All experiments were carried out in either polymix buffer A (50 mM Tris-OAc (pH 7.5), 100 mM KCl, 5 mM NH4OAc, 0.5 mM Ca(OAc)2, 5 mM Mg(OAc)2, 6 mM 2-mercaptoethanol, 0.1 mM EDTA, 5 mM putrescine and 1 mM spermidine)45 (link) or polymix buffer B (30 mM HEPES pH 7.5, 5 mM MgCl2, 50 mM NH4Cl, 5 mM 2-mercaptoethanol, 2 mM spermidine and 5 mM putrescine)46 (link). A cocktail of triplet-state quenchers (1 mM Trolox, 1 mM nitrobenzyl alcohol and 1 mM cyclooctatetraene) and an enzymatic oxygen scavenging system (protocatechuic acid (PCA)/protocatechuate-3,4-dioxygenase (PCD)) were used for smFRET experiments. Spectinomycin sulfate was purchased from MP Biomedicals. Fusidic acid sodium salt, GTP and GTPγS were from Sigma-Aldrich. GTP was further purified using a Mono Q 5/50 GL anion exchange column (GE Healthcare Life Sciences). Pyruvate kinase, myokinase and phosphoenolpyruvate (PEP) were purchased from Sigma-Aldrich. All other standard reagents were purchased from Sigma-Aldrich or VWR.
Myokinase
Manufactured by Merck Group
Myokinase is a laboratory enzyme used for the quantitative determination of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) in biological samples. It catalyzes the interconversion of these two nucleotides, which is an important step in cellular energy metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Pyruvate kinase, by Merck Group (6 mentions)
Creatine kinase, by Roche (2 mentions)
Glutamate dehydrogenase, by Merck Group (2 mentions)
D glucose, by Merck Group (2 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (2 mentions)
Pyruvate kinase from rabbit muscle, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Atp assay mix, by Merck Group (2 mentions)
Lactate dehydrogenase, by Merck Group (2 mentions)
Coash, by Sangon (2 mentions)
Pa coa, by Merck Group (2 mentions)
Gtpγs, by Merck Group (1 mentions)
Monoq 5 50 gl anion exchange column, by GE Healthcare (1 mentions)
Phosphoenolpyruvate pep, by Merck Group (1 mentions)
Fusidic acid sodium salt, by Merck Group (1 mentions)
Spectinomycin sulfate, by MP Biomedicals (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
11 protocols using myokinase
1
Buffers for Single-Molecule Experiments
2
Cell-free Protein Synthesis Assay
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The 2.5× reaction buffer was composed of amino acid mixture (0.75 mM for each amino acid), 8.125 mg/mL tRNA (Roche), 5 mM ATP, 5 mM GTP, 2.5 mM CTP, 2.5 mM UTP, 50 mM creatine phosphate, 50 μg/mL folinic acid, 125 mM HEPES–KOH 7.6, 250 mM potassium glutamate, 29.5 mM magnesium acetate, 5 mM spermidine, and 2.5 mM DTT. 10× enzyme mixture was composed of 200 μg/mL creatine kinase (Roche), 300 μg/mL myokinase (Sigma-Aldrich), 50 μg/mL nucleoside 5′-diphosphate kinase from bovine liver (Sigma-Aldrich), 20 U/μL T7 RNAP (New England BioLabs), 0.002 U/μL pyrophosphatase (Thermo Fisher Scientific) and 8 U/μL recombinant RNAse inhibitor (Takara). Reactions (final volume = 10 μL) were initiated by mixing 4 μL 2.5×reaction buffer, 1 μL 10× Enzyme mixture, 15 μg MSBC-PURE, 6 μg EF-Tu, 1.0 μM ribosomes (New England BioLabs), and 50 ng of linear DNA template. After mixing, reactions were incubated at 37 °C for 4 h. The mRFP was quantified by a platereader (Biotek Synergy H1) with the excitation at 580 nm and the emission at 610 nm using a 384-well plate (Corning Spheroid Microplate).
3
Enzymatic Synthesis of Mevalonate Pathway
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Lactate dehydrogenase (rabbit muscle), pyruvate kinase (rabbit muscle), and inorganic pyrophosphatase (Baker's yeast) were purchased from Roche Applied Science. (R, S)-[2H3]methyl-mevalonolactone, (R, S)-mevalonolactone, acetyl-CoA, glutamate dehydrogenase (bovine liver), acetyl-CoA synthetase (Baker's yeast), myokinase (rabbit muscle) and lysozyme (bovine) were purchased from Sigma. Sodium acetate (13C, 99%), sodium acetate (2H, 99%) and D2O (99%) were purchased from Cambridge Isotope Laboratories, Inc. All other chemical reagents were of the highest grades available. Plasmids pET28efTR (encodes a bi-functional enzyme, Enterococcus faecalis acetoacetyl-CoA thiolase/HMG-CoA reductase), pET28efS2 A100G (encodes Enterococcus faecalis HMG-CoA synthase), and pET28-efR (encodes Enterococcus faecalis HMG-CoA reductase) were generous provided by Prof. V. W. Rodwell [43] (link). Mevalonate kinase (Staphylococcus aureus), phosphomevalonate kinase (Streptococcus. pneumoniae), and diphosphomevalonate decarboxylase (Streptococcus. pneumoniae) were expressed and purified as described previously [46] (link), [49] (link).
