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Studies on fish liver protein synthesis—I. Isolation and characterizatin of shark liver transfer ribonucleic acid
Comp. Biochera. PhydoL, 1974, Vol. 4813,pp. 619 to 628. Pergamon Press. Printed in Great Britain
STUDIES ON FISH LIVER PROTEIN SYNTHESIS--I. ISOLATION AND CHARACTERIZATION OF SHARK LIVER TRANSFER RIBONUCLEIC ACID* M A N U E L K R A U S K O P F , t 1 A L E J A N D R O ARAYA 1 and SIMON LITVAK 2 Instituto de Bioquimica, Facultad de Ciencias, Universidad Austral de Chile, Valdivia; a and Departamento de Qulmica, Facultad de Medicina, Universidad de Chile, Santiagofl Chile (Received 14 August 1973)
Abst~et--1. The isolation of tRNA from the liver of the shark Mustdus mento is described. The procedure involves phenol extraction, chromatography on DEAE-cellulose and isopropanol fractionation. The obtained fractions were analyzed by polyacrylamide gel electrophoresis. 2. The tRNA was shown to have an intact 3' end and to accept the four amino acids tested with homologous aminoacyl-tRNA synthetases. 3. Analytical centrifuge analysis gave a sedimentation coefficient of S2o,w = 4"01. 4. Thermal denaturation profiles were compared to other tRNAs. INTRODUCTION THE ~X~OWL~GE of biomoleeular mechanisms involved in protein synthesis has been gained mainly through the study of systems which have been isolated from bacteria, mammals and plants (Allende, 1969; Lengyel & Soil, 1969; Lucas-Lenard & Lipmann, 1971). Studies on fish systems have not been properly considered in spite of the fact that they are attractive models for the study of thermally directed changes in mechanisms of macromolecular synthesis. Cell-free incorporation of amino acids by a trout liver system has been reported. Rosen et al. (1967) showed an extremely heat-labile step in the aminoacyl transfer reaction with S a l m o gairdnerii liver ribosomes, postmitochondrial supernatant and rat liver aminoacyl-tRNA. We are initiating more extensive studies on the mechanisms of protein synthesis, in organisms other than the warm-blooded vertebrates, as a new approach to understand their regulation. In the present paper we deal mainly with the isolation and characterization of transfer ribonucleie acid from the liver of the shark, M u s t e l g s mento, as a first step in the fractionation of the components involved in the process of protein biosynthesis of this poikilotherm. * This work was supported by a grant from the Research Fund of the Universidad Austral de Chile (Project 72-72). ~"To whom inquiries should be addressed (Universidad Austral, Valdivia, Chile). 619
620
MANUEL KRAUSKOPF, ALEJANDRO ARAYA AND SIMON LITVAK
W e chose a r e p r e s e n t a t i v e of t h e m o s t a n c i e n t f o r m o f c a r t i l a g i n o u s fish available, since t h e s e s t u d i e s c o u l d give s o m e i n s i g h t in t h e v a r i a t i o n s p r o d u c e d t h r o u g h t h e e v o l u t i o n a r y scale. MATERIALS AND METHODS Collection of specimens Sharks, M. mento (de Buen, 1959), were collected by midwater trawling at depths down to 120 m in the cold waters of the Peril current, near the coast of Corral, Valdivia. Liver tissue was removed from the freshly caught sharks and transported at 0°C to the laboratory where it was processed immediately, or stored at - 18°C.
Isolation of shark liver t R N A Unless otherwise specified, all procedures were carried out at 4°C. Six hundred g of frozen shark liver were minced, and 900 ml of 0.1 M Tris-HC1 buffer, p H 7"2, containing 0"1% bentonite and 0-25 M sucrose were added and homogenized batchwise, with a glassTeflon motor-driven Potter homogenizer. T h e homogenate was centrifuged for 10 min at 16,000 g and fihered through glass-wool into a separatory funnel. One vol. of phenol saturated with 0"1 M potassium acetate, p H 5'6, containing 0"1% of 8-hydroxyquinoline was added to the oil-free post-mitochondrial supernatant. It was then stirred vigorously for 1 hr at room temperature. After centrifugation at 4000 g for 1 hr the aqueous layer was collected by aspiration and precipitated by the addition of 0"1 vol. of 20% potassium acetate, p H 5"6, and 2 vol. of cold ethanol and left overnight at - 1 8 ° C . T h e precipitate was collected by centrifugation and washed twice with cold ethanol and twice with ether. T h e dried pellet was suspended in 0"1 M potassium acetate buffer, p H 5"6, centrifuged at 27,000 g for 10 min and the supernatant loaded into a DEAE-cellulose column (2 x 20 cm) previously equilibrated with the same buffer. Contaminants were eluted by use of a salt gradient, from 0"1 to 0"5 M NaC1, in 0"1 M potassium acetate buffer, p H 5.6. The t R N A fraction was eluted by changing the buffer to 1 M NaC1. T h e A260 absorbing peak was pooled and