Additional Materials General Methods and Materials. Buffers and salts [PDF]

Additional Materials General Methods and Materials. Buffers and salts of highest quality grade were purchased from Sigma unless otherwise noted. Nuclease P1, venom phosphodiesterase 1, Bacterial Alkaline Phosphatase, Lysozyme, DNase, Glutamate dehydrogenase and Nickel-NTA were purchased from Sigma. PfuUltra™ DNA polymerase was purchased from Stratagene. DTT and β-NADP+ were from Research Products International. T4 DNA ligase and restriction enzymes were from Fermentas or New England Biolabs, and Plasmid Mini-Kits were from Fermentas or Qiagen. Dialysis was performed in Slide-ALyzer™ cassettes from Pierce. Oligonucleotides were from Integrated DNA Technologies, Inc. Protein concentrations were based on the Bradford dye-binding procedure, using reagent from Bio-Rad (1). SDS-PAGE analysis utilized reagents from BioRad and was carried out using 12% gels or precast gels (10% Tris-HCl) and visualized with Coomassie Brilliant Blue. Invitrolon PDVF membranes were from Invitrogen. DNA sequencing was carried out by UF core facility or the OHSU core facility in the Department of Molecular Microbiology and Immunology. Edman degradation was carried out by the Molecular Structure Facility at the University of California, Davis. [U-14C]-L-glutamine (210 mCi/mmol), [3H]-L-asparagine (200 mCi/mmol) and [8-14C]-guanine (57 mCi/mmol) were from Moravek Biochemicals, Inc. The LIC cloning kit containing the pET32Xa plasmid was from Novagen. The mj1022 gene was custom synthesized by GenScript, M. jannaschii genomic DNA was kindly provided by Dr. Anna-Louise Reysenbach (Portland State University), Haloferax volcanii DS70, H. volcanii H26 and Halobacterium salinarium NRC-1 strains were kindly provided by Dr. Julie Maupin-Furlow.

Strains, media and growth conditions. E. coli Topo 10 (F- mcrA Δ(mrr-hsdRMSmcrBC) Φ80lacZΔM15 ΔlacΧ74 recA1 araD139 Δ(araleu)7697 galU galK rpsL (StrR) endA1 nupG) (Invitrogen), DH5α (F- φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk-, mk+) phoA supE4 thi-1 gyrA96 relA1 tonA) (Invitrogen), INV110 (F´{traΔ36 proAB lacIq lacZΔM15} rpsL (StrR) thr leu endA thi-1 lacY galK galT ara tonA tsx dam dcm supE44 Δ(lac-proAB) Δ(mcrC-mrr)102::Tn10 (TetR)) (Invitrogen), Mach1-T1R (F- ΔlacX74 hsdR(rk- mk+) Δrec1398 endA1 tonA) (Invitrogen), ET12567 (dam, dcm-13::Tn9 (CmR), hsdM, hsdS, hsdR, zjj-202::Tn10 (TetR) (2), NovaBlue (endA1 hsdR17

(rK12–

mK12+)

supE44

thi-1

recA1

gyrA96

relA1

lac

F′[proA+B+

lacIqZΔM15::Tn10(TetR)] (Novagen), and BL21 (DE3) F– ompT gal dcm lon hsdSB(rBmB-) λ(DE3 [lacI lacUV5-T7 gene1 ind1 sam7 nin5]) (Novagen) were used for cloning or protein over-production, and were routinely grown in LB medium (BD Diagnostic System) at 37 ºC. Growth media were solidified with 15g/liter agar (BD Diagnostic System) for the preparation of plates. Transformation of E. coli was performed following standard procedures (3, 4). Ampicillin (Amp, 100 μg/ml) and Kanamycin (Kan, 50 μg/ml), were used as needed. Haloferax volcanii

H26 (DS70 ΔpyrE2, (5)) and

derivatives were grown at, 45 ºC in Hv-YPC rich medium (6) (125g NaCl, 50g MgCl 6H2O, 2.5 g KSO4, 0.134 g CaCl 2H2O,5 g tryptone, and 5 g yeast extract) or a chemically defined medium Hv-Ca (6) (125g NaCl, 50g MgCl 6H2O, 2.5 g KSO4, 0.134 g CaCl 2H2O, 0.5% casamino acids) supplemented when needed 50 or 10μg/ml uracil, and 50 μg/ml 5-Fluoroorotic acid (FOA) or Novobiocin 0.2 μg/ml. H. volcanii derivatives were transformed as described by (7).

