- Email: [email protected]
[161] Demonstration of DNA-dependent amino acid incorporation
[161]
DNA-DEPENDENT CODING
791
Fingerprint Analysis of Protein Product The radioactive protein products can be analyzed in various ways. We describe here the method for fingerprinting the coat protein product together with carrier coat protein2 The general fingerprinting method has been discussed by Ingram. 11 To the reaction mixture containing a minimum of 5 X 104 cpm of 14C-labeled product, excess 12C-labeled amino acid is added and sufficient trichloroaeetic acid to give a concentration of 5%. The mixture is heated at 90 ° for 15 minutes; then the precipitate is washed with the solutions given above for the filter paper disks. To the dry precipitate 5 mg of MS2 coat protein is added, and the proteins are oxidized with performic acid. For this purpose, 10 ml of 99% formic acid is allowed to stand with 0.5 ml of 30% hydrogen peroxide at room temperature for 1 hour. To an estimated 10 mg of protein, 0.5 ml of oxidizing reagent is added. After 20 minutes at room temperature, excess water is added and the solution is lyophilized. The lyophilized protein is then dissolved at pH 10-11 with dilute KOH, and the solution is quickly brought to pH 8.0. Digestion with twice-crystallized trypsin in an amount approximately equal to 1% of the weight of protein is carried out in a pH stat at pH 8.0, and at the end of the reaction the sample is dried in a desiccator. Separation of tryptie peptides is accomplished on 18 X 22 inch Whatman 3 MM chromatography paper by electrophoresis at pH 4.5 (12 ml of pyridine, 13 ml of glacial acetic acid, 25 ml of n-butanol, made to 1 liter with water) for 2.5 hours at 30 V/cm. After being dried overnight, the paper is chromatographed (ascending) in n-butanol-acetic acidpyridine-water (75:15:50:60). (All solvents should be redistilled.) When dry, the paper can be applied to X-ray film for about 10 days to identify 14C-labeled peptides and then stained with 0.25% ninhydrin in acetone to identify the peptides of the carrier coat protein. These can be eluted and counted. ~IV. Ingram, Vol. VI [118].
[ 161 ] D e m o n s t r a t i o n of D N A - D e p e n d e n t Amino Acid Incorporation
By AUDREY STEVENS With the usual bacterial enzyme system usea to demonstrate a dependence on messenger RNA of amino acid incorporation into protein, endogenous messenger RNA is removed and DNase is added to prevent
792
NUCLEIC ACIDS INVOLVED IN PROTEIN SYNTHESIS
[161]
further messenger RNA biosynthesis. 1 In order to demonstrate a dependence of amino acid incorporation into protein on messenger RNA production from DNA, endogenous messenger RNA, DNA, and the enzyme RNA polymerase are largely removed in the preparation of the enzyme system, z,~ ~4C-labeled amino acid incorporation by the system is then stimulated by the addition of certain native DNA preparations together with the enzyme RNA polymerase.
Assay Method Principle. Enzyme fractions (ribosome and 8-hour supernatant fractions) largely freed of DNA and RNA polymerase are incubated with 14C-labeled amino acid, nineteen ~zC-labeled amino acids, ATP, CTP, UTP, GTP, a triphosphate-generating system, DNA, and RNA polymerase. 14C-labeled amino acid in acid-precipitable protein is measured after the incubation period. Reagents
Basic reaction mixture containing: phosphocreatine, 20 mM; creatine kinase 80 #g/ml; ATP, 2 mM; UTP, CTP, and GTP, each 0.50 mM; a mixture of 19 *2C-labeled ~mino acids, each 0.10 mM; mercaptoethanol, 10 mM; Tris buffer, pH 7.8, 100 mM; magnesium acetate, 20 rm~/; potassium chloride, 200 mM 14C-labeled amino acid, 5.0 mM, 1 >( 107 cpm/micromole Native DNA, 10 micromoles DNA-P per milliliter (as determined by diphenylamine reaction 4) RNA polymerase, 1000 units/ml. Highly purified RNA polymerase preparations from E s c h e r i c h i a coli ~ have routinely been used. One unit is defined as the amount of enzyme catalyzing the incorporation of 1 micromole of ATP-I~C into acid-insoluble material in 10 minutes under the assay conditions described 5 Ribosome fraction, described below, 15 mg protein per milliliter Eight-hour supernatant fraction, described below, about 4 mg protein per .milliliter Procedure (a). N o P r e i n c u b a t i o n . Mix 250 #l of the basic reaction mixture with 5 #l of l~C-labeled amino acid, 5 t~l of DNA, 10 #l of RNA
1M. W. Nirenberg and J. H. Matthaei, Proc. Natl. Acad. Sci. U.S. 47, 1588 (1961) ; see also Vol. VI [17]. W. B. Wood and P. Berg, Proc. Natl. Acad. Sci. U.S. 48, 94 (1962). C. Ning and A. Stevens, J. Mol. Biol. 5, 650 (1962). Z. Dische, Mikrochemie 8, 4 (1930). ~A. Stevens and J. Henry, J. Biol. Chem. 239, 196 (1964).
