The SRP signal sequence of KdpD

KdpD is a four-spanning membrane protein that has two large cytoplasmic domains at the amino- and at the carboxyterminus, respectively. During its biogenesis KdpD binds to the signal recognition particle (SRP) of Escherichia coli that consists of a 48-kDa protein Ffh and a 4.5S RNA. The protein is targeted to the inner membrane surface and is released after contacting the SRP receptor protein FtsY. The information within the KdpD protein that confers SRP interaction was found in the amino-terminal cytoplasmic domain of KdpD, particularly at residues 22–48. Within this sequence a Walker A motif is present at residues 30–38. To determine the actual sequence specificity to SRP, a collection of mutants was constructed. When the KdpD peptides (residues 22–48) were fused to sfGFP the targeting to the membrane was observed by fluorescence microscopy. Further, nascent chains of KdpD bound to ribosomes were purified and their binding to SRP was analysed by microscale thermophoresis. We found that the amino acid residues R22, K24 and K26 are important for SRP interaction, whereas the residues G30, G34 and G36, essential for a functional Walker A motif, can be replaced with alanines without affecting the affinity to SRP-FtsY and membrane targeting.

www.nature.com/scientificreports www.nature.com/scientificreports/ Some inner membrane proteins have a large N-terminal cytoplasmic domain, like the sensor protein KdpD with the first transmembrane segment (TMS) starting at amino acid position 400. Previous data have suggested that KdpD has its signal sequence in the cytoplasmic domain, in a short amphiphilic sequence (aa ) that targets the SRP complex to the inner membrane 20 . After targeting, the amphiphilic sequence is released from SRP and folds into an ATP binding domain. During the ongoing translation the transmembrane segments of KdpD are exiting the ribosome and can readily insert into the membrane even if YidC or SecYEG have been depleted in cells 21 . The KdpD signal sequence contains five positively charged residues, in which three are closely spaced (aa [22][23][24][25][26] and a stretch of 10 hydrophobic residues (aa [27][28][29][30][31][32][33][34][35][36] which is too short to span the membrane. The peptide contains also a Walker A motif, which is similar to classical ATP binding sites 22,23 . In the present study, we investigated in detail the involvement of the Walker A motif and the positively charged residues in the signal sequence binding to SRP and the membrane targeting using sfGFP fusion proteins. We found that when the glycine residues at positions 30, 34 and 36 are replaced by alanines the sfGFP fusion protein was still targeted to the membrane surface although these mutations destroy the Walker Box. In contrast, when the positively charged residues outside the Walker box at positions 22, 24 and 26 are replaced by glutamines membrane targeting was inhibited.

Results
Membrane targeting of N22-48-sfGFP depends on SRP. The integral sensor protein KdpD consists of two large cytoplasmic domains, located at the N-and C-terminus (1-400 and 499-894), which are separated by four closely spaced transmembrane segments (401-498). Recently, it was shown that the amino acid residues 22-48 at the beginning of the N-terminal cytoplasmic region of KdpD ( Fig. 1) serve as the SRP signal sequence 20 . In order to visualize the localization of N22-48 of KdpD we fused the N-terminal peptide  to sfGFP. To verify the involvement of SRP in the membrane targeting of the N22-48-sfGFP, we now analysed the localization of the N22-48-sfGFP in the Ffh-depletion strain MC∆Ffh and found that SRP is required for membrane targeting of N22-48-sfGFP. The MC1061 cells expressing N22-48-sfGFP were examined under the fluorescence microscope and found at the inner membrane (Fig. 2a). Likewise, when the MC∆Ffh cells were grown in presence of arabinose to express Ffh we also found the fusion protein at the surface of the inner membrane (panel b). However, when the cells were grown without arabinose and did not express Ffh (panel c, Fig. S3) most of the fluorescent signal appeared as punctuates in the cells that are indicative of aggregate formation. For controls, the sfGFP without the KdpD peptide remained in the cytoplasm under all the conditions (Fig. S1).
