Reagents were purchased from Sigma-Aldrich and used without further purification. Oligonucleotides were purchased from Integrated DNA Technologies, Inc. DNA and RNA concentrations were determined by measurements with the NanoDrop ND-1000 spectrophotometer. Radioactively labelled proteins or nucleic acids were visualised using storage phosphor screens (GE Healthcare) and a Typhoon 9400 imager (GE Healthcare).
Preparation of 5’PPP-/5’P-/5’-NAD-RNA by in vitro transcription
DNA template for Qβ-RNA was amplified by PCR. PCR products were analysed by 1 % agarose gel electrophoresis and purified using the QIAquick PCR purification kit (QIAGEN). 5’-triphosphate (PPP) Qβ-RNA was synthesized by in vitro transcription (IVT) in the presence of 1x transcription buffer (40 mM Tris, pH 8.1, 1 mM spermidine, 10 mM MgCl2, 0.01 % Triton-X-100), 5 % DMSO, 10 mM DTT, 4 mM of each NTP, 20 µg of T7 RNA polymerase (2 mg/ml) and 200 nM DNA template. The same conditions were applied for the synthesis of a mixture of α-32P-labelled 5’-NAD- and PPP-Qβ-RNAs except for the presence of 2 mM ATP, 80 µCi 32P-α-ATP and 4 mM NAD instead of 4 mM ATP. The IVT reactions were incubated at 37°C for 4 h and digested with DNase I (Roche). RNA was purified by denaturing PAGE and isopropanol-precipitation, and resuspended in Millipore water. RNA sequences are listed in Extended Data Table 1.
To convert 5’-PPP-RNAs into 5’-monophosphate RNAs (5’-P-RNAs), 250 pmol of Qβ-RNA were treated with 60 U of RNA 5’-polyphosphatase (Epicentre) in 1x polyphosphatase reaction buffer at 37°C for 70 min. Protein was removed from 5’-P-RNAs by phenol-chloroform extraction and residual phenol-chloroform removed by three rounds of diethyl ether extraction. 5’-P-RNAs were isopropanol precipitated and dissolved in Millipore water.
5’-radiolabelling of 5’-monophosphate and NAD-capped RNAs
120 pmol of 5’-P-Qβ-RNA, or 6.25 nmol of 5’-P-RNA 8-mer were treated with 50 U of T4 polynucleotide kinase (PNK) in 1x reaction buffer B and 1,250 µCi 32P-γ-ATP. The reaction was incubated at 37°C for 2 h. The resulting 5’-32P-RNA 8-mer/ 5’-32P-Qβ-RNA were separated from residual protein by phenol-chloroform extraction. The remaining 32P-γ-ATP was removed by washing with 3 column volumes of Millipore water and centrifugation in 10 kDa (Qβ-RNA) or 3 kDa (8-mer) Amicon filters (Merck Millipore) at 14,000 rpm at 4°C for four times. RNA sequences are listed in Extended Data Table 1. To convert the purified 5’-32P-RNAs into 5´-32P-NAD-capped RNAs, 800 pmol of 5´-32P-RNA 8-mer or 30 pmol of 5’-32P-Qβ-RNA were each incubated in 50 mM MgCl2 in the presence of a spatula tip of nicotinamide mononucleotide phosphorimidazolide (Im-NMN), synthesised as described in 1, at 50°C for 2 hours. RNAs were purified by washing with Millipore water and centrifugation in 10 kDa (Qβ-RNAs) or 3 kDa (8-mer) Amicon filters at 14,000 rpm at 4°C for four times. Concentrations of the 5’-32P-RNAs were measured on the NanoDrop and used to calculate the approximate concentrations of yielded 5’-NAD-capped 32P-RNAs assuming an approximate yield of the imidazolide reaction of 50 % 1. 5’-32P-ADP-ribose-RNA 8mer (ADPr-8mer) was synthesised by incubation of 4.8 µM 5’-32P-NAD-RNA 8mer and 0.08 µM ADP-ribosylcyclase CD38 in 1x degradation buffer at 37°C for 4 h. The reaction was purified by P/C/I-diethylether-extraction and filtration through 3 kDa filters washing with 4 column volumes of Millipore water.
