Construction of plasmids
Plasmid pKP219 is a low-copy mini-R1 vector carrying the lacIq gene and the LacI-regulated pA1/03/04 promoter upstream of a multiple cloning region (Pedersen and Gerdes, 1999).
pKP600a. A 4.7 kbp HindIII–SspI DNA fragment of pKP219 containing the pA1/03/04 promoter, the multiple cloning region and lacIq was inserted into pUC19 opened with HindIII and SspI creating pKP600a.
pKP600b. The 150 bp KpnI–SnaB DNA fragment of Litmus28 (New England Biolabs) containing the mcs region was inserted in pKP600a opened with KpnI and BbrPI creating pKP600b.
pKP3045. The 4.3 kbp HindIII–BglII DNA fragment of pKP600b was inserted into pBR322 opened with HindIII and BamHI leading to pKP3045. Plasmid pKP3045 has the pBR322 origin of replication and encodes bla, pA1/04/03, a multiple cloning region and lacIq.
pKP3021. Using primers BamHI-opSD-relB and relB2, relB of MG1655 was fused to an optimized Shine and Dalgarno. The resulting 0.3 kbp BamHI–SalI fragment was inserted in pKP600a opened with BamHI and XhoI, resulting in pKP3021. Primers used in PCR clonings are available upon request.
pKP3033. Using BamHI and EcoRI, the relB DNA fragment of pKP3021 was inserted into pKP219, resulting in pKP3033.
pKP3029. A PCR fragment encoding relE from MG1655 was generated using primers relE1A and relE2. The resulting 0.3 kbp DNA fragment was restricted with BamHI and XhoI and cloned into Litmus 28 yielding pKP3029.
pKP3035. The relE DNA fragment of pKP3029 was cut out with StuI and NsiI, and inserted in pBAD33 opened with HincII and PstI yielding pKP3035.
pKP3034. Using BamHI and XhoI the relE DNA fragment of pKP3029 was moved to pKP219 yielding pKP3034.
pKP3040. A DNA fragment encoding relE[R81A] (codon 81 changed from arginine to alanine) was generated by two rounds of polymerase chain reaction (PCR). PCR #1. primer –60 and mut81* with pKP3034 as template. PCR #2. primer pBADup and mut81 with pKP3035 as template. PCR #3 primer –60 and pBADup with PCR #1 and PCR #2 as templates. A BamHI–XhoI fragment generated from the product of PCR #3 was inserted in Litmus 28, yielding pKP3040.
pKP3056. The relE[R81A] encoding fragment of pKP3040 was cut out with StuI and NsiI and inserted into pBAD33 opened with HincII and PstI leading to pKP3056.
pKP3061. Using PCR primers relE1B and relE2, the relE reading frame was combined with an efficient Shine and Dalgarno sequence (SDparA). Using BamHI and XhoI, the resulting 0.3 kbp DNA fragment was inserted into Litmus28, yielding pKP3061.
pKP3067. The relE DNA fragment of pKP3061 was cut out with StuI and NsiI and inserted into pBAD33 opened with HincII and PstI, yielding pKP3067.
pKP3064. Using primers relB1-His and relB2, the 5′-end of relB was fused to an optimized SD and six histidine codons. The resulting 0.3 kbp BamHI–SalI fragment was inserted in Litmus28 opened with BamHI and XhoI leading to pKP3064.
pKP3077. The optimized-SD-His6-relB DNA fragment of pKP3064 was cut out with BamHI and SnaBI, and inserted into the expression vector pKP3045 opened with BamHI and BbrPI leading to pKP3077.
pKP3063. Using primers relE1B-his and relE2, the 5′-end of relE was fused to an optimized SD and six histidine codons. The resulting 0.3 kbp BamHI–XhoI fragment was inserted into Litmus28, yielding pKP3063.
pKP3083. The optimized-SD-His6-relE DNA fragment of pKP3063 was cut out with KpnI and SpeI, inserted into pBAD33 opened with KpnI and XbaI, thus yielding pKP3083.
pKP3032. Using BamHI and EcoRI, the optimized SD-relB DNA fragment of pKP3021 was moved to Litmus28, resulting in pKP3032.
pKP3086. The optimized SD-relB fragment of pKP3032 was cut out with BamHI and SnaBI, and inserted in pKP3045 opened with BamHI and BbrPI giving pKP3086.
pKP3102. Using primers relE1B-his and pBADdown, the 5′-end of relE[R81A] of pKP3056 was fused to an optimized SD and six histidine codons. The resulting 0.3 kbp DNA fragment was cut with HindIII and inserted into pBAD33 opened with HincII and HindIII, thus yielding pKP3102.
pKP3090. Using primers relE1A and relE6cs, the six C-terminal codons of the relE reading frame of pKP3029 were changed to code for VTVTVT. Using BamHI and XhoI, the resulting DNA fragment was inserted in Litmus28, yielding pKP3090.
pKP3097. Using primers relE1B-his and lit28cw, the 5′-end of relE[6 cs] of pKP3090 was fused to an optimized SD and six histidine codons. Using BamHI and XhoI, the resulting 0.3 kbp DNA fragment was inserted into Litmus28 leading to pKP3097.
pKP3103. The optimized SD-His6-relE[6 cs] fragment of pKP3097 was cut out with KpnI and SpeI, and inserted into pBAD33 opened with KpnI and XbaI, yielding pKP3103.
