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The Molecular Basis of Suppression in an Ochre Suppressor Strain Possessing Altered Ribosomes* by T

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The Molecular Basis of Suppression in an Ochre Suppressor Strain Possessing Altered Ribosomes* by T THE MOLECULAR BASIS OF SUPPRESSION IN AN OCHRE SUPPRESSOR STRAIN POSSESSING ALTERED RIBOSOMES* BY T. KENT GARTNER, EDUARDO ORIAS, JAMES E. LANNAN, JAMES BEESON, AND PARLANE J. REID DEPARTMENT OF BIOLOGICAL SCIENCES, UNIVERSITY OF CALIFORNIA (SANTA BARBARA) Communicated by Charles Yanofsky, January 30, 1969 Abstract.-Escherichia coli K12 2320(X)-15B has a mutation that results in ochre suppressor activity.1 This mutation concomitantly causes a decreased growth rate in rich medium, an increased sensitivity to streptomycin,l and the production of some altered 30S ribosomes which are differentially sensitive to RNase.2 The results presented below demonstrate that the molecules which cause suppression are tRNA. These observations justify the conclusions that the suppressor mutation did not occur in a structural gene for a ribosomal component, and that the decreased growth rate in rich medium, the increased sensitivity to streptomycin, and the production of altered 30S ribosomes are probably all secondary consequences of the suppressor mutation. Introduction.-Strain 15B is an ochre suppressor mutant which was isolated as a phenotypically lac+ revertant of strain 2320(X).1 Strain 15B is an atypical ochre suppressor mutant because the mutation that causes the suppression also causes the synthesis of some altered 30S ribosomes,2 a decreased growth rate in rich medium, and an increased sensitivity to streptomycin.1 Since the mu- tation that causes ochre suppressor activity in strain 15B also causes the syn- thesis of some altered 30S ribosomes, the ribosomes of strain 15B were tested for suppressor activity. The results of this investigation which are presented below demonstrate, however, that most if not all of the suppressor activity of strain 15B resides in tRNA. Materials and Methods.-Strains: Strain 15B is a lac+ derivative of 2320(X).1 Strain 2320(X) is lac- because of an ochre (UAA) codon in the beginning of the Z cistron (lac2- mutation), the structural cistron for,-galactosidase.3 Strain 15B was isolated as a spontaneous lac+ revertant of 2320(X). The mutation that causes the lac+ phenotype also causes ochre suppressor activity. Strain SBO is a su-, streptomycin-sensitive derivative of strain 2320(X).4 Two strains of the RNA bacteriophage f2, kindly supplied by Dr. N. D. Zinder, were used as sources of mRNA for an in vitro system that performs protein synthesis. One was the wild-type strain f2 and the other was mutant sus 13. Sus 13 has an amber codon in the cistron for the protein coat subunit. The amber codon replaces the normal codon for the sixth amino acid residue (from the N-terminus) of the coat protein synthesized in vivo., Both strains of f2 were grown on cells of strain S26 Rle,6 a strain carrying an amber suppressor (Su+1) which results in the insertion of serine in response to the UAG codon. This strain also was obtained from Dr. Zinder. Culture of the strains: Forty-liter batches of strains 2320(X) and SBO were grown in LC broth medium without CaC127 in a New Brunswick Fermacell Fermentor. The cultures were harvested in logarithmic phase at an approximate density of 4 X 108 cells per ml. Ten-liter batches of cells of strain 15B were grown as described previously;8 the cells harvested from each 10-liter batch were discarded if more than 10% of the population were fast-growing revertants. 946 Downloaded by guest on September 26, 2021 VOL. 62, 1969 BIOCHEMISTRY: GARTNER ET AL. 947 Wild-type f2 and sus 13 were grown in large batches and purified essentially as de- scribed by Webster et al.5 Buffers: Buffer 1: 0.02 M Tris-HCl, pH 7.8; 0.01 M Mg acetate; and 0.006 M mercaptoethanol. Buffer 2: Buffer 1 that is 0.080 M in NaCl. Buffer for dialysis of tRNA: 0.01 M Tris-HCl, pH 7.8; 0.01 M MgCl2; and 0.001 M EDTA. Preparation of S30, S100, and ribosome fractions: The S30 fraction was prepared as follows. Cells, washed in Buffer 2, were ground with alumina (Bacteriological, grade A-305, Alcoa), with approximately 2 gm of alumina per gram of cells (wet weight). DNase (Nutritional Biochemical Corp.) was added to a final concentration of 5 ug/ml and the paste was incubated at 40C for 30 min. The extract was centrifuged once at 10,400 X g for 20 min and twice at 30,000 X g for 20 min each time. The supernatant fluid from the last centrifugation (A260 of 