Supplementary Information. Single-molecule analysis reveals multi-state folding of a guanine. riboswitch

Transcription

1 Supplementary Information Single-molecule analysis reveals multi-state folding of a guanine riboswitch Vishnu Chandra 1,4,#, Zain Hannan 1,5,#, Huizhong Xu 2,# and Maumita Mandal 1,2,3,6* Department of 1 Chemistry, 2 Physics and 3 Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA. 4 Present Address: Rutgers New Jersey Medical School, Newark, NJ 07103, USA. 5 Present Address: Department of Chemistry, Swarthmore College, Swarthmore, PA 19081, USA. 6 Present Address: Single-Molecule and RNA Biology Institute, Pittsburgh, PA 15218, USA # These authors contributed equally to this work. * To whom correspondences should be addressed: maumita.mandal@gmail.com 1

2 Supplementary Results Supplementary Table 1: Structural transitions and distances. We used a serial worm-like chain model (Eq. 3) to relate the change in unfolding distances with the number of unfolding nucleobases. As shown below, the theoretical ( X) and the observed ( X obs) distances agree with each other within the error range. The helix-width (~2 nm) is subtracted or added from the net folding distance as appropriate. In the force-ramp assay, X obs= ( ) ± 1.0 nm = 29.6 ± 1.0 nm, which can be related to the unfolding of 66 ± 2.3 nts for the complete aptamer. In the CF assay, the RNA is folded in controlled steps, such that the observed distance change is directly related to the specific helix. Thus, for the E D transition, the observed X E D = 7.1 nm corresponded to 17.0 nts indicating the folding of the P2-helix, considering an inter-nucleotide distance of nm. Similarly, X D C = 7.9 nm corresponded to 18.9 nts indicating the folding of P3. Crystal structure 3 shows that P1-P3 is coaxially stacked. The arrangement of the helices can be visualized as one long stem (P1-P3), which is interrupted by a side arm (P2) and flexible junctions (J1/2, J2/3, J3/1). From our CF experiments, it is evident that the junctions and the aptamer folding is not complete until P1 organizes. Thus, we added a 2.0 nm helix-width to the final end-to-end extension change ( X B A= nm = 11.9 nm). The net adjusted distance change, X E D + X D C + X C B + X B A = 30.4 nm, which shows that the aptamer folded completely from a linear state. Data below is represented as mean standard error for n traces. The tertiary L2-L3 interactions fluctuated a 3.5 nm distance similar to the 8-nt J2/3. Molecular simulation 24 studies indicate that the junction becomes solvent exposed when extended by an average distance of 34 Å in adenine riboswitch. Because, the tertiary fluctuations did not involve any unfolding or folding of the aptamer backbone, we excluded X L2-L3 from the net adjusted distance calculation in the CF assay. Folding element/ Nucleotides Force Theoretical Observed Observed n Transition (nt) (pn) ΔX (nm) ΔXobs (nm) nucleotides (number of traces) Complete aptamer ± (Force-ramp) P2 hairpin (ED) ± 0.4 (CF) 17.0 ± 1 91 P3 hairpin (DC) ± 0.4 (CF) J2/3 (CB) ± 0.1 (CF) L2-L3 (C'B) ± 0.1 (CF) - 91 P1+ junctions (BA) ± 0.2 (CF) 11.9 ± 0.2 (adjusted)

3 Supplementary Table 2: Comparison of P2-P3-P1 kinetics in G- and A-aptamer The following Tables compare the equilibrium forces and kinetics measured by forcespectroscopy for P2, P3, and P1 in the G-aptamer (this study) and the A-aptamer 14. The respective aptamer structures are shown below 1,2. We find that the F eq for P2 and P3 helices in the G-aptamer is higher, presumably due to a higher GC content and shorter loop sizes than the A-aptamer. Moreover, the P2 and P1 helices exhibited almost similar folding kinetics in the two aptamers. However, the P3 helix in the A-aptamer displayed extremely fast kinetics. We anticipate that this difference between the two studies may be due to the experimental conditions such as salt concentrations, linker length and other instrumental variables. The A- aptamer was studied while attached to the RNA polymerase in the presence of 200 M adenine (~ 666-fold greater than K D ~ 300 nm) and Mg 2+ (4 mm). It is likely that a high ligand and salt concentration in the surrounding media provided favorable conditions for certain hairpin structures. By comparison, we have used 3mM Mg 2+ salts and ~ 200 nm guanine in our singlemolecule experiments, which is ~ 40-fold higher than the reported K D values (~ 5 nm). A comparative illustration of the folding kinetics for individual hairpins is indicated below. Folding Structure /transition This work (xpt G-Aptamer); [G] = 200 nm F eq (pn) k eq (s -1 ) Nucleotides GC% Hairpin loop size P2 (ED) ± ± (AC bulge at the base shortens the helix length from 21 to 17) P3 (DC) ± ± Closed J2/3 ~ 13 pn (CB) L2-L3 (C'B) 8.72 ± ± P1 (BA) 8.21 ± ± Folding Structure /transition pbue A-Aptamer; [A] = 200 um Greenleaf et al., Science 319, (2008) F eq (pn) k eq (s -1 ) Nucleotides GC % Hairpin loop size P ± ± P3 7.0 ± ± A-competent 5.1 ± ± complex P1 9.0 ± ±

