Bacterial strains, media and growth conditions
All cloning steps were performed in the E. coli
strain DH10B (F- mcrA Δ (mrr-hsdRMS-mcrBC)
80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL nupG). Subsequent expression experiments were conducted in E.coli
strain BL21-DE3 (Invitrogen) [F−
) gal dcm (DE3)]. Co-expression of O-mRNA and O-ribosome constructs for the in vivo
O-ribosome activity assay used the Genehog strain (Invitrogen) [F- mcrA Δ(mrr-hsdRMS-mcrBC)
80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara- leu)7697 galU galK rpsL (StrR) endA1 nupG fhuA::IS2 (confers phage T1 resistance)]. Ribosome purifications were performed from the MRE 600 strain (a natural RNase 1-deficient strain) (13
). Cultures were grown in LB or 2XTY liquid media as described in text. The media was supplemented with ampicillin at 100 μg/ml and/or kanamycin at 50 μg/ml where necessary unless described in text.
MS2-GST expression system
A mutated MS2 coat protein coding sequence containing the FG mutation (amino acid residues 68−80 deleted, WT numbering) to prevent oligomerization (14
) and the V29I mutation (wild-type numbering) to increase tag binding affinity (16
) was amplified by PCR using the primers MS2fwd (gcgcaGGATCCgcttctaactttactcagttcgttctcgtc) and MS2rev (gcgcaCTCGAGttagtagatgccggagtttgctgcgattg) bearing the XhoI (fwd), BamHI (rev) restriction sites and subcloned into the expression vector pGex 4T1 (GE Healthcare) (pBR322 origin, the ampicillin resistance and the GST gene expression given by the PTrc
promoter) using these sites resulting in a GST-MS2 construct expressed under the control of the strong inducible PTrc
To express and purify GST-MS2, cultures were grown overnight in non-inducing 2XTY liquid media and then diluted to an OD600 of 0.1 in 2 l of fresh 2XTY and grown to an OD600 of 0.6, induced with IPTG (final concentration 1 mg/ml) and incubated (16 h, 20°C, 220 rpm). Cultures were then pelleted, the pellets kept on ice and resuspended in 50 ml of ice-cold GST-MS2 lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM KCl, 2 mM DTT, 5% glycerol, 6 mM BME, 100 µM Benzamidine, 100 µM PMSF) and lysed using an emulsiflux at 4°C. The lysates were kept on ice and clarified by centrifugation (1 h, 20 000 rpm 4°C), in SS34 tubes (Beckman) and the supernatant (40−45 ml) applied to phosphate buffered solution (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM NaHPO4) pre-incubated 10 ml column of 4b glutathione−sepharose resin (GE Healthcare) (at RT with a flow rate of 0.5 ml/min, washed with 100 ml of PBS (1 ml/min) and GST-MS2 eluted with 40 ml GST-MS2 elution buffer (50 mM Tris–HCl pH 8, 10 mM reduced glutathione). The elution was then dialyzed (snakeskin dialysis tubing, Roche) in 3 l of PBS containing 20% glycerol (16 h, 4°C).The dialysis was repeated in the same volume (2 h, 4°C), and the protein separated into aliquots and flash frozen. The protein concentration was measured and the protein purity determined by SDS-PAGE gel.
Tag-ribosome expression system
The tag sequence was introduced into the O-16S by enzymatic inverse PCR. The template was pJC73-O-16S(3
) and the primers were Tagfwd (gcgcaGGTCTCAgattacccatctttactagtCTTCTTTGCTGACGAGTGGCGGACGGGTGAG), Tagrev (gcgcaGGTCTCAaatcctcatcaaaactagtCTTCTTCCTGTTACCGTTCGACTTGCATGTG), that contain BsaI sites. The PCR reaction mix was digested with BsaI and DpnI, circularized by ligation and transformed into E. coli
DH10B. pSC101*-O-ribosome-tag was created by subcloning the entire tag-O-16S sequence into pSC101*-O-ribosome (10
) (which contains the O-1623S operon under the control of the ribosomal P1P2 promoter) using the XhoI XbaI sites flanking the O-16S gene. The Ribo-X mutations (10
) were subcloned into the O-ribosome-tag construct using the unique Sca1 and Xba1 sites creating pSC101*-Ribo-X-tag.