SRP signal mutants of KdpD-N22-48-sfGFP. Mutants were generated to explore the function of the N22-48 sequence listed in Fig. 1. The positively charged residues at 22, 24 and 26 were substituted with glutamyl residues giving the 2Q and 3Q mutants. In addition to hydrophobicity, it is assumed that the presence of basic amino acid residues promote signal sequence binding to SRP 18 . The KdpD signal element contains a Walker A motif (residues 30 to 38), which is very similar to the classical ATP binding site. We constructed a mutant where the entire Walker box element was deleted (∆Walker). The influence of the residues within the Walker box on SRP-dependent membrane targeting of KdpD was investigated by a number of mutants generated in the Walker motif. The conserved lysyl residue in the Walker box was exchanged with a glutamyl in the WalkerQ mutant, the cysteine at residue 32 was substituted with a serine or valine and the conserved glycines at 30, 34 and 36 were exchanged with alanines in the Walker3A mutant. All mutant proteins were expressed in E. coli MC1061 for 30 min and analysed by SDS-PAGE (Fig. S2).   of KdpD that is involved in SRP interaction. The KdpD protein is a four-spanning membrane protein with two large cytoplasmic domains at the N-terminus and the C-terminus. The SRP signal peptide of KdpD is located in the N-terminal domain at residues 22-48 and contains five positively charged residues (red letters) and a Walker A motif (underlined). The mutations studied here are marked with a box. Site-directed mutagenesis was used to alter the amino acids in the peptide. The hydrophobicity of each mutant is indicated.
www.nature.com/scientificreports www.nature.com/scientificreports/ To analyse the effects of substituting the positively charged residues of 2Q and 3Q, the sfGFP fusion proteins were expressed in MC1061 (Fig. 3). The cells were grown to a density of 0.5 at OD 600 , induced with 1 mM IPTG and grown for 30 min at 37 °C. The cells were then applied for the fluorescence microscopy. In contrast to KdpD22-48 the fluorescence of the 2Q and 3Q proteins was detected uniformly in the cytoplasm (a, b). This shows for both mutants that the membrane targeting of the sfGFP fusion proteins was clearly inhibited. We conclude that the positively charged residues in the SRP signal are essential to target sfGFP to the membrane surface.
The residues 30 to 38 resemble the Walker motif. When these residues were deleted, the cells accumulated the sfGFP fusion protein in large aggregates at the poles of the cells (Fig. 3c). With the WalkerQ mutant where the conserved lysyl residue was substituted by a glutamyl we observed a clear fluorescence signal at the membrane (Fig. 3d).
Next, the function of the glycyl residues in the Walker box was investigated. The Walker3A mutant with the conserved glycines exchanged to alanines was expressed in MC1061 (Figs 3e, 4a) and in MC∆Ffh cells (Fig. 4b,c). When Ffh is present as is in MC1061 (Fig. 4a) and when the MC∆Ffh cells were grown with arabinose (panel b), the fluorescence was found at the membrane indicating that the mutant protein was clearly targeted to the membrane. The targeting of this mutant was also sensitive to the depletion of Ffh (panel c). We conclude that the WalkerQ and the Walker3A mutants are fully functional to interact with SRP and are targeted to the membrane surface. When SRP is depleted the targeting of these mutants was inhibited similar to the wild-type sequence (Fig. 2).  Targeting of the KdpD-GFP mutants. The KdpD-sfGFP fusion mutants 2Q (a) and 3Q (b) have 2 or 3 positively charged residues mutated to glutamines, respectively. The KdpD-sfGFP fusion mutant ΔWalker (c) and WalkerQ (d) have all residues of the Walker box deleted, WalkerQ has the conserved lysine residue 37 mutated into glutamine (d), Walker3A has the 3 glycine residues at residues 30, 34 and 36 mutated to alanines (e) and WalkerS has the cysteine residue 32 mutated to a serine (f), respectively. They were expressed in E. coli MC1061 at 37 °C and induced with 1 mM IPTG for 30 min. Except for the WalkerQ and the Walker3A mutant, the fluorescent signal was found distributed throughout the cytoplasm or in patches.
www.nature.com/scientificreports www.nature.com/scientificreports/ When the cysteine residue at 32 in the Walker box was replaced by a serine the targeting to the membrane was inhibited (Fig. 3f). This might be because the hydrophobicity of the signal sequence is reduced (Fig. 1). Replacing the serine residue by the more hydrophobic residue valine restored the membrane targeting of the mutant (Fig. 4d). Also, the targeting of the WalkerV mutant was still sensitive to the depletion of Ffh (panel f). We conclude that not the cysteine residue at position 32 is important for membrane targeting but the hydrophobicity since mutation to valine with a comparable hydrophobicity restored the membrane targeting defect of the WalkerS mutant.
