Supplementary Information

Transcription

1 Supplementary Information Single molecule FRET reveals the energy landscape of the full length SAM I riboswitch Christoph Manz, 1,2 Andrei Yu. Kobitski, 1 Ayan Samanta, 3 Bettina G. Keller 4, Andres Jäschke, 2,3 and G. Ulrich Nienhaus 1,2,5,6,*. 1 Institute of Applied Physics, Karlsruhe Institute of Technology, Wolfgang Gaede Str. 1, Karlsruhe, Germany 2 HEiKA Heidelberg Karlsruhe Research Partnership, Karlsruhe Institute of Technology, Hermannvon Helmholtz Platz 1, Eggenstein Leopoldshafen, Germany 3 Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, Heidelberg, Germany 4 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Takustr. 3, Berlin, Germany 5 Institute of Nanotechnology and Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Hermann von Helmholtz Platz 1, Eggenstein Leopoldshafen, Germany 6 Department of Physics, University of Illinois at Urbana Champaign, Urbana, Illinois 61801, USA *Corresponding author e mail: uli@uiuc.edu 1

2 Supplementary Results Supplementary Table 1. Total number of molecules analyzed for compilation of the FRET histograms in Figs. 2 and 4 and Supplementary Figs. 1 and 2. [Mg 2+ ]/ mm RS AA RS EA RS [Mg 2+ ] = 20 mm No. of molecules [Mg 2+ ]/ mm No. of molecules [SAM]/ µm No. of molecules

3 Supplementary Table 2. Peak positions of subpopulations determined by global fits of FRET histograms, and the resulting inter dye distances for riboswitch constructs RS EA and RS AA at <0.1 mm and >20 mm Mg 2+ concentration (na, not available; nd, not determined). Conformation [Mg 2+ ] < 0.1 mm [Mg 2+ ] > 20 mm E r( E ) / Å E r( E ) / Å RS EA AT AT T T RS EA /SAM SAM AT 1 na na SAM AT 2 na na SAM T 1 na na SAM T 2 na na RS AA AT AT 2 nd nd T T nd nd 3

4 Supplementary Figure 1. Mg 2+ and SAM dependent smfret histograms of SAM I riboswitch construct RS EA. The data were acquired by burst analysis of intensity time traces taken with freely diffusing molecules on the confocal microscope. (a) Histograms for selected Mg 2+ ion concentrations between 0 and 100 mm (grey bars). Colored lines represent best fit results with four states modelled by Gaussian distributions; the dashed black lines show the sum. The entire data set was fitted globally by using the following constraints: (i) For the data at 12.5 mm, peak positions were fixed to the values obtained from the HMM analysis; (ii) fractions of the T 1 state (green) for data with [Mg 2+ ] < 2 mm were restricted (within the error) to the values obtained from individual two state model fits of FRET efficiency histograms of construct RS AA at the corresponding Mg 2+ concentrations; (iii) the full width at half maximum (FWHM) values of the four subpopulations were set to be Mg 2+ independent; the fit yielded the values of 0.16, 0.2, 0.2 and 0.22 for the T 1 (green), T 2 (blue), AT 1 (violet) and AT 2 (orange) states, respectively. (b) smfret histograms at various SAM ligand concentrations between 0 and 10 µm in the presence of 20 mm Mg 2+. FRET efficiency histogram at 10 µm of SAM concentration was fitted with four Gaussian model distributions, introducing constraints on the peak positions according to the HMM result; the dashed black line shows the sum. 4

5 Supplementary Figure 2. Mg 2+ dependent smfret histograms of SAM I riboswitch construct RS AA. The data were taken on immobilized molecules at various concentrations of Mg 2+ by collecting ptirfm images with 1 s exposure time. Lines represent global fits of all histograms with a three state model using the following constraints: (i) fractions of the T 1 (green) and AT 1 (red) states were restricted (within the error) to the values from the global fit of FRET efficiency histograms of the construct RS EA with a four state model; (ii) full widths at half maximum (FWHM) were set to be Mg 2+ independent; the fit yielded values of 0.21, 0.21, and 0.28 for T 2 + AT 2 (cyan), T 1 (green), and AT 1 (violet), respectively. 5

