Supplementary Figures

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

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

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

Supplementary Tables Position X <r> / nm FWHM / nm B 3.57 0.08 1.80 C 3.25 0.08 1.46 D 3.48 0.08 1.95 E 3.70 0.08 1.88 F 3.02 0.08 0.90 Simulation 1 3.33 0.55 Simulation 2 3.48 0.81 Simulation 3 3.70 1.05 Inter-dummy-atom (Simulation 1) 3.39 Position N <r> / nm FWHM / nm A 4.86 0.08 0.88 G 4.60 0.08 0.96 H 4.73 0.08 0.71 Simulation 1 4.82 0.82 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

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

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

RESIDUE URAP 23 74 3 73 1 ZETA 0 0 0.0000 1 2 3 6 0 2 ALPHA 0 0 0.0000 2 3 6 7 0 3 BETA 0 0 0.0000 3 6 7 11 0 4 GAMMA 0 0 0.0000 6 7 11 13 0 5 DELTA 0 0 0.0000 7 11 13 73 0 6 NU2 0 0 0.0000 11 13 15 19 71 7 HOXI 0 0 0.0000 13 15 17 18 18 8 NU1 0 0 0.0000 13 15 19 21 71 9 CHI 0 0 0.0000 21 19 22 23 71 10 TAU1 0 0 0.0000 25 26 31 32 71 11 TAU2 0 0 0.0000 26 31 32 36 71 12 PSI 0 0 0.0000 31 32 36 37 71 13 OMEGA 0 0 0.0000 37 36 38 40 71 14 PHI 0 0 0.0000 36 38 40 42 71 15 XI1 0 0 0.0000 38 40 42 54 71 16 XI2 0 0 0.0000 40 42 54 55 71 17 XI3 0 0 0.0000 55 54 57 69 71 18 ETA1 0 0 0.0000 54 42 45 46 48 19 ETA2 0 0 0.0000 54 42 49 50 52 20 HOX2 0 0 0.0000 42 54 55 56 56 21 ETA3 0 0 0.0000 54 57 60 61 63 22 ETA4 0 0 0.0000 54 57 64 65 67 23 EPSI 0 0 0.0000 11 13 73 74 0 1 C3' C_ALI 0 0.0000-6.355 7.777 2.706 2 0 0 0 0 2 O3' O_EST 0 0.0000-6.756 6.585 2.068 1 3 0 0 0 3 P P_ALI 0 0.0000-6.171 5.151 2.620 2 4 5 6 0 4 OP1 O_BYL 0 0.0000-7.466 4.206 1.944 3 0 0 0 0 5 OP2 O_BYL 0 0.0000-6.337 5.081 4.096 3 0 0 0 0 6 O5' O_EST 0 0.0000-4.713 4.773 2.058 3 7 0 0 0 7

7 C5' C_ALI 0 0.0000-3.506 5.172 2.635 6 8 9 11 0 8 H5' H_ALI 0 0.0000-3.411 6.275 2.522 7 0 0 0 10 9 H5" H_ALI 0 0.0000-3.518 4.911 3.715 7 0 0 0 10 10 Q5' PSEUD 0 0.0000-3.465 5.593 3.119 0 0 0 0 0 11 C4' C_ALI 0 0.0000-2.393 4.439 1.885 7 12 13 21 0 12 H4' H_ALI 0 0.0000-2.490 4.687 0.799 11 0 0 0 0 13 C3' C_ALI 0 0.0000-0.991 4.754 2.357 11 14 15 33 0 14 H3' H_ALI 0 0.0000-0.263 4.564 1.533 13 0 0 0 0 15 C2' C_ALI 0 0.0000-0.778 3.741 3.465 13 16 17 19 0 16 H2' H_ALI 0 0.0000 0.271 3.401 3.609 15 0 0 0 0 17 O2' O_HYD 0 0.0000-1.317 4.196 4.691 15 18 0 0 0 18 HO2' H_OXY 0 0.0000-1.277 5.124 4.914 17 0 0 0 0 19 C1' C_ALI 0 0.0000-1.638 2.577 2.987 15 20 21 22 0 20 H1' H_ALI 0 0.0000-2.282 2.236 3.836 19 0 0 0 0 21 O4' O_EST 0 0.0000-2.560 3.053 2.025 11 19 0 0 0 22 N1 N_AMI 0 0.0000-0.847 1.412 2.556 19 23 29 0 0 23 C2 C_ARO 0 0.0000-0.270 1.345 1.307 22 24 25 0 0 24 O2 O_BYL 0 0.0000-0.371 2.249 0.504 23 0 0 0 0 25 N3 N_AMI 0 0.0000 0.379 0.300 0.985 23 26 0 0 0 26 C4 C_ARO 0 0.0000 0.548-0.682 1.763 25 27 31 0 0 27 C5 C_ARO 0 0.0000 0.050-0.689 3.009 26 28 29 0 0 28 H5 H_ARO 0 0.0000 0.201-1.539 3.693 27 0 0 0 0 29 C6 C_ARO 0 0.0000-0.656 0.378 3.404 22 27 30 0 0 30 H6 H_ARO 0 0.0000-1.086 0.416 4.417 29 0 0 0 0 31 S4 S_OXY 0 0.0000 1.490-2.140 1.214 26 32 0 0 0 32 C7 C_ALI 0 0.0000 2.768-1.314 0.220 31 33 34 36 0 33 H71 H_ALI 0 0.0000 2.379-0.485-0.413 32 0 0 0 35 34 H72 H_ALI 0 0.0000 3.563-0.890 0.875 32 0 0 0 35 35 Q7 PSEUD 0 0.0000 2.971-0.688 0.231 0 0 0 0 0 36 C8 C_BYL 0 0.0000 3.360-2.341-0.735 32 37 38 0 0 8