Binding of KdpD22-48 to the hydrophobic groove of SRP. To explore whether the SRP signal sequence of KdpD is recognized by the hydrophobic groove formed by the M domain of SRP, cross-linking experiments with purified SRP and synthesized KdpD peptides 22-48 were performed. Since the wildtype Ffh has a cysteine residue at position 406 in the M domain disulfide cross-linking was tested with the in vitro synthesized KdpD22-48 peptide that has a cysteine residue at position 32. In addition, two mutant Ffh proteins with a cysteine residue at position 181 in the G domain or at 423 in the M domain pocket were analysed. Both mutants were combined with a serine at 406. After mixing the proteins cross-linking with copper phenanthroline was performed for 1 h on ice. The samples were analysed on an SDS-PAGE with and without DTT, respectively (Fig. 5). When the KdpD peptide was incubated with the wildtype SRP having a cysteine residue in the M domain or at position 423 in the M domain an additional band appeared indicating that the peptide was cross-linked to SRP (lane 5, 7). The cross-link was sensitive to the reducing agent DTT (Fig. 5, lane 12,14). The shifted band did not appear when KdpD or SRP alone was incubated with the cross-linking agent. In contrast to SRP and SRP 423, the addition of the copper phenanthroline to the KdpD peptide with SRP L181C (in the G domain) showed no cross-linked band (lane 3). In conclusion, our results are consistent with the KdpD signal sequence binding in the same position of SRP as other and canonical signal sequences do 6,24 . Binding of the KdpD mutants to sRp. To test whether the observed membrane targeting abilities of the mutants correlate with binding capabilities to SRP we analysed the interaction of the 22-48 peptide with the wild-type sequence, the 3Q mutant and the Walker3A mutant with purified SRP employing microscale thermophoresis (MST).
Ribosome-nascent chains (RNC) were designed by introducing the sequence for KdpD22-74 or the mutant sequences in a TnaC stalling plasmid resulting in tryptophan dependent ribosome stalling. Since there are about 30 amino acids in the ribosomal exit tunnel the KdpD peptide 22-48 was expected to be exposed outside the tunnel, so that the accessibility is guaranteed. The different his-tagged RNCs were purified and labeled with the fluorescent dye NT-647. The interaction studies of RNCs and purified SRP were analyzed using microscale thermophoresis with a fixed concentration of 5 nM for the labeled RNCs and varying concentrations of 1 µM to 0.5 nM for unlabeled SRP. First, RNCs encoding 22-74 as nascent chain were incubated with SRP resulting in a binding event comparable with the positive control, where RNCs with amino acids 4-85 of the SRP substrate FtsQ as nascent chain were used (Fig. 6a). As a negative control, ribosomes with amino acids 2-50 of the cytoplasmic protein firefly luciferase as a nascent chain were incubated with SRP, which showed no binding in this concentration range (a). In addition, RNCs with only a short nascent chain of about 13 amino acids, mimicking ribosomes at the very beginning of translation, were used as an additional control. Like for Luc2-50-RNCs, also these short-chain ribosomes showed no binding to SRP under these conditions (a). The exchange of three conserved glycines in alanines in the Walker A motif in the KdpD nascent chain did not affect the SRP binding and was similar to that of KdpD22-74 (Fig. 6b). In contrast, the exchange of three positively charged amino acids at position 22, 24 and 26 resulted in a slightly different binding indicating a reduced affinity to SRP. The exchange of the cysteine at position 32 in the Walker A motif to a serine residue led to a very weak SRP binding resulting in the fact that no saturation could be reached in the measured concentration range. We conclude that both mutants www.nature.com/scientificreports www.nature.com/scientificreports/ that were inhibited for membrane targeting were affected in binding. Therefore, the positively charged N-terminal part of the peptide and the hydrophobicity of the core region are critical for SRP binding.