Cloning of ADP-ribosyltransferases, ADP-ribose hydrolases and target proteins
To amplify bacteriophage T4 genes modA, modB and alt, a single plaque from T4 bacteriophage revitalisation was resuspended in Millipore water and used in a “plaque”-PCR, analogous to bacterial colony PCR. The gene encoding for the ADP-ribosylhydrolase ARH1 was purchased from IDT as gblocks and amplified by PCR. E. coli genes coding for rS1 and PNPase were PCR-amplified from genomic DNA of E. coli K12, which was isolated via GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich). Nucleotide sequences are listed in Extended Data Table 2. XhoI and NcoI restriction sites were introduced during amplification using appropriate primers (Extended Data Table 3). The resulting PCR product was digested with XhoI and NcoI and cloned into pET-28c vector (Merck Millipore). After Sanger sequencing, the resulting plasmids were transformed into E. coli One Shot BL21 (DE3) (Life Technologies). The ARH1 D55,56A and rS1 mutants were generated by site-directed mutagenesis using a procedure based on the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific). The resulting plasmids were sequenced and transformed into E. coli One Shot BL21 (DE3).
AHR1, ARH1 D55A D56A and rS1 were transferred from pET28 to pTAC-vector using specific primers with restriction sites for XhoI and SphI (Extended Data Table 3). The resulting PCR product was digested with XhoI and SphI and cloned into pTAC-MAT-2 (Sigma-Aldrich). The resulting plasmids were transformed into E. coli strain B and E. coli Ced64 (kind gift from Gerhart E. Wagner, Uppsala University). All strains used and generated in this work are summarised in Extended Data Table 4
Purification of rS1 domains and variants, PNPase Domain, RNase E (1-529), Alt, NudC V157A, E174A, E177A, E178A
IPTG-induced E. coli One Shot BL21 (DE3) containing the respective plasmid were cultured in LB medium at 37°C. Protein expression was induced at OD600 = 0.8, bacteria were harvested by centrifugation after 3 hours at 37°C and lysed by sonication (30 s, 50 % power, five times) in HisTrap buffer A (50 mM Tris-HCl pH 7.8, 1 M NaCl, 1 M Urea, 5 mM MgSO4, 5 mM β-mercaptoethanol, 5 % glycerol, 5 mM imidazole, 1 tablet per 500 ml complete EDTA-free protease inhibitor cocktail (Roche)). The lysate was clarified by centrifugation (37,500 g, 30 min, 4°C) and the supernatant was applied to a 1 mL Ni-NTA HisTrap column (GE Healthcare). The protein was eluted with an imidazole gradient using an analogous gradient of HisTrap buffer B (HisTrap buffer A with 500 mM imidazole) and analysed by SDS–PAGE.
Further protein purification was achieved by size exclusion chromatography (SEC) through a Superdex™ 200 10/300 GL column (GE Healthcare) using a buffer containing 0.5 M NaCl and 25 mM Tris-HCl, pH 8. Fractions of interest were analysed by SDS–PAGE, pooled and concentrated in Amicon Ultra-4 centrifugal filters (MWCO 10 kDa, centrifugation at 2,000 rpm., 4°C). Protein concentration was measured with the NanoDrop ND-1000 Spectrophotometer. Proteins were finally stored in buffer supplemented with 50 % glycerol at -20°C.
Purification of ARH1 and ARH1 D55,56A
E. coli BL21 DE3 pET28-ARH1 and BL21-pET28-ARH1 D55A D56A were grown to an OD600 = 0.6 at 37°C, 175 rpm. Afterwards, bacteria were allowed to cool to room temperature for 30 minutes. Expression was induced with 1 mM IPTG, and bacteria were finally grown overnight at room temperature, 175 rpm. Bacteria were harvested by centrifugation, and proteins were purified analogously to rS1 variants.