4 Supplementary Figures Figure 1. A typical power spectrum and autocorrelation function (ACF) measured for a certain in the optical-tweezers instrument. (a & b) shows the power spectrum density recorded at 40 khz and 0.4 khz sampling frequencies to determine the trap stiffness. A typical trace for a certain bead #9 is shown from a total of 10 beads analyzed. For the two sampling rates, the cut-off frequencies (fc), the drag coefficient ( ) and the trap stiffness ( ) are nearly identical, as indicated. (c) The autocorrelation function (ACF) with respect to time indicates that the 0 = 0.78 msec for the bead #9. The fitted parameter τ 0 is shown in red, which sets the detection limit for the observed kinetics. For further details on the instrumental resolution see Optical Tweezers in Online Methods. 4

5 Figure 2. Three-dimensional structure for the guanine aptamer and the mutants. In optical tweezers experiment, a single-molecule of the xpt-pbux G aptamer is connected to the trap bead (ADig) and the micropipette bead (SA) via handles. The handles were attached to the RNA termini, which served as linkers to spatially separate the beads. The synthesis of RNA-DNA hybrid handles and the chemical modifications are described in Online Methods. The inset shows the crystal structure of a hypoxanthine-bound xpt-pbux aptamer 3. The altered nucleobases in the mutants m1-m8 are shown in red. The RNA backbone is highlighted in grey, and the bound hypoxanthine is shown in green. 5

6 Figure 3. Successive force-extension curves in the absence (-G) or presence of guanine (+G). (a) The 69-nt G-aptamer RNA from B.subtilis exhibited reproducible force-extension traces in successive cycles. In the plot, six consecutive force-extension curves are shown, whereby the aptamer folds into a native guanine-bound receptor conformation following each denaturation. A cooperative 1-step unfolding suggested a combined rupture of P1-P2-P3. On the other hand, the FECs in the absence of guanine exhibited small and random transitions indicating an unbound and flexible aptamer structure. Moreover, the occasional short transitions were not reproducible in successive cycles. All traces are fitted with a serial worm-like-chain (WLC) equation (Eq. 3) shown with the dashed lines. (b) The mean unfolding force in +G is observed at <F u> = 18.6 ± 1.1 pn (mean ± s.e.m., n = 110 traces from 12 molecules). The unfolding forces are highly distributive in the absence of guanine (-G). 6

7 Figure 4. The distance histograms from CF measurements at 13 pn and 8.5 pn. (a) At 13 pn, the mean extensions X ED = nm and X DC = nm (mean s.e.m, n = 96 traces) correspond to P2 and P3 folding respectively. For distance to nucleotide conversion, see Supplementary Table 1. The C 26 A 44 bulge at the base of the P2 helix interruped a complete folding, and hence a shorter distance (~ 7 nm) was observed. (b) At 8.5 pn, X C'B = nm indicated the fluctuations due to the tertiary L2-L3 interaction, while X BA = nm indicated the folding of the adjacent junctions and the P1-helix. It is noteworthy that the L2-L3 interactions and the J2/3 showed an identical distance change, ~ 3.5 nm. A difference was observed in their respective responses to the applied force. The junction J2/3 closed near 13 pn with limited or no hopping, while L2-L3 hopped frequently near 8 pn. Data from mutational strategies further confirmed the structural assignments (Figure 3). 7

8 Figure 5. Additional single-molecule data for the mutants. (a) The mutations m5 and m8 are designed to alter the P1-helix. (b) The force-extension curves (FECs) for the mutants (m1-m8) are shown. The unfolding (blue) and the refolding (red) trajectories are fitted with Eq. 3 (dashed line). All mutants, except m5, exhibited the characteristic bound-aptamer signature with unfolding near 18 pn and extension ~ 30 nm. This suggested that the ligand-binding character of the G-aptamer was retained, despite modifications. The ITC data further supported that the ligand binding was largely intact in all the m1-m8 RNA constructs (Fig.3; Supplementary Fig. 6). The m5 mutant exhibited longer extension at a higher force due to the modified P1-stem (Fig. 3i). Some mutants especially the m1, m3 and m7 required higher divalent salts or guanine in the buffer for stability. Moreover, a complete disruption of the tertiary L2-L3 interactions, where both the tetrad pairs were altered did not show any guanine binding, despite high divalent salts (data not shown). 8

9 Figure 6. Guanine binding is an exothermic reaction. (a) Table shows the dissociation constants (K D) and the thermodynamic parameters from ITC experiments. The K D is highest in m1 indicating that stable L2-L3 interactions are needed for the ligand-binding, while the strongest binding (low K D) is observed in m4 (also see Fig. 3). In m6, the K D is slightly higher than the wt, although in low nano-molar range. The m2 exhibited improved binding due to partial restoration of the tetrad base pairs. Data represents mean ± s.d. from n 3 experiments. (b) The ITC titration profiles for m1, m2 and m6 are shown. Overall, except m1 and m3, all mutants exhibited comparable free energy changes, indicating that the alterations did not abrogate ligand-binding properties. However, the effect of the mutations is distinctly observed in the overall stabilities, structural dynamics and the biological functions (Fig. 3; Fig. 5). 9