Binding of the KdpD mutants to SRP-FtsY. After the SRP-dependent membrane targeting the SRP-nascent-chain complex is expected to interact with the SRP receptor FtsY. The binding of FtsY to SRP results in conformational changes leading to a stronger binding of SRP to the nascent chain 10 . This only occurs if SRP is bound to a correct cargo. Therefore, we analyzed whether the different KdpD-nascent chains are able to bind to a closed SRP-FtsY complex containing GTP. www.nature.com/scientificreports www.nature.com/scientificreports/ showed that the SRP-FtsY complex is able to bind to the RNCs exposing residues 22-48 of KdpD (Fig. 7a). In contrast, the negative control with residues 2-50 of firefly luciferase showed no binding with the SRP-FtsY complex under these conditions (Fig. 7a). From this we conclude that RNCs with residues 22-74 of KdpD represent a correct cargo for SRP. Also, the interaction with KdpD-RNCs where the three conserved glycines in the Walker A motif were mutated in alanines showed an efficient binding to the SRP-FtsY complex (Fig. 7b). Thus, the exchange of the glycines to alanines known to be essential for the function of the Walker motif did not affect SRP-FtsY binding. In contrast, the mutant where the three positively charged amino acids at position 22, 24 and 26 were substituted with glutamines showed a weaker binding to the SRP-FtsY complex as was with only SRP (Fig. 7b). Since even in this case binding to a closed SRP-FtsY complex was observed we conclude that SRP-FtsY binding is not inhibited but is weakend after the exchange of the three positively charged amino acids. The WalkerS mutant with the lowest hydrophobicity (GRAVY = 0.17; GRAVY 22-48 = 0.29) showed the weakest binding to SRP. Therefore, the substitution of the cysteine residue with a serine residue prevents binding of the SRP-FtsY complex (Fig. 7b). Taken together, our experiments show that both, the positively charged N-terminal part of the sequence and the hydrophobicity of the core region are critical for SRP binding and, in addition, for FtsY recruitment.

Discussion
The amino-terminal region of the KdpD sensor protein has two functions. In the fully folded protein it binds ATP that might modulate the communication of KdpD with its response regulator protein KdpE 23 . ATP is bound by a classical Walker box where the Walker A motif is located at the residues 30 to 38 and a potential Walker B motif at residues 105 to 110. The second function of the amino-terminal region is early during its synthesis in the ribosome. For the targeting to the membrane the amino-terminal region of KdpD at the residues 22-48 is first contacting the SRP 20 . The RNC-SRP complex is then transported to the receptor protein FtsY to ensure that the nascent protein chain is close to the membrane surface. We show here that an artificially stalled KdpD22-74 RNC binds to SRP comparable with RNCs exposing the positive control protein FtsQ4-85, a well-studied substrate of SRP (Fig. 6) 25 . In addition, also a preassembled SRP-FtsY complex which was activated by GTP could be bound to KdpD22-74 RNC as for FtsQ4-85 RNC (Fig. 7). These results show that the residues 22-48 efficiently bind to SRP and allow the nascent chain of KdpD an early targeting to the membrane surface before translation has synthesized the full 894 long protein. The binding to SRP was analysed with microscale thermophoresis. Fluorescently labeled RNCs at 5 nM were mixed 1:1 with purified and assembled SRP (Ffh and 4.5 RNA) in different dilutions (0.5 nM to 1 μΜ) and applied to capillaries. For the binding to SRP-FtsY the assembled SRP was mixed with purified FtsY and 200 μΜ GppNHp prior to the addition of RNCs. Previous affinity determinations were performed with fluorescence equilibrium titrations using fluorescence-labeled SRP with leader peptidase RNCs 26 or by fluorescence anisotropy of fluorescein-labeled SRP with EspP RNCs 27 resulted in comparable binding events. Since the SRP-bound cargo has to pass a number of checkpoints, the binding to a preassembled SRP-FtsY complex with high affinity is an important step in the pathway 27 .