Purification of ModA
E. coli BL21 DE3 pET28-ModV were grown to an OD600 = 1 at 37°C, 175 rpm. Proteiexprssion was induced with 0.5 mM IPTG and bacteria were harvested by centrifugation after 3 hours at 37°C. Pelleted bacteria were resuspended in 50 mM NaH2PO4, pH 8, 300 mM NaCl, 1 mM DTT, 1 tablet per 500 ml complete EDTA-free protease inhibitor cocktail (Roche))and lysed by sonication (3 x 1 min, 50 % power). Lysates were centrifuged at 3,000 g, 4°C for 20 min. Sediments were washed by resuspension in 30 mL 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 100 mM NaCl, 1 M urea, 1 mM DTT, 1 tablet EDTA-free protease inhibitor (Roche), and centrifuged at 10,000 g, 4°C for 20 min. Pellets, containing inclusion bodies, were resuspended in 40 mL 100 mM Tris pH 11.6, 8 M urea, transferred to 12–14 kDa MWCO dialysis bags (Roth), and dialysed overnight against 50 mM NaH2PO4, 300 mM NaCl. Protein solutions were centrifuged at 20,000 g, 4°C for 30 min. Supernatants were batch-purified using disposable 10 mL columns (Thermo Fisher Scientific) packed with 2 mL Ni-NTA agarose (Jena Bioscience), and equilibrated with 10 column volumes (CV) of 50 mM NaH2PO4 (pH 8), 300 mM NaCl. Proteins were purified by washing the columns with 30 CV 50 mM NaH2PO4, 300 mM NaCl, 15 mM imidazole, eluted with 5 mL 50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, and concentrated in Amicon Ultra-4 centrifugal filters (MWCO 10 kDa, centrifugation at 2,000 rpm, 4°C). Proteins were finally purified by SEC as described for rS1.
Purification of ModB
E. coli BL21 DE3 pET28-ModB were grown to an OD600 = 2.0 at 37°C, 185 rpm and cooled down to 4°C while shaking at 160 rpm for at least 30 min. Protein expression was induced by the addition of 1 mM IPTG. The cultures were then incubated for 120 min at 4°C, 160 rpm and bacteria harvested by centrifugation (4,000 rpm, 4°C, 25 min). The ModB protein was purified from the supernatant as described for rS1 variants.
In vitro ADP-ribosylation and RNAylation of protein rS1 with 32P-labelled NAD, NAD-8mer and NAD-Qβ-RNA
0.3 µM protein rS1 were ADP-ribosylated in the presence of 0.25 µCi/µl 32P-NAD or RNAylated in the presence of either 0.6 µM 32P-NAD-8mer or 0.03 µM 32P-NAD-Qβ-RNA by 1.4 µM ModB and in 1x transferase buffer (10 mM Mg(OAc)2, 22 mM NH4Cl, 50 mM Tris-acetate pH 7.5, 1 mM EDTA, 10 mM β-mercaptoethanol, 1 % glycerol) at 15°C for at least 120 minutes. 5 µL samples were taken after 0 (before addition of ModB), 1, 2, 5, 10, 30, 60 and 120 minutes and mixed with 5 µL 2x Laemmli buffer to stop the reaction. Reactions were assessed by 12 % SDS-PAGE and gels stained in Instant Blue solution (Sigma-Aldrich) for 10 min. Radioactive signals were visualised using storage phosphor screens (GE Healthcare) and a Typhoon 9400 imager (GE Healthcare). Intensities of radioactive bands were quantified using ImageQuant 5.2.
