Supplementary Figures

Transcription

1 Supplementary Figures Supplementary Figure 1: Primary DEER data for the complexes of the RsmE protein dimer bound to two stem-loop RNAs singly spin-labelled at positions A to H (corresponding distance distributions are shown in Fig. 1b, c). Left: Primary time domain DEER data (black) and the background fit functions (red). Right: DEER form factors (black) and their best model free fits (red) obtained by Tikhonov regularization with DeerAnalysis. 1

2 Supplementary Figure 2: Primary DEER data for three selected distance distributions of the full RsmZ(1-72)/RsmE-dimer 1:3 complex (corresponding distance distributions are shown in Fig. 2c). (a) Primary time domain DEER data (black) with 3D background fits (red). (b) DEER form factors (black) and their best model free fits (red) obtained by Tikhonov regularization with DeerAnalysis. 2

3 Position A: UGUCGA(tU)GGAUAGACA Position B: CAGCCAUCAAGGA(tU)GAUGG Position C: CGCAGGA(tU)GCGAUU Position D: CAUCAGGA(tU)GAUGA Position E: UCAGGA(tU)AU Position F: UGUCGACGGA(tU)AGACA Position G: CAGCCAUCA(tU)GGACGAUGG Position H: UCA(tU)GGACAU Supplementary Figure 3: Overview of all 4-thiouridine modified RNAs used. On the top, the secondary structure of RsmZ is shown with all the 8 spin-labelling positions indicated. The sites of RNase H cleavage (to obtain the unlabelled RNA fragments from the full-length RNA precursor) and subsequent ligations (of the spin-labelled and unlabelled RNA fragments) are shown in cyan double bars. 3

4 Supplementary Tables Position X <r> / nm FWHM / nm B C D E F Simulation Simulation Simulation Inter-dummy-atom (Simulation 1) 3.39 Position N <r> / nm FWHM / nm A G H Simulation Inter-dummy-atom 5.00 Supplementary Table 1: Mean distances and full width at the half maximum (FWHM) parameters of experimental and back-calculated distance distributions for model systems consisting of two singly spin-labelled RNAs bound to one RsmE protein homo-dimer (see Fig. 1). Mean distances were calculated as a Gaussian mean distance parameter for the main peak in a two Gaussian approximation of the distance distribution. FWHM is the full width at the half maximum for each distance distribution with only the main peak taken into account. For the samples D and F the main peak was assumed to be symmetric and the FWHM parameter was approximated as twice the distance from the left half maximum point to the maximum of the distance distribution. Simulation 1: For the thiouridine residue to which the spin label is attached, the same NOE restraints as for the unmodified complex were used (see experimental part). Simulation 2: To account for the increased flexibility of 4

5 nucleotide X of the common A(N)GGAX motif, the upper limit restraints for all the intermolecular NOEs involving the thio-uridine residue at nucleotide X were increased by 2.0 Å (Fig. 1d, positions B, C, D, F). Simulation 3: In addition to the intermolecular NOEs also the intra RNA NOEs involving the thio-uridine residue were increased by 2.0 Å (Fig. 1d, position E). The inter-dummy-atom distance is describing the distance between two dummy atoms placed into the geometrical centers of the simulated distance distributions ( radical clouds ) of the model systems consisting of two singly spinlabelled RNAs bound to one RsmE protein homo-dimer. The restraints taken to fix the dummy atoms into the centers of the corresponding radical clouds are given in Supplementary Table 2. 5

6 Supplementary Table 2: Typical constraint files for fixing the dummy atoms to the centers of the radical clouds. Shown are the constraint files for fixing the dummy atoms to spin label position G (representing nucleotide N) or position B (representing nucleotide X). Both spin label positions B and G are located on SL2 and are therefore contacting the same RsmE protein dimer (Fig. 1a). 6

7 RESIDUE URAP ZETA ALPHA BETA GAMMA DELTA NU HOXI NU CHI TAU TAU PSI OMEGA PHI XI XI XI ETA ETA HOX ETA ETA EPSI C3' C_ALI O3' O_EST P P_ALI OP1 O_BYL OP2 O_BYL O5' O_EST

8 7 C5' C_ALI H5' H_ALI H5" H_ALI Q5' PSEUD C4' C_ALI H4' H_ALI C3' C_ALI H3' H_ALI C2' C_ALI H2' H_ALI O2' O_HYD HO2' H_OXY C1' C_ALI H1' H_ALI O4' O_EST N1 N_AMI C2 C_ARO O2 O_BYL N3 N_AMI C4 C_ARO C5 C_ARO H5 H_ARO C6 C_ARO H6 H_ARO S4 S_OXY C7 C_ALI H71 H_ALI H72 H_ALI Q7 PSEUD C8 C_BYL

9 37 O8 O_BYL N9 N_AMI H9 H_AMI C10 C_ALI H10 H_ALI C11 C_ALI Q15 PSEUD Q16 PSEUD C15 C_ALI H151 H_ALI H152 H_ALI H153 H_ALI C16 C_ALI H161 H_ALI H162 H_ALI H163 H_ALI QQ11 PSEUD N12 N_AMO O12 O_EST HO12 H_OXY C13 C_ALI Q17 PSEUD Q18 PSEUD C17 C_ALI H171 H_ALI H172 H_ALI H173 H_ALI C18 C_ALI H181 H_ALI H182 H_ALI

10 67 H183 H_ALI QQ13 PSEUD C14 C_ALI H141 H_ALI H142 H_ALI Q14 PSEUD O3' O_EST P P_ALI Supplementary Table 3: CYANA library for URAP residue. The URAP residue corresponds to the 4-thiouridine residue to which the 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) spin label is attached. 10