The early targeting of KdpD at the membrane surface ensures that the 4 transmembrane segments can readily insert when they appear at the ribosome tunnel exit 28 . Previous studies have shown that the insertion of KdpD into the inner membrane can occur even in the absence of SecYEG or YidC 21 . Most likely, the cotranslational membrane insertion directly into the bilayer is possible because the periplasmic loops are very short with 4 and 10 residues, respectively (Fig. 1).
C-tailed membrane proteins share with KdpD the fact that a large hydrophilic domain is released from the ribosome before a membrane anchoring segments is exposed at the tunnel exit. We have recently shown that the membrane targeting and insertion of the C-tail protein SciP involves SRP and the SRP receptor 29 . Similar as in KdpD the TMS of SciP is far from the N-terminus (residues 184-206). We found that two short hydrophobic www.nature.com/scientificreports www.nature.com/scientificreports/ regions at residues 12-20 and 62-71 of SciP have the potential to interact with SRP. When these peptides were fused with sfGFP the fusion protein was targeted the membrane surface. We conclude that the amino-terminal sequences found in SciP and KdpD are functional SRP signal sequences and are located close to the N-terminus in membrane proteins that contain large cytoplasmic domains. In both cases, the SRP signal sequence is separate from the first membrane spanning segment and therefore allows an early binding of SRP before the membrane segments are translated by the ribosome.
The function of the SRP-signal sequence can be tested in a fusion protein with the green fluorescent protein GFP 20 . Here, we show that the assay can be improved when the super folding GFP (sfGFP) is used for the fusion. The fluorescence in cells can be observed already 20 min after induction. With this assay we tested a collection of mutants that affected the function of the 22-48 sequence as a SRP signal and/or Walker A element. The key lysine of the Walker A element cannot be replaced by another amino acid without affecting the function of the Walker element 30 . When we substituted the key lysine with a glutamine in the WalkerQ mutant, or when the conserved glycine residues at position 30, 34 and 36 in the Walker3A mutant were replaced by alanine residues, membrane localization was not affected (Fig. 3d,e). Likewise, the binding events of the Walker3A mutant to SRP and to SRP-FtsY corresponded to the binding we had obtained with the wild-type signal sequence of   (Figs 6, 7). Therefore, these modified sequences fully function as a SRP targeting signal.
The deletion of the Walker A element (residues 30-38) resulted in a patchy appearance of the fusion protein in the cells (Fig. 3c). A similar phenotype was observed for the wild-type sequence when Ffh was depleted (Fig. 2c). We assume that the patches form because of aggregation in the cytoplasm. Interestingly, the sfGFP itself did not form such aggregates, even when Ffh was depleted (Fig. S1). It is surprising that small 19 to 28 residues long N-terminal extensions of sfGFP have such a big effect on the solubility. The binding affinity of the WalkerS mutant to SRP was the lowest we had measured and there was no binding to SRP-FtsY. It is possible that the lower hydrophobicity of the cysteine is critical in allowing that the sequence is still able to bind to SRP. The malfunctioning of this mutant in membrane targeting was supported by the fluorescence microscopy (Fig. 3f). Interestingly, mutation of the cysteine residue into the more hydrophobic valine restored membrane targeting indicating that not the cysteine residue but the hydrophobicity is critical for SRP binding (Fig. 4d).
Finally, the positively charged residues at 22, 24 and 26 were investigated for membrane targeting and SRP binding. The sfGFP fusion protein showed that the fluorescence was evenly distributed in the cytoplasm suggesting that the binding to SRP is affected. Indeed, the binding to SRP and to SRP-FtsY was lower compared to the wild-type signal sequence of 22-48 respectively (Figs 6, 7). This shows that the positively charged residues play an important role for the interaction with SRP.
Taken together, this study shows that a SRP signal sequence is not restricted to a transmembrane segment but can be localized in a cytoplasmic region. The results obtained with the mutants of the signal sequence underline the importance of the positively charged N-terminal part and the hydrophobic C-terminal part of the signal sequence to allow the interaction with the M-pocket of SRP and membrane targeting.

Construction of SRP-signal mutants of KdpD-N22-48-sfGFP.