Kinetics of RNAylation in the presence of high NAD concentrations
Kinetic experiments for RNAylation in the presence of different NAD concentrations were performed using conditions for RNAylation and ADP-ribosylation as mentioned above. The kinetics were performed in 0.15 µM 32P-NAD-RNA 8-mer with either 0.125 µM, 12.5 µM, 125 µM or 1.25 mM NAD corresponding to an approximate molar excess of NAD over 32P-NAD-RNA 8-mer of 1-, 100-, 1,000- and 10,000-fold. The reactions temperatures were pre-adjusted to 15°C. 5 µL samples were taken after 0 (before addition of ModB), 1, 5, 10, 30, 60 and 120 minutes and mixed with 5 µl 2x Laemmli buffer to stop the reaction. Samples were analysed by 12 % SDS-PAGE analysis. 10 µL of a reference in 1x Laemmli buffer were applied to each SDS-polyacrylamide gel containing 1.91 pmol of protein rS1 modified by 6.74 pmol ModB in 1x transferase buffer and 0.15 µM 32P-NAD-RNA 8-mer. Intensities of radioactive bands were quantified using ImageQuant 5.2. Relative RNAylation values were computed by the division of the individual intensity values by the intensity of the reference.
Investigation of self-RNAylation of ribosomal protein rS1
In scales of 20 µL reactions, 3.6 µM of 32P-ADPr-8mer were either incubated with 2.6 µM of protein rS1, 3.9 µM of ModB or both 2.59 µM of protein rS1 and 3.89 µM of ModB in 1x transferase buffer. As a positive control, equal amounts of protein rS1 and ModB were incubated with 0.6 µM of 32P-NAD-8mer. All reactions were incubated at 15°C for 60 minutes. Samples were taken before the addition of ModB (0 minutes), after 60 minutes of incubation and stopped by the addition of 1 volume 2x Laemmli buffer each. The reactions were analysed by 12 % SDS-PAGE and autoradiography imaging.
RNAylation of protein rS1 with Qβ-RNA and specificity for the 5’-NAD-cap
0.05 µM 32P-NAD-Qβ-RNA, 0.15 µM 5’-32P-Qβ-RNA or 0.15 µM 5’-32PPP-Qβ-RNA were incubated with 2.3 µM of protein rS1 and 1.4 µM of ModB each in the presence of 1x transferase buffer at 15°C for 60 minutes. Samples were taken before the addition of ModB (0 minutes) and after 60 minutes of incubation and reactions stopped by the addition of 1 volume 2x Laemmli buffer. Reactions were analysed by 10 % SDS-PAGE applying a reference of rS1-ADPr (32P) in 1x Laemmli buffer and autoradiography imaging.
Preparation of RNAylated and ADP-ribosylated rS1 for enzymatic treatments
ADP-ribosylation or RNAylation reactions performed with radio-labelled substrates were washed and equilibrated in 1x transferase or 1x degradation buffer for further enzymatic treatment. Therefore, the reactions were washed with 4 column volumes of the corresponding buffer via centrifugation at 10,000 x g, 4°C in 10 kDa Amicon filters.
Nuclease P1 digest of protein rS1 RNAylated with Qβ-RNA (rS1-Qβ-RNA)
19 µL of rS1/rS1-Qβ-RNA (32P) mixture equilibrated in 1x transferase buffer were incubated with either 1 µL of nuclease P1 or 1 µL of Millipore water at 37°C for 60 minutes. Samples were taken before the addition of enzyme (0 minutes) and after 60 minutes of incubation and reactions stopped by addition of 1 volume 2x Laemmli buffer. Reactions were analysed by 10 % SDS-PAGE using rS1-ADPr (32P) as a reference and autoradiography imaging.
Digest of rS1-8mer and rS1-ADPr by NudC and alkaline phosphatase
28 µL of rS1/rS1-8mer (32P) mixture or rS1/rS1-ADPr (32P) mixture equilibrated in 1x degradation buffer (12.5 mM Tris-HCl, pH 7.5, 25 mM NaCl, 25 mM KCl, 5 mM MgCl2) were either incubated with 2.3 µM of NudC and 1 U of alkaline phosphatase (FastAP, Thermo Scientific) or 2 µL of Millipore water as a negative control at 37°C. Samples were taken before the addition of enzymes/Millipore water and after 60 minutes of incubation. Reactions were stopped by adding 1 volume 2x Laemmli buffer, and additional samples were taken and resuspended in 1 volume 2x PAGE Loading Dye. Samples were analysed by either 12 % SDS- or denaturing 20 % PAGE using 32P-NAD-8mer and 5’-32P-8mer as references. Autoradiography imaging was applied to all analyses.