To express and purify actively translating tagged O-ribosomes the pSC101*-O-ribosome-tag or pSC101*-Ribo-X-tag constructs were co-transformed with O-MBP-GST (10
), plated onto LB-KA (containing 50 μg/ml kanamycin and 50 μg/ml ampicillin). The cultures were grown overnight (16 h, 20°C, 220 rpm) in 2XTY-KA, diluted to an OD600
of 0.1 in 4 L of fresh 2XTY-KA, grown to an OD600
of 0.9 (16 h, 20°C, 220 rpm). The cultures were then cooled at 4°C for 2 h, pelleted, and resuspended in 30 ml of ribosome lysis buffer (20 mM Tris–HCl pH 7.5, 100 mM NH4
Cl, 10 mM MgCl2
, 0.5 mM EDTA, 6 mM BME, 100 µM Benzamidine, 100 µM PMSF). Cells were lysed using an emulsiflux at 4°C. The lysate were kept on ice and clarified by centrifugation (1 h, 20 000 rpm 4°C, in SS34 tubes (Beckman) and the supernatant (25 ml) were purified for the ribosome fraction by applying it to an ice-cold 35 ml sucrose cushion (20 mM Tris–HCl pH 7.5, 500 mM NH4
Cl, 10 mM MgCl2
, 0.5 mM EDTA, 1.1 M sucrose, 6 mM BME, 100 µM Benzamidine) in a Ti45 ultracentrifuge tube (Beckman) and the ribosome fraction sedimented by centrifugation (18 h, 37 000 rpm 4°C) to reveal a glassy ribosome pellet. This pellet was used for affinity purification of tagged ribosomes or further processed for storage as described below. The glassy pellet was rinsed with ice-cold ribosome lysis buffer and then resuspended in 2 ml ribosome lysis buffer by application of the buffer and allowing the glassy pellet to dissolve on ice. The ribosomes were diluted in ribosome binding buffer (20 mM Tris–HCl pH 7.5, 100 mM NH4
Cl and 10 mM MgCl2,
6 mM BME, 100 µM Benzamidine, 100 µM PMSF) to a total volume of 10 ml, giving a ribosome concentration in 7−14 mg/ml range (determined using the conversion 1 A260
= 62 mg). The ribosomes were split into aliquots and flash frozen for affinity purification.
RF1 expression system
To express RF1, the RF1expression plasmid from the PURE expression system (18
) was transformed into BL21-DE3 and the cultures grown overnight (16 h, 20°C, 220 rpm) in 2XTY liquid media. The culture was diluted to an OD600
of 0.1 in 2 L of fresh 2XTY, grown to an OD600
of 0.9 (16h, 20°C, 220 rpm) and cooled at 4°C for 2 h. Cultures were pelleted and resuspended in 50 ml RF1 lysis buffer (50 mM Na2
pH 8, 300 mM NaCl), and lysed at 4°C using an emulsiflux. The supernatant was further diluted with RF1-lysis buffer to a total volume of 150 ml and applied (4°C, 5 ml/min) to a 50 ml Ni-chelating fast flow sepharose column (GE Healthcare). The column was washed with 150 ml of RF1-wash buffer (50 mM Na2
, pH 8 300 mM NaCl, 10 mM imidazole) and eluted using a gradient of imidazole (upto 250 mM imidazole) using RF1-elution buffer (50 mM Na2
pH 8, 300 mM NaCl, 250 mM imidazole), the elution was collected in 5 ml fractions. RF1 containing fractions were pooled and further purified using a 300S gel filtration column: the pooled fractions were diluted into 300 ml of RF1 buffer (5 mM HEPES pH 7.5, 50 mM KCl, 100 mM NH4
Cl, 10 mM MgCl2
) and separated into 5 ml fractions using a 5 ml/min flow rate. RF1 was precipitated by slowly stirring in ammonium sulphate to a final concentration of 50% and incubating (16 h, 4°C). The precipitated proteins were resuspended in 500 µl of RF1 buffer containing 50% glycerol.