[151]
DNA-DEPENDENT CODING
793
polymerase, 30/~I of ribosome fraction, and 200 ~l of 8-hour supernatant fraction. Incubate for 45 minutes at 37 ° . At the end of the incubation period, add to each mixture 2 ml of 5% trichloroacetic acid. Collect the precipitates by centrifugation and wash by suspension and centrifugation with 2 ml of 5% trichloroacetic acid in the cold and then with 2 ml at 95 ° for 15 minutes. Protein precipitates are then collected on Millipore filters and counted as described by Nirenberg. 1 There is some incorporation of amino acid in the absence of added DNA and RNA polymerase due to endogenous messenger RNA remaining in the enzyme fractions. The degree of stimulation of amino acid incorporation by DNA must be determined by comparing the amount of 14C-labeled amino acid incorporated into protein using the above reaction mixture with that of a reaction mixture lacking DNA. Procedure (b). Preincubation Technique. Mix 250 #l of the basic reaction mixture with 30 t~l of ribosome fraction and 200 t~l of the 8-hour supernatant fraction. Incubate the mixture for 10 minutes at 37 ° and then add (while the mixture is still in the 37 ° bath) 5 ~l of 14C-labeled amino acid, 5 t~l of DNA, and 10 ~l of RNA polymerase. Continue the incubation for 35 minutes. The preincubation leads to removal of some endogenous messenger RNA, and the amount of ~4C-labeled amino acid incorporation in the absence of DNA and RNA polymerase is lower than when procedure (a) is followed. Preparation of Ribosome and Supernatant Fractions
Step 1. Growth o] Bacteria. Escherichia coli, Salmonella typhimurium, and Alicaligenes ]aecalis have been successfully used. The bacteria are grown as described by Nirenberg and Matthaei. 1 The cells are collected by centrifugation at 0 ° and washed twice with a solution containing 50 mM Tris buffer, pH 7.8, 10 mM magnesium acetate, 20 mM potassium chloride, and 1 mM mercaptoethanol (T-M buffer). Step 2. Preparation o/30,000 g Supernatant Fraction. All operations are carried out at 0-4 °. Ten grams of cells are ground (precooled mortar and pestle) with 25 g of alumina (Fisher Scientific Company, A-542), and the paste is extracted with 50 ml of T-M buffer. The suspension is centrifuged, first for 20 minutes at 10,000 g, and then the supernatant solution is again centrifuged for 30 minutes at 30,000 g. The sedimented material is discarded and the supernatant solution (30,000 g supernatant fraction) is used for the preparation of ribosome and 8-hour supernatant fractions as described below. Step 8. Preparation o] Ribosome Fraction. The 30,000 g supernatant fraction is centrifuged for 90 minutes at 100,000 g, and the ribosome pellet is collected and treated in a manner similar to that described by
794
NUCLEIC ACIDS INVOLVED IN PROTEIN SYNTHESIS
[162]
Lengyel et at. 6 It is suspended in T-M buffer containing 0.1% Triton X-100 (manufactured by Rohm and Haas) and 0.2M sucrose to a protein concentration of 10 mg/ml. Two-milliliter portions are layered on 9.5 ml of T-M buffer containing 0.3 M sucrose in polyethylene tubes for the 40 rotor of the Spinco preparative centrifuge. The tubes are centrifuged for 3 hours at 100,000 g and the ribosome pellet is again collected. It is resuspended in T-M buffer and centrifuged for 90 minutes at 100,000 g. The final pellet is suspended in T-M buffer to a protein concentration of 15 mg/ml and dispensed into small tubes and stored at --20 °. Step 4. Preparation o] 8-Hour Supernatant Fraction. The 100,000 g supernatant fraction resulting from the first centrifugation of step 2 is centrifuged for 8 hours