37 O8 O_BYL 0 0.0000 4.496-2.739-0.655 36 0 0 0 0 38 N9 N_AMI 0 0.0000 2.479-2.770-1.690 36 39 40 0 0 39 H9 H_AMI 0 0.0000 1.519-2.426-1.642 38 0 0 0 0 40 C10 C_ALI 0 0.0000 2.804-3.787-2.693 38 41 42 69 0 41 H10 H_ALI 0 0.0000 3.908-3.874-2.835 40 0 0 0 0 42 C11 C_ALI 0 0.0000 2.207-5.178-2.399 40 45 49 54 0 43 Q15 PSEUD 0 0.0000 0.368-5.056-1.730 0 0 0 0 53 44 Q16 PSEUD 0 0.0000 3.272-6.122-1.072 0 0 0 0 53 45 C15 C_ALI 0 0.0000 0.756-5.079-1.881 42 46 47 48 0 46 H151 H_ALI 0 0.0000 0.714-4.547-0.904 45 0 0 0 43 47 H152 H_ALI 0 0.0000 0.310-6.085-1.711 45 0 0 0 43 48 H153 H_ALI 0 0.0000 0.081-4.535-2.575 45 0 0 0 43 49 C16 C_ALI 0 0.0000 3.050-5.925-1.348 42 50 51 52 0 50 H161 H_ALI 0 0.0000 2.647-6.945-1.154 49 0 0 0 44 51 H162 H_ALI 0 0.0000 4.106-6.044-1.683 49 0 0 0 44 52 H163 H_ALI 0 0.0000 3.064-5.376-0.380 49 0 0 0 44 53 QQ11 PSEUD 0 0.0000 1.820-5.589-1.401 0 0 0 0 0 54 N12 N_AMO 0 0.0000 2.307-5.848-3.692 42 55 57 0 0 55 O12 O_EST 0 0.0000 1.573-6.939-3.796 54 56 0 0 0 56 HO12 H_OXY 0 0.0000 1.957-7.539-4.410 55 0 0 0 0 57 C13 C_ALI 0 0.0000 2.100-4.854-4.748 54 60 64 69 0 58 Q17 PSEUD 0 0.0000 0.408-5.019-5.709 0 0 0 0 68 59 Q18 PSEUD 0 0.0000 3.542-5.060-6.043 0 0 0 0 68 60 C17 C_ALI 0 0.0000 0.760-4.989-5.502 57 61 62 63 0 61 H171 H_ALI 0 0.0000 0.638-4.162-6.239 60 0 0 0 58 62 H172 H_ALI 0 0.0000 0.700-5.944-6.070 60 0 0 0 58 63 H173 H_ALI 0 0.0000-0.115-4.951-4.817 60 0 0 0 58 64 C18 C_ALI 0 0.0000 3.242-5.016-5.775 57 65 66 67 0 65 H181 H_ALI 0 0.0000 3.224-6.028-6.239 64 0 0 0 59 66 H182 H_ALI 0 0.0000 3.162-4.267-6.595 64 0 0 0 59 9

67 H183 H_ALI 0 0.0000 4.239-4.886-5.296 64 0 0 0 59 68 QQ13 PSEUD 0 0.0000 1.975-5.040-5.876 0 0 0 0 0 69 C14 C_ALI 0 0.0000 2.199-3.471-4.066 40 57 70 71 0 70 H141 H_ALI 0 0.0000 2.808-2.734-4.636 69 0 0 0 72 71 H142 H_ALI 0 0.0000 1.189-3.021-3.933 69 0 0 0 72 72 Q14 PSEUD 0 0.0000 1.999-2.878-4.285 0 0 0 0 0 73 O3' O_EST 0 0.0000-0.874 6.089 2.732 13 74 0 0 0 74 P P_ALI 0 0.0000 0.517 6.632 3.409 73 0 0 0 0 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