Qrr2 was present at 1.4 µM in each case. On the right, the upper panels show a ribbon representation of Qrr2, predicted by iFoldRNA (26,27), superimposed with.
Table S1. DNA Primers Primer Name Y25Df Y25Dr K56Af K56Ar Qrr1S1 Qrr1AS1 Qrr1S2 Qrr1AS2 Qrr2S1 Qrr2AS1 Qrr2S2 Qrr2AS2 Qrr3S1 Qrr3AS1 Qrr3S2 Qrr3AS2 Qrr4S1 Qrr4AS1 Qrr4S2 Qrr4AS2
Sequence (5’ to 3’) gtc gtg aac gta tcc ctg ttt cta tcg acc ttg tga acg gca tta aac tgc aag g cct tgc agt tta atg ccg ttc aca agg tcg ata gaa aca ggg ata cgt tca cga c gct gaa gaa cac agt aaa cca aat ggt tta cgc gca tgc gat ttc tac tgt ggt tcc tg cag gaa cca cag tag aaa tcg cat gcg cgt aaa cca ttt ggt tta ctg tgt tct tca gc gtg acc cgc aag ggt cac cta gcc aac tga cgt tgt tag tga ata atc gaa tga gtc tat tgg ctg tta ttt gtg aac att gat tat tca cta aca acg tca gtt ggc taa tac gac tca cta tag ggt gac ccg caa ggg tcac aaa aaa ata gcc aat aga atg agt cta ttg gct gtt att tgt gaa c ggt gac cct tgt taa gcc gag ggt cac cta gcc aac tga cgt tgt tag tg cgc gat tgg ctt ata tat gat gtg aac aat act att cac taa caa cgt cag ttg gct agg taa tac gac tca cta tag ggt gac cct tgt taa gcc gag aaa aaa ata gcc aat cgc aag aac cgc gat tgg ctt ata tat gat gtg aac ggg tga ccc tta att aag ccg agg gtc acc tag cca act gac gtt gtt ag gat tgg ctg ata aaa caa atg tga aca att tca ttc act aac aac gtc agt tgg cta ggt g taa tac gac tca cta tag ggt gac cct taa tta agc cga g aaa aaa gcc aat cac aaa agg gtg att ggc tga taa aac aaa tgt gaa caa ttt cat tc gtg acc ctt cta agc cga ggg tca cct agc caa ctg acg ttg tta gtg aac acc gtg tga ttg gcc gtc tat aag tgt gaa caa tgg tgt tca cta aca acg tca gtt gg taa tac gac tca cta tag ggt gac cct tct aag ccg agg aaa aaa aag gcc aac cac aag aag tgt gat tgg ccg tct ata agtgtg
Description Mutagenic primers to construct distal face RNAbinding mutant of V. cholerae Hfq (Y25D) Mutagenic primers to construct proximal face RNA-binding mutant of V. cholerae Hfq (K56A) Gene synthesis primers to construct Qrr1 template for in vitro transcription
Gene synthesis primers to construct Qrr2 template for in vitro transcription
Gene synthesis primers to construct Qrr3 template for in vitro transcription
Gene synthesis primers to construct Qrr4 template for in vitro transcription
Qrr1 - 99 nucleotides
Qrr2 - 111 nucleotides
C A G A G CC G C G A UU U G U A U C U U U C U A G 3’ CU U C G 5’ U UA GACU U A G C G U CG AA UA CG A U CC AG C GC A C UA A C U G AU A GU UA UA A A GC UA GC AU AU UG AU A A U A C 0
A U U A G G C C UU G A C G C G C G A U C U G U U C U G U U U G A UU U GC CGGU C G A U C G 5’ 3’ UU UCG A U A GG UC CCA A AG A G U A C U A C U A A A U GU C U C U U A G C AU G G UA A C GC AU AU UG AU U G U A
1
Qrr3 - 110 nucleotides UU A A A A U G U C C G UC U U A U C G G G CA U G AC CC G A U GU U UC C U U U G U A A G U C GG U CG C A 3’ C C U 5’ U G A C A U A G U U C U U C G U A G UU A U UU A G CA U C U UA G G GC A C AU AU U GU G A AU A
Qrr4 - 110 nucleotides A G C
AU C U C G UC A G C G C G A U G U C C U U U U G U U A G U UU GU CA CU C G G A U 3’ U C G UC GU U 5’ U CG CA A C C A GG C G A G C A C U A A A U GU U U C U U A C A CU G G G C UA A GC AU AU CG AU U C C A
Figure S1. Secondary structure of Qrrs1-4 Secondary structures were predicted using the Vienna RNAfold server (RNA stuff @ tbi.univie.ac.at). Nucleotides are coloured according to the probability of being present in the shown secondary structure with blue corresponding to a probablility of zero an red to a probability of one. The RNA sequences shown include the three additional guanosines that were added to the 5’ end of the RNAs in this work in order to ensure efficient transcription by T7 RNA polymerase. The structures are similar to those reported by Lenz et al. (6) suggesting that the addition of these nucleotides does not significantly alter the secondary structures.