Tryptic digest of rS1-8mer and rS1-ADPr
19 µL of both rS1/rS1-8mer (32P) mixture and rS1/rS1-ADPr (32P) mixture in 1x degradation buffer were incubated with either 0.2 µg Trypsin (Sigma, EMS0004, mass spectrometry grade) or Millipore water as a negative control at 37°C. Samples were taken before the addition of Trypsin/Millipore water (0 minutes) and after 120 minutes of incubation. Reactions were stopped by adding 1 volume 2x Laemmli buffer to samples and were analysed by 12 % SDS-PAGE and autoradiography imaging.
Chemical removal of ADP-Ribosylation and RNAylation in vitro
Aliquots from washed and equilibrated ADP-ribosylated (1 µL) and RNAylated (2 µL) (32P) rS1 were treated with either 10 mM HgCl2 or 500 mM NH2OH 2,3 at 37°C for 1h. Enzymatic reactions were stopped by addition of 2x Laemmli-Buffer and analysed by 12 % SDS-PAGE.
Enzymatic removal of ADP-Ribosylation and RNAylation in vitro
Aliquots from washed and equilibrated ADP-ribosylated (1 µL) and RNAylated (2 µL) rS1 (32P) were treated with 0.5 U endonuclease P1 (Sigma-Aldrich) 4 or 0.95 µM ARH1 or ARH3 (human, recombinant, Enzo Life Science) 5 in the presence of 10 mM Mg(OAc)2, 22 mM NH4Cl, 50 mM HEPES, 1 mM EDTA, 10 mM β-mercaptoethanol and 1 % (v/v) glycerol in a total volume of 20 µL at 37°C for 1 h. Enzymatic reactions were stopped by the addition of 2x Laemmli-Buffer and analysed by 12 % SDS-PAGE.
Inhibition of RNAylation and ADP-ribosylation with 3-methoxybenzamide
20 µL reactions of 1.4 µM ModB and 2.3 µM protein rS1 with either 1 µM of 32P-NAD-8mer or 3 µM 5’-32P-8mer were incubated in the presence of 2 mM 3-methoxybenzamide (50 mM stock in DMSO) or in the absence of the inhibitor (DMSO only) at 15°C 6. Samples were taken before the addition of ModB (0 minutes) and after 60 minutes of incubation with ModB. Reactions were stopped by the addition of 1 volume 2x Laemmli buffer and analysed by 12 % SDS-PAGE.
Cultivation of E. coli B strain and T4 phage infection
Pre-cultures of E. coli B strain pTAC-rS1 were incubated in LB medium with 100 µg/mL ampicillin at 37°C, 185 rpm overnight. For main cultures, 150 mL LB medium with 100 µg/mL ampicillin were inoculated with pre-culture to an OD600 = 0.1. At OD600 = 0.4 protein expression was induced by the addition of 1 mM IPTG. At an OD600 = 0.8, cultures were either infected with bacteriophage T4 at an MOI 10 (20 mL phage solution) (DSM 4505, Leibniz Institute DSMZ). For the negative control, 20 mL LB medium were added to the culture. Cultures were incubated for 20 min at 37°C, 240 rpm. Bacteria were harvested by centrifugation at 4,000 x g at room temperature for 15 min. Pellets were stored at -80°C.
