation of the functionally relevant kinkturn motif is not further influenced by the conformational change that occurs upon glycine binding. Conclusion. Here we have demonstrated the feasibility and utility of applying EPR studies to spectroscopically follow conformational dynamics of the large 232 nt VC glycine riboswitch kink-turn motif. Utilizing this approach, site-specific riboswitch folding from the ligand free to ligand bound state was monitored in addition to the influence of ion-induced RNA folding and establishes a foundation for our continued studies of characterizing interaptamer conformational flexibility, as well as, the ability to obtain distance measurements for structural refinement. We 18 ACS Paragon Plus Environment

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believe this work complements well established methods such as in-line probing (ILP) and selective 2ʹ-hydroxyl acylation analyzed by primer extension (SHAPE) to study RNA dynamics, as well as, spectroscopic methods such as FRET and NMR. However, SDSL EPR methods uniquely differentiates itself from other techniques in its ability to distinguish disparate dynamics within regions of a single nucleotide, as well as, the ability to assess conformational equilibria within an RNA folding pathway with no biomolecular size limitation. Thus, this works sets a solid foundation for future efforts aimed at structural refinements for large RNAs or more detailed investigations about changes in secondary and tertiary interactions in large dynamic RNAs as a function of environment/ligand. It is noteworthy that SDSL EPR does not have an upper molecular size limitation, advocating the utilization of this methodology for large proteinnucleic acid complexes or other RNA-RNA interactions.

Acknowledgments. We would like to thank Peter Qin and Carol Fierke for helpful discussions regarding RNA SDSL and ligation strategies, as well as, the Herschlag lab for generously providing T4 DNA ligase plasmid. We would also like to thank Gunnar Jeschke for updating the MMM software to allow for modeling of RNA structures with various spin labels and thank Dr. Christian Altenbach and Dr. Wayne Hubbell for graciously providing Labview based software for baseline correction and normalization of spectra

Supporting Information. Supporting materials may be accessed free of charge online at hppt://pubs.acs.org and are available for RNA sequence information, statistical analysis of significant changes for varying riboswitch environmental conditions, CW EPR simulations, DEER data processing, and MMM rotamer analysis details.

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Strobel, S. A., and Cech, T. R. (1995) Minor groove recognition of the conserved G.U pair at the Tetrahymena ribozyme reaction site, Science 267, 675-679. Soukup, G. A., and Breaker, R. R. (1999) Relationship between internucleotide linkage geometry and the stability of RNA, RNA 5, 1308-1325. Regulski, E. E., and Breaker, R. R. (2008) In-line probing analysis of riboswitches, Methods Mol. Biol. 419, 53-67. Schroeder, K. T., McPhee, S. A., Ouellet, J., and Lilley, D. M. J. (2010) A structural database for k-turn motifs in RNA, RNA 16, 1463-1468. Lilley, D. M. (2014) The K-turn motif in riboswitches and other RNA species, Biochim. Biophys. Acta 1839, 995-1004. Lilley, D. M. J. (2012) The structure and folding of kink turns in RNA, Wiley Interdiscip Rev RNA 3, 797-805. Liu, J., and Lilley, D. M. (2007) The role of specific 2'-hydroxyl groups in the stabilization of the folded conformation of kink-turn RNA, RNA 13, 200-210. Stoll, S., and Schweiger, A. (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR, J. Magn. Reson. 178, 42-55. Klare, J. P., and Steinhoff, H. J. (2009) Spin labeling EPR, Photosynth Res 102, 377-390. Langen, R., Cai, K. W., Altenbach, C., Khorana, H. G., and Hubbell, W. L. (1999) Structural features of the C-terminal domain of bovine rhodopsin: A site-directed spinlabeling study, Biochemistry 38, 7918-7924. Huang, H., and Cafiso, D. S. (2008) Conformation and Membrane Position of the Region Linking the Two C2 Domains in Synaptotagmin 1 by Site-Directed Spin Labeling, Biochemistry 47, 12380-12388. Budil, D. E., Lee, S., Saxena, S., and Freed, J. H. (1996) Nonlinear-least-squares analysis of slow-motion EPR spectra in one and two dimensions using a modified LevenbergMarquardt algorithm, J. Magn. Reson., Ser A 120, 155-189. Freed, J. H. (1976) Esr Studies of Spin Probes in Anisotropic Media, ACS Symp. Ser., 115. Barnes, J. P., Liang, Z., McHaourab, H. S., Freed, J. H., and Hubbell, W. L. (1999) A multifrequency electron spin resonance study of T4 lysozyme dynamics, Biophys. J. 76, 3298-3306. Goody, T. A., Melcher, S. E., Norman, D. G., and Lilley, D. M. (2004) The kink-turn motif in RNA is dimorphic, and metal ion-dependent, RNA 10, 254-264. Freed, D. M., Lukasik, S. M., Sikora, A., Mokdad, A., and Cafiso, D. S. (2013) Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex, Biochemistry 52, 2638-2648. Cai, Q., Kusnetzow, A. K., Hubbell, W. L., Haworth, I. S., Gacho, G. P., Van Eps, N., Hideg, K., Chambers, E. J., and Qin, P. Z. (2006) Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe, Nucleic Acids Res. 34, 4722-4730. Daldrop, P., and Lilley, D. M. (2013) The plasticity of a structural motif in RNA: structural polymorphism of a kink turn as a function of its environment, RNA 19, 357364. Huang, L., and Lilley, D. M. (2013) The molecular recognition of kink-turn structure by the L7Ae class of proteins, RNA 19, 1703-1710. Klein, D. J., Schmeing, T. M., Moore, P. B., and Steitz, T. A. (2001) The kink-turn: a new RNA secondary structure motif, EMBO J. 20, 4214-4221. McPhee, S. A., Huang, L., and Lilley, D. M. J. (2014) A critical base pair in k-turns that confers folding characteristics and correlates with biological function, Nat Commun 5. Duss, O., Yulikov, M., Allain, F. H., and Jeschke, G. (2015) Combining NMR and EPR to Determine Structures of Large RNAs and Protein-RNA Complexes in Solution, Methods Enzymol. 558, 279-331. Yang, Y., Ramelot, T. A., McCarrick, R. M., Ni, S., Feldmann, E. A., Cort, J. R., Wang, H., Ciccosanti, C., Jiang, M., Janjua, H., Acton, T. B., Xiao, R., Everett, J. K., 23 ACS Paragon Plus Environment