at 100,000 g, and the upper two-thirds of the resulting supernatant solution is removed by aspiration and called the 8-hour supernatant fraction. The fraction is dispensed into tubes and stored at --20 ° . Remarks. Native DNA from the T bacteriophages 7 are very active in the system. Furth, Kahan, and Hurwitz s have also described the activity of pneumococcal DNA and slime mold DNA in a similar system. Most bacterial DNA preparations are inactive, or only slightly active. Ribosome and 8-hour supernatant fractions have been stored for as long as one month, but variable losses in activity occur. 6 p. Lengyel, J. F. Speyer, and S. 0choa, Proc. Natl. Acad. Sci. U.S. 47, 1936 (1961). 7j. D. Mandell and A. D. Hershey, Anal. Biochem. 1, 66 (1960). 8j. j. Furth, F. M. Kahan, and J. Hurwitz, Biochem. Biophys. Res. Commun. 9, 337 (1962).
[ 162] T e c h n i q u e s f o r D e m o n s t r a t i n g Protein Synthesis
DNA-Dependent
B y B. NISMAN
The lack of efficiency of many systems in carrying out de novo specific protein synthesis may arise from methods of preparation that damage fundamental spatial configurations inside the active particles and possibly disrupt polyenzymal functional units. We have looked 1 for a mild method of fractionating bacterial cells in an endeavor to avoid serious damage to the main cell components. It has been known for some time 1 that a mild detergent, digitonin, does not severely inhibit oxidative phosphorylation activity in cell particles. We have lysed spheroplasts of 'A. L. Lehaiag~r~ Harvey I.,ecture~ ~er. 49, 176 (1955),
DNA-DEPENDENT CODING
791
Fingerprint Analysis of Protein Product The radioactive protein products can be analyzed in various ways. We describe here the method for fingerprinting the coat protein product together with carrier coat protein2 The general fingerprinting method has been discussed by Ingram. 11 To the reaction mixture containing a minimum of 5 X 104 cpm of 14C-labeled product, excess 12C-labeled amino acid is added and sufficient trichloroaeetic acid to give a concentration of 5%. The mixture is heated at 90 ° for 15 minutes; then the precipitate is washed with the solutions given above for the filter paper disks. To the dry precipitate 5 mg of MS2 coat protein is added, and the proteins are oxidized with performic acid. For this purpose, 10 ml of 99% formic acid is allowed to stand with 0.5 ml of 30% hydrogen peroxide at room temperature for 1 hour. To an estimated 10 mg of protein, 0.5 ml of oxidizing reagent is added. After 20 minutes at room temperature, excess water is added and the solution is lyophilized. The lyophilized protein is then dissolved at pH 10-11 with dilute KOH, and the solution is quickly brought to pH 8.0. Digestion with twice-crystallized trypsin in an amount approximately equal to 1% of the weight of protein is carried out in a pH stat at pH 8.0, and at the end of the reaction the sample is dried in a desiccator. Separation of tryptie peptides is accomplished on 18 X 22 inch Whatman 3 MM chromatography paper by electrophoresis at pH 4.5 (12 ml of pyridine, 13 ml of glacial acetic acid, 25 ml of n-butanol, made to 1 liter with water) for 2.5 hours at 30 V/cm. After being dried overnight, the paper is chromatographed (ascending) in n-butanol-acetic acidpyridine-water (75:15:50:60). (All solvents should be redistilled.) When dry, the paper can be applied to X-ray film for about 10 days to identify 14C-labeled peptides and then stained with 0.25% ninhydrin in acetone to identify the peptides of the carrier coat protein. These can be eluted and counted. ~IV. Ingram, Vol. VI [118].