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Instrumentation. PCR was carried out with a Perkin-Elmer 2400 thermocycler or an Eppendorf Mastercycler ep gradient S. Radioactivity was quantitated using a Beckman LS 6500 Liquid Scintillation Counter. UV-Vis spectroscopy was conducted with a Cary 100-Bio UV-Vis spectrophotometer. HPLC analysis was carried out with an Hitachi L7100 multisolvent system equipped with an L-4500 photo diode array detector and controlled via the Hitachi ConcertChrome HPLC software.

A Molecular Dynamics

Typhoon 9200 variable mode imager with ImageQuant 5.2 software was used for phosphorimaging.

Construction and checking of the H. volcanii ΔtgtA2 derivatives All PCR reactions were done as described in (8) using PfuUltra™ and all constructs were verified by sequencing. The 0.8 kb region upstream of the H. volcanii tgtA2 gene was amplified

with

the

following

primers:

tgtA2HvUPF

(5’-

TCTAGAGGTGATTACCACGCGCAAG-3’) containing a XbaI site and tgtA2HvUPR (5’-GGATCCGCGTGAACCTCGAAGTAGT-3’) containing a BamHI site. The PCR product was ligated into pCRTOPO4 Blunt (Invitrogen) yielding plasmid pNAB110. The 0.8 kb fragment downstream of the gene was amplified in a similar fashion using the following primers: tgtA2HvDSol1 (5’-AATGGATCCTCGAAGGGTAATTCGG-3’) containing

a

BamHI

restriction

site

and

AATGCGGCCGCAAGCTTCGGCAGTCGCGTGTTGATG-3’)

tgtA2HvDSol2 containing

(5’a

NotI

restriction site. The amplified fragment was then digested with NotI and BamHI and ligated into the corresponding sites of pNAB110. The resulting plasmid (pNAB130) was

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digested with XbaI and NotI restriction enzyme to liberate the 1.6 kb fragment containing the concatenated upstream and downstream fragments that was ligated into pTA131 (5). The resulting plasmid (pNAB135) was passaged through ET12567 before transformation of H. volcanii. Generation and checking of the H. volcanii H26 ΔtgtA2 strain (VDC5203) was done as described by El Yacoubi et al (8) both by PCR and Southern analysis. The primers used for checking for the presence/absence of the target gene (or inside primers) were RTtgtA2 ol1 (5’-TTCGACCAGCAGTACAGTTT-3’) and RTtgtA2 ol2 (5’AACGCCACGTCCGAAAAGA-3’). The primers used to check for insertion at the correct

location

(or

outside

TCGACTCCGTCACGCTCCAC-3’)

primers) and

were

ChktgtA2

ChktgtA2

ol1 ol2

(5’(5’-

CGGCGGAAGTCGTCCCGGATA-3). The primers to amplify the probe for the Southern hybridization were SBHVtgtA2.ol1 (5’-TGTGGTCTCGACATGGCACTC-3’) and SBHvtgtA2.ol2 (5’-CGGCGGACGACCTCTGGATG-3’).

Cloning of the Halobacterium salinarium tgtA2 gene for complementation. The H. salinarium NRC-1 tgtA2 (VNG1957G) gene was PCR amplified from genomic DNA. The following primers, corresponding to the 5’- and 3’- regions of the genes, were used: HstgtA2.ol1 (5’ -GCGGCATATGACTGAGTACTTCGAGATCCA- 3’), HstgtA2.ol2 (5’ – GCGGGCTCAGCTTATCGTTCGACGCAGTGCC- 3’). PCR products were produced and purified as described in (9), and inserted into pCR2-1 using the TATM technology (Invitrogen) and transformed into Topo10. The primary structure of the resulting construct pGPP089 was confirmed by sequencing. pGPP089 was then digested with NdeI/BlpI, the 1751 bp fragment corresponding to the tgtA2Hs gene was gel purified as

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described in (9), ligated into pJAM202 (10), digested with the appropriate endonucleases and transformed into Topo10 cells. The resulting construct, pGPP099 was passaged in INV110 cells to increase transformation efficiency (11) before being transformed into the H. volcanii strain VDC5203 (ΔpyrE2 ΔtgtA2) to give strain VDC3224 (ΔpyrE2 ΔtgtA2 pGPP099) ). pJAM202c (kind gift of Dr. Maupin-Furlow), a derivative that contains no insert was transformed in H26 and VDC5302 to give strains VDC3225 (ΔpyrE2 ΔtgtA2 pJAM202c) and VDC3226 (ΔpyrE2 pJAM202c) respectively.