All oligonucleotides used in this study are listed in the Supplementary Table S1 and the used plasmids in the Supplementary Table S2. The amino acid residues 22 to 48 of KdpD were amplified flanking the PCR product with a HindIII and BamHI recognition site. The PCR product was then cloned in a pMS119EH derivate containing the sfGFP gene (own collection) using the restriction enzymes HindIII and BamHI. The different SRP-signal mutants of KdpD-N22-48-sfGFP were constructed by a PCR-based mutagenesis of N22-48-sfGFP. Substitution of the basic residues at position 22 and 24 of N22-48-sfGFP into glutamine resulted in the mutant pMS-KdpD22-48-2Q-sfGFP (named 2Q). Substitution of three of the closely spaced basic amino acid residues in N22-48-sfGFP were substituted into glutamines resulted in the mutant pMS-KdpD22-48-3Q-sfGFP (named 3Q). Substitution of lysine 37 within the Walker A motif with a glutamine resulted in mutant pMS-KdpD22-48-WalkerQ-sfGFP (named WalkerQ). To construct mutant pMS-KdpD22-48-∆Walker-sfGFP (named ∆Walker), amino acids 30-38 were deleted with site-directed mutagenesis. To change the conserved motif of the Walker box into a no Walker-motif a site-directed mutagenesis was done to generate pMS-KdpD22-48-Walker3A-sfGFP (named Walker3A). Substitution of cysteine 32 within the Walker A motif with a serine or valine resulted in mutant pMS-KdpD22-48-WalkerS-sfGFP (named WalkerS) and mutant pMS-KdpD22-48-WalkerV-sfGFP (named WalkerV). The coding regions of all constructs were verified by DNA sequence analysis.
The plasmid encoding the first 50 amino acids of the cytoplasmic protein firefly luciferase was generated by amplification from plasmid pUC19-T7-Luc 50 (kindly provided by Shu-ou Shan; Caltech). The PCR product was flanked with an EcoRI and a NcoI recognition site producing compatible sticky ends with MfeI and NcoI digested plasmid resulting in plasmid pMS-Luc2-50-TnaC. The plasmid encoding the first 85 amino acids of FtsQ (pEM36-3C) was kindly provided by R. Beckmann, Munich.
Fluorescence microscopy. Strains were grown overnight at 37 °C, diluted in fresh LB medium and grown to an OD 600 of 0.5. IPTG was then added to a final concentration of 1 mM. The cells were incubated for 20 min (MC∆Ffh) or 30 min (MC1061) at 37 °C under continuous shaking. The cells were treated as described 29 and collected by centrifugation, washed twice with LB medium and resuspended in 2 mM EDTA, 50 mM Tris-HCl, pH 8.0. The cell suspension (3 µL) was applied to a polylysine-coated cover glass (Sigma-Aldrich) and examined immediately by fluorescence microscopy with the Zeiss AxioImager M1 fluorescence microscope. Emission was detected with filters specific for GFP. Analysis was done by using the AxioVision Software (Zeiss). The expression of the different KdpD22-48-sfGFP mutants was analysed on a 12% SDS-PAGE (after TCA precipitation with 10% TCA) and immunoblotting with an α-GFP and α-rabbit antibody.
Purification of Ffh and FtsY. E. coli Ffh was purified essentially as described by Seitl 37  www.nature.com/scientificreports www.nature.com/scientificreports/ 25 mM MgCl 2 , 2 mM L-tryptophane, 0.1% DDM) and the His-tagged ribosomes were eluted with 3 mL buffer B RNC + 150 mM imidazole and 3 mL buffer B RNC with 300 mM imidazole in 1 mL fractions. The elution fractions were loaded onto a linear sucrose gradient (10-40% sucrose in buffer B RNC ) and centrifuged at 30 000 rpm for 3.5 h in a Beckman SW40 rotor. The gradient was collected in 1 mL fractions, the ribosome containing fractions were identified measuring the absorbance at 260 nm, pooled and centrifuged at 40 000 rpm for 4 h in a Beckman Ti60 rotor. The ribosomal pellet was resuspended in buffer C RNC (20 mM Hepes pH 7.2, 50 mM KOAc, 5 mM Mg(OAc) 2 , 2 mM L-tryptophane) and stored at −80 °C.