[ 161 ] D e m o n s t r a t i o n of D N A - D e p e n d e n t Amino Acid Incorporation
By AUDREY STEVENS With the usual bacterial enzyme system usea to demonstrate a dependence on messenger RNA of amino acid incorporation into protein, endogenous messenger RNA is removed and DNase is added to prevent
792
NUCLEIC ACIDS INVOLVED IN PROTEIN SYNTHESIS
[161]
further messenger RNA biosynthesis. 1 In order to demonstrate a dependence of amino acid incorporation into protein on messenger RNA production from DNA, endogenous messenger RNA, DNA, and the enzyme RNA polymerase are largely removed in the preparation of the enzyme system, z,~ ~4C-labeled amino acid incorporation by the system is then stimulated by the addition of certain native DNA preparations together with the enzyme RNA polymerase.
Assay Method Principle. Enzyme fractions (ribosome and 8-hour supernatant fractions) largely freed of DNA and RNA polymerase are incubated with 14C-labeled amino acid, nineteen ~zC-labeled amino acids, ATP, CTP, UTP, GTP, a triphosphate-generating system, DNA, and RNA polymerase. 14C-labeled amino acid in acid-precipitable protein is measured after the incubation period. Reagents
Basic reaction mixture containing: phosphocreatine, 20 mM; creatine kinase 80 #g/ml; ATP, 2 mM; UTP, CTP, and GTP, each 0.50 mM; a mixture of 19 *2C-labeled ~mino acids, each 0.10 mM; mercaptoethanol, 10 mM; Tris buffer, pH 7.8, 100 mM; magnesium acetate, 20 rm~/; potassium chloride, 200 mM 14C-labeled amino acid, 5.0 mM, 1 >( 107 cpm/micromole Native DNA, 10 micromoles DNA-P per milliliter (as determined by diphenylamine reaction 4) RNA polymerase, 1000 units/ml. Highly purified RNA polymerase preparations from E s c h e r i c h i a coli ~ have routinely been used. One unit is defined as the amount of enzyme catalyzing the incorporation of 1 micromole of ATP-I~C into acid-insoluble material in 10 minutes under the assay conditions described 5 Ribosome fraction, described below, 15 mg protein per milliliter Eight-hour supernatant fraction, described below, about 4 mg protein per .milliliter Procedure (a). N o P r e i n c u b a t i o n . Mix 250 #l of the basic reaction mixture with 5 #l of l~C-labeled amino acid, 5 t~l of DNA, 10 #l of RNA
1M. W. Nirenberg and J. H. Matthaei, Proc. Natl. Acad. Sci. U.S. 47, 1588 (1961) ; see also Vol. VI [17]. W. B. Wood and P. Berg, Proc. Natl. Acad. Sci. U.S. 48, 94 (1962). C. Ning and A. Stevens, J. Mol. Biol. 5, 650 (1962). Z. Dische, Mikrochemie 8, 4 (1930). ~A. Stevens and J. Henry, J. Biol. Chem. 239, 196 (1964).