Cloning of mj1022. The mj1022 gene corresponding to tgtA2 in Methanocaldococcus jannaschii was initially cloned from genomic DNA and inserted into a pET32 vector to enable expression of the recombinant protein as an N-terminal His6-Trx-fusion protein. However, heterologous over-expression of the native gene in E. coli BL21(DE3) was associated with generally low levels of recombinant protein and the apparent production of prematurely terminated proteins, presumably due to the prevalence of rare codons in mj1022. The expression profile was only marginally improved by expression in Rosetta™ cells, which are engineered to over-express a number of specific tRNA to compensate for the presence of rarely used codons in E. coli. These results were in marked contrast to our experience with mj0436, which encodes the arcTGT enzyme in M. jannaschii (12). To overcome these problems a synthetic gene was prepared codonoptimized for expression in E. coli, and this was cloned into several expression vectors harboring N- and C-terminal His6 fusion tags. The M. jannaschii mj1022 gene was amplified from both genomic DNA and a custom synthesized gene (GenScript) via PCR and cloned with both N- and C-terminal His6-fusions using the following primers:

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Gen1022F 5’-GGTATTGAGGGTCGCATGCTCGAACCAATTG-3’ Gen1022R 5’-AGAGGAGAGTTAGAGCCTCAGCTTTTAACATTTC-3’ Syn1022F 5’-GGTATTGAGGGTCGCCATATGCTGGAACCG-3’ Syn1022R 5’-AGAGGAGAGTTAGAGCCGGATCCTTACG-3’ CterF 5’-GAAAACCATATGCTGGAACCGATTGCGTACG-3’ CterR 5’-GTGGTGCTCGAGCGATTTAACATTGCGAATATTC-3’. Primers for amplification of mj1022 from genomic DNA (Gen1022) and the synthetic gene (Syn1022) were designed for subsequent LIC cloning in pET32Xa with an Nterminal His6-Trx-fusion. The primers for amplification of the synthetic gene with a Cterminal His6-fusion (Cter) contained the restriction sites (underlined) for NdeI and XhoI to allow directional cloning into a pET30 vector. The genes were amplified from genomic DNA (20 ng) or synthetic template (10 ng) with PfuUltra™ DNA polymerase using the following program: 95 oC (5 minutes), 30 cycles of 95 oC (30s), 60 ˚C (genomic DNA), 55 ˚C (synthetic gene, N-terminal fusion), or 57 oC (synthetic gene, C-terminal fusion) (30s), and 72 oC (2 minutes), followed by a final extension at 72 oC for 10 minutes and hold at 4 oC. Amplification of the genes for the construction of N-terminal fusions were followed by insertion of the PCR products into pET32Xa following the manufacturers instructions to give pVCII-120 (native gene) and pVCIII-28 (synthetic gene). Amplification of the gene for construction of the C-terminal fusion was followed by restriction digestion of the PCR product with NdeI/XhoI, gel purification (1% agarose), and ligation into NdeI/XhoI digested pET30 using T4 DNA ligase to give pVCIV-42. Plasmid DNA was isolated from all three constructs and the primary structure of the genes confirmed by sequencing.

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Overproduction and purification of recombinant MjTgtA2. The plasmids containing mj1022 were transformed into E. coli BL21(DE3) for expression of the variously tagged recombinant proteins. Cultures of transformed cells were grown at 37 ˚C in LB/kan with shaking (250 rpm) until an OD600 of 0.9 was obtained, IPTG was added to a final concentration of 0.25 mM, and the cells were grown for an additional 4½ hours. The cells were collected by centrifugation at 7,500 g for 15 minutes at 4 oC and frozen with liquid nitrogen. The cells were stored at -80 oC until needed.

Frozen cell paste was suspended to 250 mg/mL in 50 mM Hepes (pH 7.0), 0.75 M NaCl, 2 mM βME, and 1 mM PMSF. Lysozyme was added to a final concentration of 250 µg/mL and the cells incubated at 37 ˚C for 30 minutes, followed by 3 intervals of freeze thaw cycles. DNase was added to a final concentration of 10 µg/mL and the cells were left at 37 oC for an additional 30 minutes. The cell lysate was centrifuged at 20,000g for 30 minutes at 4 ˚C, and the cell free extract recovered and filtered (0.45 µm MCE). The CFE was heated at 60 ˚C for 15 minutes followed by centrifugation for 20 minutes at 20,000g; this cycle was repeated at 75 ˚C. The heat-treated CFE was loaded onto a column containing Ni- NTA equilibrated in lysis buffer, and washed with 10 volumes of lysis buffer without PMSF and containing 25 mM imidazole. The recombinant protein was eluted with 250 mM imidazole in lysis buffer without PMSF. The eluant was concentrated to 2 mL and dialyzed overnight against 4 L of lysis buffer without imidazole or PMSF. To remove the affinity tag the Trx-His6 construct (20 mg) was incubated with Factor Xa (20 μg) at 4 ˚C in 100 mM Tris (pH 8.0), 0.5 M NaCl, 1 mM CaCl2, and 1 μM

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DTT for ~20 hrs. The cleaved recombinant protein was then recovered after passage over a Ni-NTA column.