Tag MS2-GST affinity measurements
The activity of MS2-GST for binding the tag was assayed by fluorescence anisotropy using the chemically synthesized, fluorescently labelled tag RNA (5′ fluorescein GCGAGGATTACCCGC). The change in fluorescence anisotropy was measured (excitation wavelength 494 nm, emission 521 nm) with the incremental addition of 10 µM Tag RNA to a 200 µM solution of MS2-GST in ribosome buffer (supplemented with 0.1 mg/ml bovine serine albumen). The measurements were performed in a quartz cuvette with mixing. The change in fluorescence anisotropy (binding) with increasing RNA tag concentration was plotted and the Kd value extracted from a best-fit.
Tag-O-ribosome in vivo translational activity
To determine the effect of the tag on O-ribosome activity we transformed pSC101*-O-ribosome or pSC101*-O-ribosome-tag into Genehog E. coli
containing the O-Cat reporter (10
). The cultures were grown overnight in LB liquid media supplemented with 50 μg/ml kanamycin and 25 μg/ml tetracycline, diluted 10-fold into LB supplemented with 12.5 μg/ml kanamycin and 6.25 μg/ml tetracycline, grown to mid log phase (3 h, 37°C, 220 rpm) and then plated onto LB-agar supplemented with 12.5 μg/ml kanamycin and 6.25 μg/ml tetracycline and a range of chloramphenicol concentrations (0–1000 μg/ml) and incubated (overnight, 37°C).
Tagged O-ribosome affinity purification
Four milligrams of purified GST-MS2 was diluted in 40 ml of PBS and bound to 10 ml of glutathione sepharose beads (in PBS) by incubating with gentle mixing for 30 min at RT. The beads were pelleted by centrifugation (5 min, 500 rpm, 4°C) and washed; twice with 40 ml of PBS and twice with 40 ml of ribosome binding buffer (20 mM Tris–HCl pH 7.5, 100 mM NH4Cl and 10 mM MgCl2, 6 mM BME, 100 µM benzamidine, 100 µM PMSF).
The sucrose cushion purified glassy ribosome pellet was resuspended in 40 ml of ribosome binding buffer, added to the pre-chilled beads and incubated (rocked gently for at 4°C for 1 h). The beads were then washed three times with 40 ml of ribosome binding buffer at 4°C; the wash steps separated by 45 min. The final bead pellet was resuspended in 10 ml of ribosome buffer (20 mM Tris–HCl pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 6 mM BME, 100 µM Benzamidine, 100 µM PMSF) + 3 mM RNA tag and incubated (18 h, gently rolling, 4°C).
The synthesized RNA Tag sequence [5′PGCGAGGAUCACCCGC, a known competitive ligand for MS2 (16
)] was dissolved in Tris–HCl pH 7.5. The tag was folded by heating to 98°C and slow cooling to RT, it was then diluted in ribosome binding buffer to a concentration 3 mM and used to elute the bound ribosomes 10 ml of the 3 mM RNA tag solution (4°C) was added to the washed beads and incubated (18 h, gentle rolling, 4°C). The eluted ribosomes were separated from the beads by pelleting the beads (20 min, 4000 rpm, 4°C). Any remaining beads were removed from the supernatant by filtering through a 0.2 µM filter.
The tagged O-ribosomes were concentrated using a 30 000 Da molecular weight cut-off Amicon concentrator (Millipore), pre-equilibrated with ribosome buffer + 5% Tween, by centrifugation (4°C, 2000 rpm) to a final volume of ~500 µl, split into aliquots and flash frozen.