In vitro 4.5S RNA synthesis and SRP reconstitution.
To get a functional SRP the purified Ffh protein was reconstituted with the in vitro synthesized 4.5S RNA as described by Seitl 37 . Therefore, plasmid pUC18-4.5S RNA (kindly provided by Irmgard Sinning, Heidelberg) was used where the 4.5S RNA sequence was placed downstream of the T7 promoter. First, the plasmid was linearized with BamHI and gel purified. For in vitro transcription with the HiScribe ™ T7 High Yield RNA Synthesis Kit (NEB) 1 µg linearized plasmid DNA was used. After incubation for 16 h at 37 °C the 4.5S RNA was purified using the RNA Clean & Concentrator TM −25 Kit (Zymo Research), analyzed on a 2% agarose gel in 1x Tris-borate-EDTA buffer and stored at −80 °C. Prior to the reconstitution, the RNA was heated to 75 °C for 2 min and chilled on ice for 1 min. Ffh and 1.5-fold molar excess of 4.5S RNA were mixed in MST-buffer (20 mM HEPES pH 7.2, 50 mM KOAc, 5 mM Mg(OAc) 2, 2 mM L-tryptophane, 0.05% Tween-20) and incubated for 10 min at 20 °C.
Labeling of RNCs. The different RNCs were labeled with the cysteine-reactive fluorescent dye NT-647 and the RED-maleimide labeling kit (NanoTemper Technologies). The RNCs were adjusted to a concentration of 2 µM in 100 µL buffer C RNC and mixed with 3-fold molar excess of the dye in 100 µL labeling buffer provided in the kit. After incubation for 30 min at RT in the dark the labeling reaction was purified to remove the free dye using the column provided in the kit. The concentration of the labeled RNCs after purification was calculated measuring the absorbance at 260 nm and they were stored at −80 °C.

Microscale thermophoresis.
For the interaction studies between the RNCs and SRP, the labeled RNCs were adjusted to 10 nM in MST buffer. With the reconstituted SRP a series of 1:1 dilutions was prepared in MST buffer with a concentration ranging from 1 µM to 0.49 nM. The ligand dilutions were mixed with one volume of labeled RNCs resulting in a RNC concentration of 5 nM. After incubation for 5 min on ice, the dilutions were filled in Monolith NT Premium Treated capillaries (NanoTemper Technologies) and measured using the Monolith NT.115 instrument. During measurement the temperature was kept constant at 22 °C. Thermophoresis was measured with 5 s laser off, 20-30 s laser on and 5 s laser off, a LED power of 40-60% and the MST Power "Low". The data of three independently pipetted measurements were merged and analyzed using the software MO.Affinity Analysis v2.3 (NanoTemper Technologies) using the manual evaluation (Cold region start/end: −1 s/0 s; Hot region start/end: 5.01 s/10.08 s).
For the interaction studies between the RNCs and SRP-FtsY a preincubated closed SRP-FtsY complex was used. Therefore, the reconstituted SRP was mixed with 4-fold molar excess of FtsY in MST buffer containing 200 µM of the non-hydrolysable GTP-analogue GppNHp. After incubation for 10 min at 25 °C the stable SRP-FtsY complex was incubated on ice. For measurements a series of 1:1 dilutions was prepared in MST buffer with 200 µM GppNHp with a complex concentration ranging from 500 nM/250 nM to 0.25/0.12 nM. The ligand dilutions were mixed with one volume of labeled RNCs resulting in a RNC concentration of 5 nM. After incubation for 5 min on ice, the dilutions were filled in Monolith NT capillaries and measured using the Monolith NT.115 instrument. During measurement the temperature was kept constant at 22 °C. Thermophoresis was measured with 5 s laser off, 20-30 s laser on and 5 s laser off, a LED power of 60% and the MST Power "low". The data of three independently pipetted measurements were merged and analyzed using the software MO.Affinity Analysis v2.3 (NanoTemper Technologies) using the manual evaluation (Cold region start/end: −1 s/0 s; Hot region start/end: 5.01 s/10.02 s).