Expression from pVCIII-28 and pVCIV-42, which encode N- and C-terminal His6 fusions respectively, in E. coli BL21(DE3) was robust, and provided ~25 mg of recombinant protein per liter of culture (Fig. S4A in SI Appendix). However, we discovered that purified protein obtained after Ni-NTA affinity chromatography was typically contaminated with one or more other bands (Fig. S4A in SI Appendix). In the case of the His6-Trx-fusion protein several large bands ~15 KDa less than the fusion were observed as well as a small band that was ~15 KDa, while in the samples of C-terminal His6 fusion protein a single additional band ~7 KDa smaller than the fusion was observed. When these samples were kept at temperatures above freezing (4 ˚C to 25 ˚C) over a period of days to weeks all of the fusion protein converted to the smaller bands. The presence of a variety of protease inhibitors did not prevent or influence the cleavage reaction, suggesting that the protein was undergoing self-cleavage. We also observed that the protein, either as a fusion, the Factor Xa cleaved product, or the self-cleaved product, was prone to precipitation, and required at least 0.5 M NaCl to remain in solution.

Determination of self-cleavage sites in C- and N-terminal fusions of MjTgtA2. To allow the N- and C-terminal recombinant fusion proteins to self-cleave the Trx-His6MjTgtA2 and MjTgtA2-His6 fusion constructs (~100 µg each) were incubated in 100 mM Hepes (pH 7.0), 0.5 M NaCl, and 2 mM BME at 4 ˚C for up to 4 weeks. The proteins were then electrophoresed on a precast 10% acrylamide/Tris.HCl gel. Electroblotting

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onto an Invitrolon membrane was carried out for 1 hour at 22 V according to the Manufacturer’s instructions using NuPAGE transfer buffer. The membrane was then stained with coomassie blue followed by washing with 50% methanol, air-drying, and specific bands (Fig. S4A in SI Appendix) analyzed by Edman sequencing. N-terminal sequencing revealed that while cleavage occurred in 2 specific regions, one within the fusion domain and a second ~75 residues into the N-terminal domain, N-terminal heterogeneity was observed due to multiple cleavage sites within each region (Fig. S4B in SI Appendix).

Preparation of PreQ0-tRNASer. Unmodified RNA transcripts corresponding to H. volcanii tRNASer were generated as previously described (12). The expression and purification of M. jannaschii TGT was carried out as previously described (12). PreQ0 was loaded into the tRNASer transcript in a TGT-catalyzed reaction comprised of 50 mM succinate (pH 5.5), 20 mM MgCl2, 100 mM KCl, 2 mM DTT, 100 µM tRNASer, M. jannaschii TGT (10 µM), and 1 mM preQ0 in a total volume of 1 mL. After 45 minutes at 80 ˚C, the reaction was terminated by the addition of one-tenth volume of 2 M NaOAc (pH 4.0) followed by one volume of water saturated phenol and one fifth volume chloroform:isoamyl alcohol (49:1). After vortexing for 20s, the solution was centrifuged in a swinging bucket rotor at 700g for 20 minutes. The aqueous phase was recovered and applied directly to a Quick-Sep column containing 2 mL of Sephadex G-25. The sample was centrifuged at 700g in a swinging bucket rotor for 1.5 min to separate the unreacted preQ0 from the preQ0-tRNASer. The preQ0-tRNASer was precipitated from the eluant after the addition of 3 volumes of ethanol and cooling at -20 ˚C for 2 hours. The solution was

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centrifuged at 14,000g for 20 minutes at 4 ˚C, the supernatent removed, and the RNA pellet washed with 70% cold ethanol. After centrifugation again at 14,000g the supernatant was removed and the preQ0-tRNASer was resuspended in 3 mM citrate (pH 6.3) and stored at -20 ˚C. In order to quantify preQ0 incorporation into tRNASer a control reaction was run in which [8-14C]-guanine was first loaded into the tRNA using M. jannaschii arcTGT under standard conditions and the specific radioactivity of the labeled tRNA measured after isolation from the reaction. The [8-14C]-guanine in the tRNASer was then replaced with preQ0 in a second reaction with M. jannaschii TGT under the conditions above, and subsequent measurements of the isolated tRNASer confirmed that no radioactivity remained, confirming the quantitative incorporation of preQ0.