[151]
DNA-DEPENDENT CODING
793
polymerase, 30/~I of ribosome fraction, and 200 ~l of 8-hour supernatant fraction. Incubate for 45 minutes at 37 ° . At the end of the incubation period, add to each mixture 2 ml of 5% trichloroacetic acid. Collect the precipitates by centrifugation and wash by suspension and centrifugation with 2 ml of 5% trichloroacetic acid in the cold and then with 2 ml at 95 ° for 15 minutes. Protein precipitates are then collected on Millipore filters and counted as described by Nirenberg. 1 There is some incorporation of amino acid in the absence of added DNA and RNA polymerase due to endogenous messenger RNA remaining in the enzyme fractions. The degree of stimulation of amino acid incorporation by DNA must be determined by comparing the amount of 14C-labeled amino acid incorporated into protein using the above reaction mixture with that of a reaction mixture lacking DNA. Procedure (b). Preincubation Technique. Mix 250 #l of the basic reaction mixture with 30 t~l of ribosome fraction and 200 t~l of the 8-hour supernatant fraction. Incubate the mixture for 10 minutes at 37 ° and then add (while the mixture is still in the 37 ° bath) 5 ~l of 14C-labeled amino acid, 5 t~l of DNA, and 10 ~l of RNA polymerase. Continue the incubation for 35 minutes. The preincubation leads to removal of some endogenous messenger RNA, and the amount of ~4C-labeled amino acid incorporation in the absence of DNA and RNA polymerase is lower than when procedure (a) is followed. Preparation of Ribosome and Supernatant Fractions
Step 1. Growth o] Bacteria. Escherichia coli, Salmonella typhimurium, and Alicaligenes ]aecalis have been successfully used. The bacteria are grown as described by Nirenberg and Matthaei. 1 The cells are collected by centrifugation at 0 ° and washed twice with a solution containing 50 mM Tris buffer, pH 7.8, 10 mM magnesium acetate, 20 mM potassium chloride, and 1 mM mercaptoethanol (T-M buffer). Step 2. Preparation o/30,000 g Supernatant Fraction. All operations are carried out at 0-4 °. Ten grams of cells are ground (precooled mortar and pestle) with 25 g of alumina (Fisher Scientific Company, A-542), and the paste is extracted with 50 ml of T-M buffer. The suspension is centrifuged, first for 20 minutes at 10,000 g, and then the supernatant solution is again centrifuged for 30 minutes at 30,000 g. The sedimented material is discarded and the supernatant solution (30,000 g supernatant fraction) is used for the preparation of ribosome and 8-hour supernatant fractions as described below. Step 8. Preparation o] Ribosome Fraction. The 30,000 g supernatant fraction is centrifuged for 90 minutes at 100,000 g, and the ribosome pellet is collected and treated in a manner similar to that described by
794
NUCLEIC ACIDS INVOLVED IN PROTEIN SYNTHESIS
[162]
Lengyel et at. 6 It is suspended in T-M buffer containing 0.1% Triton X-100 (manufactured by Rohm and Haas) and 0.2M sucrose to a protein concentration of 10 mg/ml. Two-milliliter portions are layered on 9.5 ml of T-M buffer containing 0.3 M sucrose in polyethylene tubes for the 40 rotor of the Spinco preparative centrifuge. The tubes are centrifuged for 3 hours at 100,000 g and the ribosome pellet is again collected. It is resuspended in T-M buffer and centrifuged for 90 minutes at 100,000 g. The final pellet is suspended in T-M buffer to a protein concentration of 15 mg/ml and dispensed into small tubes and stored at --20 °. Step 4. Preparation o] 8-Hour Supernatant Fraction. The 100,000 g supernatant fraction resulting from the first centrifugation of step 2 is centrifuged for 8 hours at 100,000 g, and the upper two-thirds of the resulting supernatant solution is removed by aspiration and called the 8-hour supernatant fraction. The fraction is dispensed into tubes and stored at --20 ° . Remarks. Native DNA from the T bacteriophages 7 are very active in the system. Furth, Kahan, and Hurwitz s have also described the activity of pneumococcal DNA and slime mold DNA in a similar system. Most bacterial DNA preparations are inactive, or only slightly active. Ribosome and 8-hour supernatant fractions have been stored for as long as one month, but variable losses in activity occur. 6 p. Lengyel, J. F. Speyer, and S. 0choa, Proc. Natl. Acad. Sci. U.S. 47, 1936 (1961). 7j. D. Mandell and A. D. Hershey, Anal. Biochem. 1, 66 (1960). 8j. j. Furth, F. M. Kahan, and J. Hurwitz, Biochem. Biophys. Res. Commun. 9, 337 (1962).
[ 162] T e c h n i q u e s f o r D e m o n s t r a t i n g Protein Synthesis
DNA-Dependent
B y B. NISMAN
The lack of efficiency of many systems in carrying out de novo specific protein synthesis may arise from methods of preparation that damage fundamental spatial configurations inside the active particles and possibly disrupt polyenzymal functional units. We have looked 1 for a mild method of fractionating bacterial cells in an endeavor to avoid serious damage to the main cell components. It has been known for some time 1 that a mild detergent, digitonin, does not severely inhibit oxidative phosphorylation activity in cell particles. We have lysed spheroplasts of 'A. L. Lehaiag~r~ Harvey I.,ecture~ ~er. 49, 176 (1955),