6 Supplementary Figure 3. Validation of hidden Markov models. (a) Time window dependent FRET efficiency histograms with 10, 50 and 250 ms bin widths of the experimentally determined data set (black line) and its 95% confidence interval (region between the grey lines) and the HMM prediction (red line). (b) Residence time distributions of the individual states calculated from the maximumlikelihood paths. The different states are colored as in Figure 3. 6

7 Supplementary Figure 4. Mg 2+ dependence of fractional areas and peak positions of the Gaussian distributions resulting from global fits of smfret histograms. Legends assigning the data to specific states are given in the insets. Lines through the data points are fits of either fractional subpopulations or peak positions, X, with a Hill equation X([Mg 2+ ]) = X(0) + ΔX/(1+[Mg 2+ ] 0.5 /[Mg 2+ ]), where [Mg 2+ ] 0.5 is the transition midpoint concentration. (a) RS EA construct; the fractional populations show exchanges between T 2 and AT 2 with [Mg 2+ ] 0.5 = (1.5 ± 0.6) mm and between T 1 and AT 1 with [Mg 2+ ] 0.5 = (7.5 ± 0.5) mm. The peak positions of all subpopulations shift to higher FRET efficiencies with [Mg 2+ ] 0.5 = (8 ± 1) mm, although to a different extent. (b) RS AA construct; dashed lines depict the fractional populations of RS EA ; the solid cyan line represents the sum of the orange and blue dashed lines. The peak positions of the T 1 and AT 1 states shift markedly ([Mg 2+ ] 0.5 = (8 ± 1) and (15 ± 2) mm, respectively). Also shown are the average peak positions of 0.09 and 0.07 FRET efficiency at low (< 1.5 mm, blue) and high (> 1.5 mm, orange) Mg 2+ concentration for the sum of the T 2 and AT 2 states. Error bars represent uncertainties given by the non linear least squares fitting software (OriginPro 2015). 7

8 Supplementary Figure 5. Alternative secondary structure model of the B. subtilis yitj SAM I riboswitch (169 nt fragment) in the anti terminator (AT) conformation. Base pairing in the putative P1 and P1 regions is indicated by dots. Donor (Cy3) and acceptor (Cy5) dye labeling sites of the two labeled constructs (RS AA and RS EA ) are shown as green and red stars, respectively. Biotin is represented by a black diamond at the 3 end. 8

9 Supplementary Figure 6. Schematic diagram of the confocal microscope with single molecule sensitivity. Abbreviations: AOTF acousto optical tunable filter, APD avalanche photodiode, DM dichroic mirror, F optical filter, L lens, M mirror, P pinhole, PC personal computer, T telescope lens. Details are described in Methods. 9

10 Supplementary Figure 7. Schematic depiction of the AOTF based programmable beam splitter of the confocal microscope. It consists of four acousto optical tunable filters (AOTF, Model # , Crystal Technology, Palo Alto, CA) and allows us to excite the sample with one of four wavelengths in a computer controlled fashion by using a multi function data acquisition card (PCI 6713, National Instruments, Austin, TX). AOTF1 deflects the incoming laser beam into the first order diffraction maximum emitted along the optical axis of the objective. The other three AOTFs are required to compensate for dispersion and birefringence effects introduced by AOTF1. Shown are the excitation and emission light paths through the four AOTFs. Arrows mark the common usage direction for coupling into the zeroth order. Circles with crosses mark the top and empty circles the bottom of the AOTF crystals. 10

11 Supplementary Figure 8. Schematic diagram of the ptirf microscope setup. Abbreviations: AOTF acousto optical tunable filter, BS beam splitter, DM dichroic mirror, F optical filter, L lens, M mirror. The close up schematic shows RNA molecules immobilized by using BSAbiotin/streptavidin/biotin RNA. For details, see Methods. 11