pH and temperature dependence of MjTgtA2 activity. The pH dependence of MjTgtA2 was investigated using the tri-buffer system composed of 100 mM Tris/Mes/Acetate (pH 4.0 – 9.0), 0.5 M NaCl, 20 mM MgCl2, 1 mM DTT, 10 µM [U14

C]-glutamine, 20 µM preQ0-tRNASer, and MjTgtA2 (7.8 µg). The activity of MjTgtA2

was also determined in several discreet buffers, including Hepes, phosphate, citrate, and acetate. Assays were carried out at 40 ˚C, initiated with the addition of MjTgtA2, and terminated after 10 minutes with the addition of 1/5 volume concentrated NH4OH. Quantification of [U-14C]-glutamate formation was carried out as described above. The temperature dependence of the reaction was determined in the presence of 100 mM Hepes (pH 7.0), 0.5 M NaCl, 20 mM MgCl2, 1 mM DTT, 10 µM [U-14C]-Gln, 20 µM preQ0-tRNASer, and MjTgtA2 (7.8 µg). Assays were carried out as described above, with temperatures from 30 ˚C to 80 ˚C.

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Quaternary structure of TgtA2. X-ray analysis of P. horikoshii arcTGT demonstrated that this enzyme exists as a dimer in the biologically active form. Given the homology observed between arcTGT and TgtA2 we hypothesized that the latter would also exhibit a dimer structure, and subjected MjTgtA2 to analytical gel filtration to test this hypothesis. The molecular masses of recombinant fusion and cleaved MjTgtA2 were determined by gel filtration on a BioSepSEC-S4000 column (300 mm x 7.8 mm, Phenomenex Inc.). The MjTgtA2 proteins and molecular weight standards were eluted with a mobile phase of 50 mM Phosphate pH 7.2, 0.5 M NaCl at a flow rate of 1 ml/min. The void volume (V0) and total volume (VT) were measured with blue dextran (2000 KDa) and potassium ferricyanide (330 Da). The standards used in the calibration included carbonic anhydrase (29 KDa), BSA (66 KDa), alcohol dehydrogenase (150 KDa), β-amylase (200 KDa), apoferritin (443 KDa) and thyroglobulin (669 KDa). KD values were calculated according to the equation KD=(VeV0)/(VT-V0) where Ve is the elution volume the relevant protein, V0 is the void volume and VT the total volume. The KD values of the protein standards were then plotted vs log MW to generate a standard curve.

Both the Trx-His6 fusion and the protein produced from self-cleavage exhibited elution times (Fig. S4C in SI Appendix) consistent with the molecular weight of a dimer (154 and 122 kDa for the fusion and cleaved proteins, respectively), demonstrating that like arcTGT, TgtA2 also functions as a dimer. However, while the fusion protein exhibited a sharp peak in the elution profile, the protein produced from self-cleavage exhibited a

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much broader peak, consistent with looser subunit interactions in the cleaved protein and suggesting that loss of a portion of the N-terminal domain was accompanied by reduced dimer stability.

References 1.

Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-54.

2.

MacNeil DJ et al. (1992) Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111, 61.

3.

Miller JH (1972) Experiments in Molecular Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

4.

Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor).

5.

Allers T, Ngo H, Mevarech M & Lloyd RG (2004) Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes. Appl Environ Microbiol 70, 943.

6.

Kauri T, Wallace R & Kushner DJ (1990) Nutrition of the halophilic archaebacterium, Haloferax volcanii. Syst Appl Microbiol 13, 14–18.

7.

Cline SW, Schalkwyk LC & Doolittle WF (1989) Transformation of the archaebacterium Halobacterium volcanii with genomic DNA. J Bacteriol 171, 4987-4991.

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8.

El Yacoubi B et al. (2009) A Gateway platform for functional genomics in Haloferax volcanii: deletion of three modification genes. Archaea 2, 211-219.

9.

El Yacoubi B et al. (2006) Discovery of a new prokaryotic type I GTP cyclohydrolase family. J Biol Chem 281, 37586-93.

10.

Kaczowka SJ & Maupin-Furlow JA (2003) Subunit topology of two 20S proteasomes from Haloferax volcanii. J Bacteriol 185, 165-174.

11.

Holmes ML, Nuttall SD & Dyall-Smith ML (1991) Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase. J Bacteriol 173, 3807-3813.

12.

Bai Y et al. (2000) Hypermodification of tRNA in Thermophilic archaea. Cloning, overexpression, and characterization of tRNA-guanine transglycosylase from Methanococcus jannaschii. J Biol Chem 275, 28731-8.

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