Characterization of affinity purified tag-ribosomes
The purity of the affinity purified tagged O-ribosome was assayed by reverse transcription (12
). The template rRNA was prepared by taking a 50–100µg aliquot of concentrated eluted ribosomes or ribosomal RNA extracted using Trizol (Invitrogen), according to the manufactures instructions. The RT primer (GGCCATGATACTTGAC) used was gel purified and subsequently 32
P labelled (5 µl reaction mixture consisting of 5 µM of RT primer, 3.36 pmols γ-32
P ATP, 1× PNK buffer (NEB) and 3 U polynucleotide kinase (NEB) and incubated at 37°C for 45 min followed by 65°C for 15 min). One microlitre of this labelled primer mixture and 1 µg of template was then added to a 50 µl reaction mixture consisting of 1× superscript 3 buffer (Invitrogen), 5 mM DTT, 100 mM ddATP, dGTP, dTTP, dCTP. Annealing was performed by incubating the mixture at 94°C for 2 min followed by a temperature gradient down to 25°C at 0.1°C/s, 200 U Superscript 3 (Invitrogen) was then added and extension preformed by incubating at 50°C 16 h. The completed RT reaction was mixed with a equal volume of formamide running buffer (5% sucrose, 0.1% bromophenol blue, formamide) incubated at 85°C for 15 min and 2–5 µl loaded onto a denaturing gel [10% formamide, 20% acylamide, 1× TBE (90 mM Tris–Borate 2 mM EDTA)] run at 37 W for 2.5 h using 1× TBE running buffer. The gel was imaged using a Typhoon scanner and the intensity of the bands analyzed using the Image Quant TL program (GE Health Care).
The integrity of the eluted ribosomes was determined by non-denaturing electrophoresis [gel: 0.5% agarose, 3% acylamide, 1× TBM (89 mM tris base, 89 mM boric acid 1 mM MgCl2)]. The gel was pre-run (1 h, 4°C, 100 V) in 1× TBM running buffer, the running buffer changed for fresh running buffer, and 0.1–2 OD260 of the eluted ribosomes [diluted in a equal volume of non-denaturing loading buffer (20% sucrose, 1× TBM, 0.1% bromophenol blue)] loaded. Gels were run for 2 h, (4°C, 100 V) before visualizing the nucleic acids by incubating in 10 µg/ml ethidium bromide for 10 min and then imaging under UV.
Analytical sucrose gradients were also used to look at the integrity of purified ribosomes. An analytical sucrose gradient was prepared with 15% and 30% (w/v) sucrose in ribosome buffer. These two solutions were added to a gradient pourer to pour even 13 ml 15–30% sucrose gradients in 40Ti tubes. The gradients were cooled to 4°C and then 2 OD260 of the ribosome preparations were carefully added to the very top layer of the gradients. The gradients were spun (20 h, 20 500 rpm, 4°C) and slowly removed from the bottom of the tubes through a fine needle. The OD254 was measured along the gradient to give the gradient profile.
Preparation of tRNAfMet
H]fMet for the RF1 termination assay (19
To prepare tRNAfMet-[3H]fMet the tRNAfmet was first aminoacylated with [3H]Met. A 200 µl reaction mix consisting of 1× fMet reaction buffer (20 mM Tris–HCl pH 7.5, 7 mM MgCl2, 150 mM KCl), 12 µM L-[3H]Met, 4 mM ATP (pH 7), 80 µM tRNAfmet and 1 µM MetRS was incubated (15 min, 37°C). The reaction was driven to completion by quenching with a high concentration of cold fMet; so the reaction mix was then supplemented with 15 µmol of fMet, a volume of reaction buffer to keep the buffer concentration constant and 0.4 µMoles of ATP and incubated (15 min, 37°C).