Qrr2 Qrr3 Qrr4
Distal and proximal face double mutant (Y25D K56A) 0 15 30 45 60 75 90 105 120 135 150 200
Proximal face mutant (K56A) 0 15 30 45 60 75 90 105 120 135 150 200
0 15 30 45 60 75 90 105 120 135 150 200
Distal face mutant (Y25D)
Qrr1
0 15 30 45 60 75 90 105 120 135 150 200
Wild-type
[Protein] (nM) Higher order complexes 1:1 complex Free RNA
Higher order complexes 1:1 complex Free RNA
Higher order complexes 1:1 complex Free RNA
Higher order complexes 1:1 complex Free RNA
Figure S2. VcHfq binding to Qrrs 1-4 Representative EMSAs for wild-type VcHfq, a distal site mutant (Y25D), a proximal site mutant (K56A) and a distal and proximal site double mutant (Y25D K56A) binding to Qrr1, Qrr2, Qrr3 and Qrr4. RNAs were present at 30 nM in each case and the concentration of VcHfq present is indicated above the lane. The mobilities of free Qrr RNA, the 1:1 VcHfq:Qrr complex and higher order complexes are indicated on the right of the gels.
a
C+16
6041.06
Qrr2
Relative intensity (%)
100
50
0
C+15
6448.87
+17
C
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000
m/z
b
C+16
6046.99
Qrr3
Relative intensity (%)
100
C+17 C+15
50
6451.71
M+8
1271.86 0
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000
m/z
c
C+16
6011.69
Qrr4
Relative intensity (%)
100
C+15
6414.57 50
0
C+17
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000
m/z
Figure S3. Stoichiometry of VcHfq-Qrr complexes Non-denaturing MS spectra of VcHfq with Qrr2 (a), Qrr3 (b) and Qrr4 (c). VcHfq and the respective Qrr were mixed a equimolar concentrations. Peak series corresponding to the 15+ to 17+ charge states of the 1:1 VcHfq (hexamer):Qrr complexes are labelled with C in each spectrum. (a) VcHfq:Qrr2 1:1 complex (theoretical mass: 96,714.8 Da; experimental mass: 96,615 Da). (b) VcHfq:Qrr3 1:1 complex (theoretical mass: 96,291.6 Da; experimental mass: 96,750 Da). A smaller peak series, centred on the 8+ charge state, corresponds to a small amount of monomeric VcHfq and is labelled with M (theoretical mass 10,163.6 Da; experimental mass: 10,164.9 Da). (c) VcHfq:Qrr4 1:1 complex (theoretical mass: 96,278.6 Da; experimental mass: 96,185 Da).
a
C+16 M+8
Relative intensity (%)
100
Qrr1
C+15 50
M
+7
C+17
M+9
0
1000 2000 3000 4000 5000 6000 7000 8000 9000 1000011000
m/z
b
C+16
Relative intensity (%)
100
Qrr2
C+17 M+8 C+15
50 M+7
M+9
0
1000 2000 3000 4000 5000 6000 7000 8000 9000 1000011000
m/z
c
M+8
Qrr3
Relative intensity (%)
100 C+16
C+17 50
0
M+9
C+15
M+7
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000
m/z
d
C+16
Qrr4
Relative intensity (%)
100 C
M+8
+17
C+15 50 M+9
0
M+7
1000 2000 3000 4000 5000 6000 7000 8000 9000 1000011000
m/z
Figure S4. Collision induced dissociation of VcHfq-Qrr complexes CID MS spectra of (a) VcHfq-Qrr1, (b) VcHfq-Qrr2, (c) VcHfq-Qrr3 and (d) VcHfq-Qrr4 complexes. The peak series corresponding to the VcHfq:Qrr 1:1 complex is labelled with C and the peak series corresponding to free VcHfq (monomer) is labelled with M in each spectrum.