Purification of His-tagged rS1 from infected E. coli strain B pTAC-rS1
Bacterial pellets were resuspended in 10 mL buffer A and lysed via sonication (1 x 5 min, cycle 2, 50 % power). Lysates were centrifuged at 37,500 g, 4°C for 30 min. The supernatant was filtered through 0.45 µm filters. rS1 from bacteriophage T4-infected or non-infected E. coli B strain was purified from the supernatant by gravity Ni-NTA affinity chromatography. 1 mL of Ni-NTA agarose slurry was added to a 10 mL propylene column and equilibrated in buffer A. The supernatant was loaded onto the column twice. The column was washed with a mixture of 95 % buffer A and 5 % buffer B containing 29.75 mM imidazole. Protein was eluted from the column with 10 mL buffer B.
His-tagged-protein rS1 from T4-infected or uninfected E. coli B strain pTAC-rS1 was washed with two filter volumes of 1x degradation buffer (12.5 mM Tris-HCl, pH 7.5, 25 mM NaCl, 25 mM KCl, 5 mM MgCl2) by centrifugation in 10 kDa Amicon Ultra-4 centrifugal filters at 5,000 g, 4°C and concentrated to a final volume of 120 µL. The fractions were analysed by 12 % SDS-PAGE analysis, and the gel was stained in Instant Blue solution for 10 min and imaged immediately.
Purification of endogenous rS1 from infected E. coli strain B
For isolation of endogenous protein rS1, ribosomes were first isolated as described in 7. Bacterial pellets of T4 infected and non-infected E. coli B strain were resuspended in resuspension buffer (20 mM MgCl2, 200 mM NH4Cl, 20 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 6 mM β-mercaptoethanol) and cells were lysed by sonication. The lysate was cleared by ultracentrifugation at 30,000 x g, 4°C, 30 min. 15 mL of cleared supernatant were loaded onto 1 volume sucrose cushion (20 mM MgCl2, 200 mM NH4Cl, 20 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 6 mM β-mercaptoethanol, 30 % (w/v) sucrose) in glass ultracentrifuge tubes, which were centrifuged at 67,000 x g, 18°C for 24 hours. The supernatant was discarded, the ribosomal pellets washed with rS1 wash buffer (20 mM Mg(OAc)2, 1 M NH4Cl, 10 mM Tris-HCl pH 7.6, 7 mM β-mercaptoethanol) carefully and resuspended in 4 mL of rS1 wash buffer.
Endogenous rS1 was then purified from resuspended ribosomes using self-synthesised poly(U)-Sepharose 8. Synthesis of poly(U)-Sepharose was performed according to 9 with slight modifications. 700 mg of cyan bromide-activated Sepharose beads were resuspended in 30 mL 0.1 M NaCl, 2 mM Tris-HCl pH 7.5 and incubated at 4°C for 2 hours under rotation. Beads were pelleted by centrifugation at 15,567 x g for 3 minutes and the supernatant removed. Beads were then resuspended in 30 mL of 0.2 M 4-morpholine-ethanesulfonic acid (MES) and pelleted again to discard the supernatant. For coupling of poly-uridine (poly(U)) material to the beads, 10 mg of poly(U) material (Santa Cruz) resuspended in 5 mL of 0.2 M MES pH 6.0 were added to the equilibrated, activated Sepharose beads and the suspension incubated at 4°C overnight. The resulting poly(U)-Sepharose was equilibrated in rS1 wash buffer by washing with 4 polypropylene column volumes (10 mL). 6 mL of poly(U)-Sepharose suspension were obtained for isolation of endogenous rS1 from ribosomes. To specifically isolate rS1, 3 mL of the beads were mixed with 4 mL of the isolated ribosomes and incubated at 4°C for 30 minutes. The bead-protein mixture was subjected to gravity affinity purification with 10 mL polypropylene columns. Ribosomes were washed with 4 column volumes of rS1 wash buffer and endogenous rS1 eluted with 10 mL rS1 elution buffer (20 mM Mg(OAc)2, 1 M NH4Cl, 10 mM Tris-HCl pH 7.6, 7 mM β-mercaptoethanol, 7 M urea). Afterwards, beads were re-equilibrated in rS1 wash buffer and the washing and elution steps were repeated once. Eluates of the same sample were pooled, concentrated and analysed by SDS-PAGE.