600-800) was frozen and stored at - 20'C until used. The S100 and ribosome fractions were prepared from an S30 as follows. The S30 was dialyzed for 6 hr at 4°C against Buffer 2 which was 1 M in NH4Cl. The dialyzed S30 was then centrifuged for 1.5 hr at 4°C at 269,000 X g in a type-65 rotor in a Spinco L2-65B ultracentrifuge. The supernatant fluid of the 269,000 X g centrifugation (S100) was dialyzed against Buffer 2 for 8 hr at 4°C, frozen, and stored in small aliquots at - 20°C until used (A260 of 250-300). The pellet obtained in the 269,000 X g centrifugation (ribosome fraction) was resuspended in Buffer 2, dialyzed for 8 hr against Buffer 2, diluted to an A260 of 500 with Buffer 2, frozen, and stored at -20°C in small aliquots until used. Preparation of tRNA: tRNA was prepared by the method of Zubay.9 The tRNA preparations for both 2320(X) and 15B used in this study had a glycine acceptor activity of 0.4 mumole of glycine/10 A260 units of tRNA. Concentrated S100 for charging: S100 was prepared as described above except that the S100 was dialyzed against Buffer 2 containing one-tenth the concentration of all the com- ponents found in the normal buffer. The S100 was heated at 45°C for 30 min, prior to concentration. The heated S100 was clarified by centrifugation twice at 30,000 X g for 20 min. The heated, clarified S100 was lyophilized. A volume of distilled water equal to one-tenth the original volume of the 8100 was added to the Iyophilized S100 to ac- complish approximately a tenfold concentration. This concentrated S100 was used as the source of activating enzymes for the charging experiment. Charging oftRNA: Charging occurred at 43.5°C during a 30-min incubation period in a 12-ml reaction mixture which contained 3.5 ml of a lOX concentrated S100, 1.6 ml of tRNA (510 A260 units), 62 ,ug of creatine phosphokinase, and the following ingredients at the final concentrations indicated: 1.2 mM MgCl2, 4.2 mM Mg acetate, 22 mM ATP, 0.16 mM GTP, 64 mM Tris-HCl (pH 7.6), 16 mM mercaptoethanol, 14 mM creatine phosphate, and 38 mM KCl. After 30 min of incubation, the tRNA was extracted from the charging reaction mixture with an equal volume of 88% phenol and then precipitated from the upper layer with ethanol-KCl. The precipitate was collected by centrifugation, and the pellet was dissolved in 0.1 M Na acetate, pH 4.7, in preparation for the periodate treatment. Stripping: The tRNA was stripped by dissolving the tRNA in 0.2 M Tris (John Car- bon, personal communication) to a concentration of 100 A260 units/ml. The solution was incubated for 30 min at 37°C. The stripped tRNA was precipitated three times with ethanol-KCl and finally dissolved in 0.1 M Na acetate, pH 4.7, in preparation for the periodate treatment. Periodate treatment: One ml of a solution of 0.1 M Na acetate, pH 4.7, containing 100 A260 units of either charged or stripped tRNA, was mixed with 0.5 ml of a 0.1 M solution of NaIO4. This mixture was incubated in the dark for 20 min at room temperature. After the 20-min incubation period, excess glucose was added to inactivate the remaining perio- date. The RNA was precipitated three times with ethanol-KCl and finally dissolved in H20 to a concentration of 200 A260 units/ml. The solution of tRNA was dialyzed for 18 hr against the dialysis buffer described previously. Downloaded by guest on September 26, 2021 948 BIOCHEMISTRY: GARTNER ET AL. PROc. N. A. S. Protein synthesis: Protein synthesis occurred at 34'C5 during an incubation period of 30 min in a volume of 150,ul that had the following concentrations of ingredients :10 0.002 M ATP, 0.00017 l GTP, 0.042 M Tris-HCl (pH 7.9), 0.015 Al creatine phosphate, 0.0125 Ml Mg acetate, 0.014 M mercaptoethanol, 0.04 M KCl, and 0.17 M for each amino acid other than valine. The incubation medium also contained 0.825 Mug of creatine phosphokinase, 10 Mug of leucovorin, 2 A260 units of mRNA, 0.2 Mc of '4C-valine (uniformly labeled at a specific activity of 200-250), and either 11 A260 units of S30 (for the S30 incorporating sys- tem) or 2.5 A260 units of ribosomes, 2 A260 units of tRNA, and 8 A260 units of S100 (for the S100 incorporation system). At the end of the 30-min incubation period, 100 Ml from each reaction mixture was placed on a Whatman 3-MM filter paper disc, and the discs were treated as described by Mans and Novelli."1 Results.-The suppressor activity of 15B was investigated with an in vitro protein synthesizing system. This system uses as messenger RNA the RNA from either the wild-type RNA phage f2 or sus 13, an amber mutant derivative of f2.
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