Biochemistry

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84.

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Page 25 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Tables. Table 1. Summary of Experimental Distances for Varying Environmental Conditions Environment

Most Probable

Average

FWHM

Span

Distance (± 0.2 Å)

(± 0.2 Å)

(± 0.3 Å)

(± 0.2 Å)

R

42.1

37.9

18.3

38

RK

37.6

35.7

14.9

31

RKM

37.0

34.6

14.5

35

RKMG

36.7

34.3

13.8

34

Mut-RKMG

39.7

31.0

26.9

32


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figures and Figure Legends. Figure 1. (A) Secondary structure of the 232 nt VC glycine riboswitch with the kink-turn motif boxed in black, the P0 duplex, also known as, the leader-linker interaction boxed in red, and the P1 duplex boxed in purple. (B) Kinkturn sequence showing the chosen nucleobase labeling sites for CW EPR studies and the (C) SDSL reaction scheme for nucleobase labeling with IAP at 4-thiouridine modified sites. (D) Doubly labeled R5 sites for DEER experiments and (E) SDSL reaction scheme for R5 backbone labeling at phosphorothioate modified sites.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 2. Crystal structures of the (A) 23S rRNA of Haloarcula marismortui (PDB:3CC2) (kink-turn 42) and (B) SAM riboswitch (PDB:2GIS) with the kink-turn motif boxed and enlarged for each displaying the (C) stacked nucleobase at position L2 in yellow and (D) the solvent exposed nucleobase at position L3 in red. Most probable IAP spin label rotamer as determined by multiscale modeling of macromolecular systems (MMM) software (http://www.epr.ethz.ch/software) attached to the (E) stacked nucleobase and (F) solvent exposed nucleobase. Green color indicates prospective stacking partners for L2 and cyan color represents the spin label attachment point.