To identify ADPr-modification sites on rS1, 100 µg of in vitro or in vivo modified-rS1 were digested with trypsin (sequencing grade modified, Promega) overnight at a 1:20 (w/w) ratio at 37°C. The digest was stopped by adding 0.5 % trifluoroacetic acid and 5 % acetonitrile (v/v), followed by desalting using acommercially available pre-packed C18 column for acidic RP C18 desalting (Harvard Apparatus, Microspin C18 Column) and enriched using TiO2 as described elsewhere for protein-RNA cross-link enrichment 10. Next, the sample was subjected to LC-MS analysis using a Dionex UltiMate 3000 UHPLC+ focused system (Thermo Scientific) with a C18 analytical column (75 µm × 300 mm, ReproSil-Pur 120 C18-AQ, 1.9 µm, Dr. Maisch GmbH, packed in house). Peptides were separated by RP C18 chromatography on a 58 min multi-step gradient (flow rate 0.3–0.4 µl min− 1). Eluting peptides were analysed with an orbritrap mass spectrometer (QExactive HF-X or Orbitrap Lumos Fusion, Thermo Scientific) with the following settings: MS1 spectra in profile mode (resolution of 120k), MS2 spectra in centroid mode (resolution of 30k); isolation window set to 1.6 m/z and dynamic exclusion set to 7 s. Raw data were searched in an open search using PD2.2 (SEQUEST search engine) to identify possible adduct masses that could correspond to RNA or NAD. Precursor mass tolerance was set to 700 Da and fragment mass tolerance was set to 0.02 Da. We only identified ribose-5-phosphate as a shorter peptide modification reliably with an adduct mass of mz = 212.0086 and validated results in stringent searches with precursor mass tolerance set to 7 ppm and fragment mass tolerance set to 10 ppm. Identified peptides were filtered on PSM level to an FDR of 1 %.
To identify larger adduct masses than ribose-5-phosphate that escaped LC-MS detection, we performed MALDI-TOF experiments. Modified rS1 peptides were prepared as described above at basic pH. Formic acid was substituted with 10 mM ammonium hydroxide in all RP-C18 buffers. Eluted sample was mixed with 2,5-dihydroxy benzoic acid (DHB) and then spotted onto an MTP stainless steel plate in a double layer fashion. MS data was acquired on a Bruker Autoflex Speed MALDI-TOF instrument in positive reflectron mode for survey scans or LIFT mode for pseudo MS2. The instrument was calibrated against BSA peptides and then recalibrated against wt rS1 peptides to 20 ppm mass error. Record spectra were baseline corrected but not smoothened.
Proteins were separated by 10 % SDS-PAGE and gels were equilibrated in transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine, 20 % [v/v] methanol). 0.2 µm polyvinylidene difluoride (PVDF) membranes (GE Healthcare) were activated in methanol for 1 min and equilibrated in transfer buffer. Proteins were transferred from gels to PVDF membranes in a semi-dry manner at 300 mA for 1.5 h, if not indicated differently. After the transfer, membranes were dehydrated by soaking in methanol and washed 2x with TBS-Tween (TBS-T; 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05 % [v/v] Tween® 20). Afterwards 10 mL blocking buffer (5 % [w/v] milk powder in TBS-T) were added to the membranes and incubated at room temperature for 1 h. In order to detect ADP-ribosylated proteins, membranes were incubated with 0.1 ng/mL primary antibody MABE1016 (Merck) in 10 mL washing buffer (1 % [w/v] milk powder in TBS-T) at 4°C overnight 11. Membranes were washed and incubated with 10 mL of a 1:10,000 dilution of the horseradish-peroxidase-(HRP)-goat-anti-rabbit-IgG secondary antibody (Advansta) in washing buffer at room temperature for 1 h. Afterwards, membranes were washed with PBS. Proteins with detected modifications were visualised by chemiluminescence using the SignalFire ECL Reagent or the SignalFire Elite ECL Reagent (Cell Signaling Technology) according to the manufacturer’s instructions.