The charged tRNAfmet-Met was formylated by the addition of 4 µl of formyl donor (preparation is described below) (N5H10 methenyl-tetrahydrofolic acid) and 5 µM of formylase. This reaction mix was then incubated (15 min, 37°C).
The formyl donor was prepared by dissolving 100 mg folinic acid calcium salt in 8 ml of 50 mM β-mercaptoethanol, adding 880 µl of 1 M HCl and incubating for 3 h. This is frozen and before use, 100 µl thawed on ice and 10 µl of 1 M Tris–HCl pH 8 and 10 µl 1 M KOH added, vortexed and incubated at RT for 20 min and any precipitation removed by centrifugation.
The tRNAfMet-[3H]fMet mixture was then phenol/chloroform (pH 4.3) extracted, ethanol precipitated and resuspended in 50 µl of tRNA storage buffer (10 mM Ammonium acetate pH 5, 50 mM KCl). Finally the aminoacylated tRNA was desalted using a Sephadex G25 column (GE), pre-equilibrated with tRNA storage buffer, by centrifugation at 7000g for 2 min. The specific activity was calculated by measuring the RNA concentration (OD260, Mr 25 000 Da) and the scintillation counts (1 µl of purified tRNAfMet-[3H]fMet was added to 3.8 ml of scintillation fluid [Fluoran-safer 2 (BDH)] in triplicate and 3H counted (Beckham LS6000SC).
In vitro RF1 termination assay
The initiation complex/pre-termination complex was assembled as described (19
), by incubation (37°C, 20 min) of a reaction mix of 1 µM purified ribosome, 2 µM O-mRNA amber (5′P GCGGCCGCUUUCAUAUCCCUCCGCAAAUGUAGUUU) or mRNA amber (5′P AGGAGGUGAGGUAUGUAGUUU), 2 µM tRNAfMet
H]fMet in ribosome buffer (20 mM Tris–HCl pH 7.5, 100 mM NH4
Cl, 10 mM MgCl2
, 6 mM BME, 100 µM Benzamidine, 100 µM PMSF) (either 50 or 100 µl). 0.1 µg/ml BSA was added to the ribosome buffer for the reactions and RF1 dilutions.
Termination was then induced by the addition of RF1 (19
), initial experiments were performed with saturating concentration of RF1 and [3
H]fMet release measured by filter binding. RF1 was added to the 50 or 100 μl pre-termination reaction mix to a final concentration of 5 µM, a control was also prepared where no RF1 was added (incubated in parallel to control for spontaneous tRNAfMet
H]fMet hydrolysis). The incubation was continued at 37°C, 15 μl aliquots were then removed at 1, 5 and 20 min, added to 1 ml of ice-cold ribosome buffer, separated into three replicates and added to a nitrocellulose filter pre-soaked in ribosome buffer, washed three times with 900 μl of ribosome buffer, air dried and added to 3.8 ml of scintillation fluid and the bound 3
H counted. This measures the amount of ribosome bound [3
Subsequent experiments were performed with a range of RF1 concentrations and measured [3H]fMet release. The pre-termination complex was assembled as described above and split into 18 μl aliquots in a row of a 96-well plate, with the next row containing 2 μl of RF1 at various concentrations in ribosome buffer. A no RF1 control containing only ribosome buffer was also prepared. The plate was incubated at 37°C for 2 min to equilibrate the solution to the reaction temperature. To start the reaction the pre-termination complex was added to the RF1 samples using a multi-channel pipette and mixed rapidly. After 10 s, 200 μl of ice-cold 5% TCA (trichloroacetic acid) was added to quench the reaction and precipitate the complex and unhydrolyzed tRNAfMet-[3H]fMet leaving hydrolyzed [3H]fMet in solution. 200 µl of the quenched reaction was removed and added to 800 μl ice-cold 5% TCA and mixed thoroughly. The sample was then spun (20 min, 25 000g, 4°C) and the top 650 µl removed split into three replicates, added to 3.8 ml scintillation fluid and 3H counted.