pSC228. chpAI was PCR amplified from CF1648 with primers chpAI optimized SD–BamHI and mazE2–SalI. The PCR product was digested with BamHI and SalI and ligated to BamHI-, XhoI-treated pNDM220. pSC228 produces ChpAI upon addition of IPTG.
pSC3326. chpAK was PCR-amplified from CF1648 with primers chpAK1 optimized SD and chpAK2. The PCR product were digested with PstI and HindIII and ligated to pBAD33 digested with PstI and HindIII. pSC3326 produces ChpAK upon addition of arabinose.
pSC33. pBAD33 was stabilized by insertion of the hok/sok system from plasmid R1. pKG1022 was digested with HincII and the fragment containing hok/sok and the gene coding for kanamycin resistance was inserted into pBAD33 digested with Eco47III.
pSC3035. pKP3035 was stabilized by insertion of the hok/sok system from plasmid R1. pKG1022 was digested with HincII and the fragment containing hok/sok and the gene coding for kanamycin resistance was inserted into pKP3035 digested with Eco47III.
pSC3056. pKP3056 was stabilized by insertion of the hok/sok system from plasmid R1. pKG1022 was digested with HincII and the fragment containing hok/sok and the gene coding for kanamycin resistence was inserted into pKP3056 digested with Eco47III.
Rates of protein and DNA syntheses
Cells were grown at 37°C in AB minimal medium plus 0.5% glycerol and an amino acid mixture to an OD450 of 0.5. The cultures were then diluted 10 times. At OD450, approximately, IPTG was added to a final concentration 2 mM to induce relE or chpAK transcription. Samples of 0.5 ml were taken at the time-points indicated and added to 5 μCi of [35S]-methionine (protein synthesis) or 2 μCi methyl-3H-thymidine (DNA synthesis). In addition, a sample was taken for OD450 measurement. After 1 min of incorporation, samples were chased for 10 min with 0.5 mg of unlabelled methionine or thymidine. The samples were harvested and resuspended in 200 μl of cold 20% TCA and centrifuged at 20 000 g for 30 min at 4°C. The samples were washed twice with 200 μl of cold 96% ethanol and precipitates transferred to vials. Radioactivity was counted in a liquid scintillation counter. The amount of incorporated radioactivity per OD450 (specific rate of synthesis) was plotted against time.
Translation assay with purified RelE proteins
Purified RelE was tested for inhibition of translation in an E. coli S30 extract (Promega). Each reaction mix contained the following solutions: 6 μl of S30 premix, 4.5 μl of S30 extract and 1.5 μl of amino acid mix without methionine (1 mM). The assay was started by adding 1 μl of purified RelE (diluted in polymix buffer, 20% glycerol) followed by incubation for 5 min at 37°C. After this preincubation, 1 μl of 3 μM (3 pmol) test mRNA (hok mRNA from plasmid R1), 1 μl of [35S]-methionine and 1 μl of polymix buffer or 1 μl of purified RelB in polymix buffer were added. After an additional 25 min at 37°C, the reactions were precipitated with acetone and analysed using SDS–PAGE.
Purification of proteins
RelE was purified from strain BL21/pKP3077 (pA1/O4/O3:: his6::relB)/pKP3067 (pBAD::relE). A 10 ml overnight culture was diluted into 2 l of 2× YT medium containing 50 μg ml−1 of chloramphenicol (Cml), 100 μg ml−1 of ampicillin (Amp) and 1 mM IPTG, and was cultured at 30°C. At OD600≈ 0.4, arabinose (0.2%) was added. After 4 h, the culture was harvest by centrifugation. From the cell pellet, a His-RelB– RelE complex was purified on Talon according to the manufacturer's (Clontech’s) prescription for native purification. From the Talon-bound protein complex, RelE was washed out using the standard binding buffer for denaturing purification containing 8 M urea. This fraction of RelE was concentrated with Centriplus centrifugal filter YM-3 (Millipore). His-RelE, His-RelE (R81A) and His-RelEcs6 were purified from BL21/pKP3086 (pA1/O4/O3::relB) also carrying pKP3083 (pBAD::his6::relE), pKP3102 (pBAD::his6::relER[R81]A) or pKP3103 (pBAD::his6::relEcs6) respectively. The procedure was the same as for purification of RelE except that RelB protein was washed out with the denaturing binding buffer, and the His-tagged RelE proteins were collected with standard denaturing imidazole elution buffer. Imidazole was removed from the His-tagged RelE proteins by dialysis. All four proteins were further purified with Hi Trap SP HP 1 ml ion exchange columns (Amersham Pharmacia), with stepwise increase of NaCl in a buffer of pH 7 according to manufacturer but with the modification that all buffers contained 8 M urea. Finally, the purified proteins were refolded by dialysing against the following buffers for 12 h each: (i) 1× PBS, pH 7.4, 5 mM DTT, 0.1% Triton X-100; (ii) 1× PBS, pH 7.4, 5 mM DTT; (iii) 1× polymix buffer (5 mM magnesium acetate, 5 mM ammonium chloride, 95 mM potassium chloride, 0.5 mM calcium chloride, 5 mM potassium phosphate, pH 7.4, 1 mM DTT); and (iv) 1× polymix buffer, 20% glycerol. The final concentration of the purified proteins was typically between 0.2 and 0.02 μg μl−1 estimated from SDS–PAGE gels. The authenticity of the proteins was verified by matrix-assisted laser desorption-ionization (MALDI)-MS of proteins extracted from SDS gels.