If proteins in SDS-PAGE gels needed to be visualised before blotting, a 2,2,2-trichloroethanol (TCE) staining method 12 was used. Resolving gels were supplemented with 0.5 % (v/v) TCE. For visualisation, gels were activated by ultraviolet transillumination (300 nm) for 60 s. Proteins then showed fluorescence in the visible spectrum.
Quantification of RNAylation
rS1 proteins were isolated from E. coli strain B pTAC rS1 infected or non-infected with bacteriophage T4. 1.5 µM rS1 were digested with 1 µM ARH1 in the presence of 12.5 mM Tris-HCl pH 7.5, 25 mM NaCl, 25 mM KCl and 5 mM MgCl2. 1.5 µM rS1 were digested with 0.5 U endonuclease P1 in 100 mM Mg(OAc)2, 220 mM NH4Cl, 500 mM HEPES pH 7.5, 10 mM EDTA, 100 mM β-mercaptoethanol and 10 % glycerol. Digests were incubated at 37°C for 2 hours. Afterwards, digests were ethanol-precipitated by the addition of 9 volumes of ethanol and precipitated by centrifugation (14,000 rpm) at 4°C for 1 h. Protein pellets were resuspended in 10 µL 1x Laemmli buffer and analysed via Western blot. ADPr-modifications were detected by the primary antibody MABE1016 (Merck) as described above.
Reprobing of PVDF membranes with sodium azide
Following the visualisation of the chemiluminescence signal, the PVDF membranes were washed with Dulbecco’s PBS. Subsequently, the membranes were incubated in blocking solution (5 % [w/v] milk powder in TBS-T) with 1 mM sodium azide for 3 hours at 4°C. Afterwards, the membranes were washed in TBS-T 6 times, for 5 minutes each. Inactivation of HRP was visualised by chemiluminescence using the SignalFireTM ECL Reagent or the SignalFireTM Elite ECL Reagent.
Detection of the FLAG-tagged rS1
PVDF membranes were incubated with a 1:10,000 dilution of the anti-FLAG-M2 antibody (Thermo Fisher Scientific) in a washing solution at 4°C overnight. The membranes were washed 3 times with washing solution and incubated in a 1:10,000 dilution of the HRP-goat-anti-mouse-IgG (Advansta) for 1 h at room temperature. FLAG-tagged rS1 was visualised by chemiluminescence as described above.
T4 Phage infection of E. coli strain B and Ced64 with ARH1WT and ARHD55,56A plasmids
E. coli B strain or E. coli Ced64 containing the plasmid pTAC ARH1WT or pTAC ARH1D55,56A were inoculated in 40 mL LB medium supplemented with 100 µg/mL ampicillin at OD600 = ~ 0.1 and incubated at room temperature, 150 rpm. At OD600 = 0.3, the expression of ARH1WT or ARH1D55,56A was induced with 1 mM IPTG. At OD600 = 0.8, cultures were infected with T4 at a MOI 10. After 0, 5, 10, 30, 60, 90, 110, 140 and 200 minutes, the OD600 values of the cultures were measured. At the indicated time points, 100 µL aliquots were taken, and bacteriophage T4 infections stopped by the addition of 2x Laemmli buffer. The aliquots were analysed via Western Blotting.
Visualisation of ARH1 WT and ARH1D55,56A via Western Blotting
The proteins were transferred onto a PVDF membrane as described above. Following the exact same treatment, the membranes were incubated in a 1:2,000 dilution of 6x-His Tag Monoclonal Antibody (HIS.H8) in 10 mL of washing solution. The following day, horseradish-peroxidase-(HRP)-goat-anti-mouse-IgG (Advansta, San Jose, USA) was used as a secondary antibody. His-tagged proteins were visualised by chemiluminescence as described above.
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