4
Fluorescence-Based Kinase Assay
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RES was purchased from Tocris Bioscience (Minneapolis, MN, USA). CUR, 3-isobutyl-1-methylxanthine (IBMX), DMSO, d -glucose, ATP assay mix, calmodulin from bovine heart, myokinase (adenylate kinase) from rabbit muscle, pyruvate kinase from rabbit muscle, cAMP, ATP, AMP, CTP, PEP, dithiothreitol (DTT), Protease Inhibitor Cocktail, and all other chemicals used in this study were obtained from Sigma.
5
Purification of Metabolic Enzymes
6
Detailed Experimental Protocol for Ribosomal Proofreading
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All experiments were done in polymix buffer (Ehrenberg et al. 1990 ; Shoji et al. 2006 (link)), unless otherwise noted. Purified E. coli MRE600 tRNAs (tRNAfMet, tRNAMet, tRNAPhe, tRNATyr, tRNALys, and tRNAGlu) were purchased from Chemical Block and aminoacylated as described (Walker and Fredrick 2008 (link)). For initial selection and proofreading experiments, mRNA was transcribed in vitro from pGENE32-based plasmids and gel-purified as described (Fredrick and Noller 2002 (link)). The mRNAs used for proofreading experiments have the sequence 5′-(N)42AAGGAAAUAAAAAUGNNNGUAUACAAAUCU(N)67-3′, where NNN corresponds to UUU, CUU, UAC, or UAG. Messages for the miscoding experiments of Figure 3 were synthesized by Sigma-Aldrich and have the sequence 5′-AAGGAAAUAAAAAUGNNNGUAUACAAAUCU-3′, where NNN is the indicated A site codon. Ribosomes, translation factors, and phenylalanyl-tRNA synthetase were purified as described (McClory et al. 2010 (link)). Tyrosyl-, lysyl-, and glutamyl-tRNA synthetases were purified with Talon Cobalt Affinity Resin (Clontech) from overexpression strains JW1629, JW2858, and JW2395, obtained from the ASKA collection (National BioResource Project-E. coli at the National Institute of Genetics, Japan). Pyruvate kinase and myokinase were purchased from Sigma-Aldrich.
7
Bacterial cultivation and chemical reagents
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The bacteria strains and plasmids used in this study are listed in Table S4. Escherichia coli strains carrying and expressing the cloned genes were cultivated at 37°C in lysogeny broth (LB) medium supplemented with 50 µg/mL kanamycin. Analytically pure chemical PAA was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), the PAA derivatives from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) including 2-hydroxyphenylacetate (2HPA), 3-hydroxyphenylacetate (3HPA), 4-hydroxyphenylacetate (4HPA), 3,4-dihydroxyphenylacetate (3,4DHPA), as well as BA and its derivatives 2-hydroxybenzoate (2HBA), 3-hydroxybenzoate (3HBA), 4-hydroxybenzoate (4HBA), 3,4-dihydroxybenzoate (3,4DHBA), and phosphoenol pyruvate. The CoASH and ATP were obtained from Sangon Biotech Co., Ltd. (Shanghai, China), and the PA-CoA from Sigma Chemical (St. Louis, MO, USA). The biochemical reagents myokinase, pyruvate kinase, and lactate dehydrogenase were all purchased from Sigma Chemical.
8
Bacterial cultivation and chemical reagents
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The bacteria strains and plasmids used in this study are listed in Table S4. Escherichia coli strains carrying and expressing the cloned genes were cultivated at 37°C in lysogeny broth (LB) medium supplemented with 50 µg/mL kanamycin. Analytically pure chemical PAA was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), the PAA derivatives from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) including 2-hydroxyphenylacetate (2HPA), 3-hydroxyphenylacetate (3HPA), 4-hydroxyphenylacetate (4HPA), 3,4-dihydroxyphenylacetate (3,4DHPA), as well as BA and its derivatives 2-hydroxybenzoate (2HBA), 3-hydroxybenzoate (3HBA), 4-hydroxybenzoate (4HBA), 3,4-dihydroxybenzoate (3,4DHBA), and phosphoenol pyruvate. The CoASH and ATP were obtained from Sangon Biotech Co., Ltd. (Shanghai, China), and the PA-CoA from Sigma Chemical (St. Louis, MO, USA). The biochemical reagents myokinase, pyruvate kinase, and lactate dehydrogenase were all purchased from Sigma Chemical.
9
Resveratrol and Curcumin Bioactivity Analysis
10
Mycobacterial Peptide Formation Assay
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For Met-Leu dipeptide formation, an initiation complex was formed with mycobacterial 70S ribosome variants (1 μM to 5 μM), AUG-CUG-UAA XR7 mRNA (20 μM) and [3H]fMet-tRNAfMet (1 μM), initiation factors (2 μM) at 37 °C. In parallel, an elongation mix was prepared which contained M. smegmatis EF-Tu (20 μM), EF-Ts (20 μM), tRNALeu (100 μM), Leu amino acid (0.5 mM), Leu-tRNA synthetase (1 unit per μL), and GTP (1 mM) at 37 °C. Both initiation complex and elongation mix containing energy pump adenosine 5′-triphosphate (1 mM), phosphoenolpyruvate (10 mM), pyruvate kinase, and myokinase (Sigma). The reaction was started by mixing equal volumes of the initiation complex and elongation complex at 37 °C and was quenched after 10 s by adding 17% formic acid. The peptides were isolated from the ribosome complex by KOH treatment and further analyzed by high-performance liquid chromatography (HPLC) equipped with a radioactivity detector.
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