VcHfq-Qrr1 (SAXS)
VcHfq-Qrr2 (SAXS)
VcHfq-Qrr3 (SAXS)
P(r) (arbitrary units)
b
log I(q) (arbitrary units)
a
VcHfq-Qrr2 (SAXS)
VcHfq-Qrr3 (SAXS) VcHfq-Qrr1 (SAXS)
VcHfq-Qrr4 (SAXS)
VcHfq-Qrr4 (SAXS)
0
0.05
0.10
0.15
0.20
0
40
q (Å ) -1
80
120
r (Å-1)
160
200
240
c
90º proximal face
90º distal face
Figure S5. Comparison of VcHfq-Qrr complexes (a) SAXS scattering curves for VcHfq-Qrr1 (circles), VcHfq-Qrr2 (triangles), VcHfq-Qrr3 (squares) and VcHfq-Qrr4 (crosses). The VcHfq-Qrr1 scattering curve is the same as in Figure 5a but is shown again here for comparison. Back-transformed distance distribution functions, P(r), are shown for VcHfq-Qrr1 (orange), VcHfq-Qrr2 (magenta), VcHfq-Qrr3 (green) and VcHfq-Qrr4 (blue). (b) P(r) functions for the SAXS data of VcHfq-Qrr1 (orange), VcHfq-Qrr2 (magenta), VcHfq-Qrr3 (green) and VcHfq-Qrr4 (blue). The VcHfq-Qrr1 P(r) function is the same as in Figure 5b but is shown again here for comparison. Since some evidence of aggregation was evident for the VcHfq-Qrr3 complex, a qmin of 0.019 Å-1 was chosen to obtain a P(r). (c) Ab initio model of the VcHfq-Qrr2 complex generated in MONSA (29,30) and visualized in PyMOL. VcHfq is shown as grey spheres and the Qrr2 sRNA as magenta spheres. The proximal and distal faces labels are derived from the data for the RNA-binding mutants presented in Figure S2.
a
Free Qrr2
8
∆ε (deg-1 cm2 dmol-1)
6
VcHfq:Qrr2 (0:1) Qrr2 (20ºC)
4
90º
VcHfq:Qrr2 (1:1) Qrr2 (44ºC)
90º
Qrr2 in complex with VcHfq
2 Qrr2 (92ºC) 0 240
250
260
270
280
290
300
310
320
90º
Wavelength (nm)
90º
-2
-4
c
8
∆ε (deg-1 cm2 dmol-1)
VcHfq:Qrr4 (0:1) Qrr4 (20ºC)
VcHfq:Qrr3 (0:1) Qrr3 (20ºC)
6
4
6
VcHfq:Qrr3 (1:1) Qrr3 (44ºC)
2 Qrr3 (92ºC) 0 240
8
250
260
270
280
290
300
310
320
∆ε (deg-1 cm2 dmol-1)
b
VcHfq:Qrr4 (1:1) Qrr4 (36ºC)
4
2 Qrr4 (92ºC) 0 240
250
260
270
280
290
300
310
320
Wavelength (nm)
Wavelength (nm) -2
-2
-4
-4
Figure S6. The conformation of the RNA changes upon complex formation (a) On the left are the CD spectra for VcHfq-Qrr2 complexes (magenta lines) at 20ºC at VcHfq:Qrr2 ratios of 0:1 (solid magenta line) and 1:1 (dotted magenta line) and for free Qrr2 sRNA (black lines) at 20ºC (solid black line), 44ºC (dotted black line) and 92ºC (dashed black line). Qrr2 was present at 1.4 µM in each case. On the right, the upper panels show a ribbon representation of Qrr2, predicted by iFoldRNA (26,27), superimposed with SUPCOMB (28) on the ab initio model of the sRNA (spheres) generated from the SAXS data of free Qrr2 with DAMMIF (24) and averaged with DAMAVER and DAMFILT (25). The lower panels show the ab initio model of the sRNA, as it appears in complex with VcHfq, generated from the SAXS data of the complex with MONSA (29,30). All models were visualized with PyMOL. (b) CD spectra for VcHfq-Qrr3 complexes (green lines) at 20ºC at VcHfq:Qrr3 ratios of 0:1 (solid green line) and 1:1 (dotted green line) and for free Qrr3 sRNA (black lines) at 20ºC (solid black line), 44ºC (dotted black line) and 92ºC (dashed black line). Qrr3 was present at 1.8 µM in each case. (c) CD spectra for VcHfq-Qrr4 complexes (blue lines) at 20ºC at VcHfq:Qrr4 ratios of 0:1 (solid blue line) and 1:1 (dotted blue line) and for free Qrr4 sRNA (black lines) at 20ºC (solid black line), 36ºC (dotted black line) and 92ºC (dashed black line). Qrr3 was present at 1.6 µM in each case.