Biochemistry

A

+ RNA KCl MgCl2 Glycine h(+1)

+ + -

+ + + -

B

+ + + +

0.8

U2 U3 Stacked Base Exposed Base

0.7

h(0) h(+1)/h(0)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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h(-1)

U2

0.6 0.5 0.4

τcorr = 0.4 ns

τcorr = 1.3 ns

τcorr = 1.3 ns

τcorr = 1.3 ns

0.3

RNA KCl MgCl2 Glycine

+ -

+ + -

+ + + -

+ + + +

+ -

+ + -

+ + + -

+ + + +

U3 20 G

τcorr = 0.4 ns R

τcorr = 0.9 ns RK

τcorr = 1 ns

τcorr = 1 ns RKMG

RKM

Figure 3. (A) 100G X-Band CW EPR spectra for site U2 (top) and site U3 (bottom) as environmental conditions change (salts and ligand added). Spectra are scaled to equal intensity of the central transition so that changes in the h(+1) resonance line can be more readily observed. Dashed lines are guides for the eyes. Correlation times obtained from simulation using an isotropic model are reported next to each spectrum. (B) Values of h(+1)/h(0) for sites U2 and U3 for the R, RK, RKM and RKMG environments. Data points represent the average values of three separate measurements where the error is represented by the size of the data point. Changes among RK, RKM, RKMG are not significant at the 95% confidence level. Changes between R and RK are significant at the 99.9% for both sites.


Page 29 of 36

A

B R

R RK

0

RK

P(r)

Echo Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

RKM

RKM

RKMG

RKMG

1

2

3

20

τ (μs)

30

40

50

60

Distance (Å)

Figure 4. (A) Background corrected and long-pass filtered DEER modulation curves with the corresponding overlaid TKR fit (sold gray line) for each environmental condition. Curves are vertically offset for clarity. (B) Corresponding distance profiles for each environmental condition. Curves are vertically offset for clarity. The vertical lines represent the most probable distances of 42.1Å in the RNA in water only (R – black dashed) and 36.7 Å in conditions known to promote full folding of the VC glycine riboswitch (RKMG – black solid). Full details of data processing are given in the Supporting Information.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 36

For table of contents use only: Graphic for the Table of Contents. (ToC Graphic).

Conformational Flexibility and Dynamics of the Internal Loop and Helical Regions of the KinkTurn Motif in the Glycine Riboswitch by Site-Directed Spin Labeling Jackie M. Esquiaqui,1 Eileen M. Sherman,2 Jing-Dong Ye,2 Gail E. Fanucci,1*


Page 31 of 36

1 2 3 4 5A 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

B

C

GU Site 2: U3 3bA G3n Site 1: U2 Stacking Solvent 1bA G1n Base -3n GA 3′ Exposed L3 U G G A A C UGC C U U G U U 5′ L2 L1 -3b

D

P1

O N

Kink-turn motif

3b

3n

1b L3 L2

1n -3n L1

-3b

P0 Duplex Leader Linker Interaction

GU 3b A G3n A G 1bG A1n -3n 3′ G G A AC L3 U U L2 GC C U U G U U 5′ L1

Distance?

-3b

N

O

ACS Paragon Plus Environment

S

IAP

NH N O

O H O

I

O

N N O

N O

O

H OH

O

O NH

N O

H H O H S P O O

N O

O

O H O

H OH

E O

O

H N

H N

S

O

R5 ON

NH N

I

O

ON

O

H H O H S P O O

O

Biochemistry

1 2 3 4 5 A 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 B 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

C

Page 32 of 36

E

3ʹ

5ʹ

5ʹ Stacked base Stacking partners Exposed base Spin label attachment

D

F

3ʹ

3ʹ

5ʹ

Stacked base Stacking partners 5ʹ Exposed base Spin label attachment


3ʹ

IAP Spin label Attached

IAP Spin label Attached

Page 33 of 36

1 2 3 4 RNA 5 KCl MgCl2 6 Glycine 7 8 9 h(+1) 10 11 U2 12 13 14 τ = 0.4 ns corr 15 16 17 18 19 20 21 U3 22 23 τ = 0.4 ns corr 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

Biochemistry

+ -

+ + + -

+ + -

B

+ + + +

U2 U3 Stacked Base Exposed Base

h(0) h(-1)

τcorr = 1.3 ns

τcorr = 1.3 ns

τcorr = 1.3 ns

RNA KCl MgCl2 Glycine 20 G

R

τcorr = 0.9 ns RK

τcorr = 1 ns

RKM

τcorr = 1 ns RKMG


+ -

+ + -

+ + + -

+ + + +

+ -

+ + -

+ + + -

+ + + +

Biochemistry

A

B R

R RK

RK

P(r)

Echo Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 36

RKM

RKM

RKMG

RKMG

τ (μs)

Distance (Å)


Page 35 of 36

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Biochemistry


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

82x36mm (300 x 300 DPI)


Page 36 of 36

Conformational Flexibility and Dynamics of the Internal Loop and

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