precipitated with 2 vol. of cold ethanol. Finally, the precipitate collected by centrifugation was dissolved in 0"3 M sodium acetate, p H 7"0, and subjected to isopropanol precipitation at room temperature, as described by Rogg et al. (1969). Hydrolysis of the aminoacyl residues from the t R N A thus isolated was accomplished by incubation in 0"5 M Tris-HC1, p H 8"7, for 1 hr at 37°C. T h e discharged t R N A was recovered by ethanol precipitation. Assay of amino acid acceptor activity Crude shark liver aminoacyl-tRNA synthetase was prepared as described by Nishimura & Weinstein (1969) for the rat liver system. T h e enzyme was stored in 0"1 M Tris-HC1, p H 7'2, 5 m M MgCI~ and 50% glycerol at - 1 5 ° C . Amino acid acceptance assays were performed essentially as previously described (Henes et al., 1969). Mixtures (0'1 mi) containing 0.1 M 3,3-dimethylglutaric acid buffer, p H 7"0, 10 m M magnesium acetate, 10 m M KC1, 12 m M A T P , 0"02 m M l~C-amino acid, shark liver t R N A and enzyme, were incubated for 30 rain at 30°C. The amounts of t R N A and enzyme were adjusted to give complete charging of the tRNA. Reactions were stopped with cold 5 % trichloroacetic acid, and the precipitate was collected on Millipore filters (HA, 0"45/~m), washed three times with cold trichloroacetic acid and dissolved in Bray's solution (Bray, 1960) for counting at 77 per cent efficiency. Gel electrophoresis Polyacrylamide gel electrophoresis was performed according to the method of Peacock & Dingman (1967). Runs of 10% disc gel were made at 4 mA/gel either at 4°C or at room
SHARK LIV-~ tRNA
621
temperature. The stained gels (Peacock & Dingman, 1967) were scanned at 615 nm in a Gilford Model 2400 spectrophotometer using the Model 2410-S linear transport system.
Assay of A M P and CMP incorporating activity of tRNA A 1-ml reaction mixture containing 50 mM Tris-HCl buffer, pH 8"5, 10 mM MgC12, 2 mM fl-mercaptoethanol, 5 mM 8H-ATP or 3H-CTP (1.7 x 10 ~ counts/rain), 13 A~6o units of tRNA (M. mento) and 5-15/zg of purified Escherichia coil CTP (ATP)-tRNA nucleotidyltransferase (CCA-enzyme) was incubated at 37°C (Carre et al., 1970). At different lengths of time 100-/~1 samples were withdrawn and the reaction stopped by the addition of 0.1 mg of bovine serum albumin and 0"2 ml of 50% triehloroaeetie acid containing 3 % of nucleosides triphosphate. The precipitates were filtered through glass fiber discs, washed with 5% trichloroacetic acid and counted in a toluene PPO,* POPOP scintillation mixture. Venom phosphodiesterase digestion of tRNA was accomplished essentially as described byMiller et aL (1970). The partially digested tRNA was recovered in the void volume from a Sephadex G-25 column (1 x 45 cm) with a 1-cm layer of acid-washed silicic acid on top, by elution with 0"1 M potassium acetate, pH 4"5. The A,e0 peak was precipitated overnight with 2 vol. of ethanol at - 20°C. The precipitate was collected by centrifugation, washed twice with cold ethanol, dried and dissolved in distilled water. Thermal denaturation studies The variation of absorbancy at 260 nm as a function of temperature was measured in a Beckmann DU spectrophotometer equipped with a thermostatically controlled cuvette block. Heating was produced by means of a circulating water-bath which was manually controlled. Sedimentation coefficient The sedimentation velocity boundary profile of the shark liver tRNA diluted in 0"1 M KCI, was measured in a Beckmann Model E analytical ultracentrifuge, at 56,000 rev/min at 20°C. The time between scans was 16 min. This work was kindly performed by Mr. Daniel Luk, from the Roche Institute of Molecular Biology in Nutley, N.J. Materials 14C-Valine (41 mCi/m-mole), 14C-methionine (60 mCi/m-mole) and 14C-phenylalanine (10 mCi/m-mole) were obtained from Amersham/Searle. t*C-Alanine (63 mCi/m-mole) was from CEA--France and aH-ATP and aH-CTP from Schwarz BioResearch. Snake venom phosphodiesterase (E.C. 3.1.4.1), B grade, was from Calbiochem. E. coli (strain B) tRNA was from Schwarz BioResearch. RESULTS AND D I S C U S S I O N Isolation of the shark liver t R N A T h e different R N A families obtained through the main fractionation steps during the isolation of M . mento liver t R N A were studied by gel electrophoresis. Figure la shows the absorbancy tracing of the fraction obtained by elution with 1 M NaC1 of the bulk R N A f r o m a DEAE--cellulose column as detailed under Materials and Methods. T h e major peak which migrates 3 cm f r o m the origin under the given conditions is the t R N A . Figure la shows that this step in the purification procedure is not enough, since higher molecular weight R N A s are * Abbreviations used: PPO, 2,5-diphenyloxazole; POPOP, 4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene; EDTA, ethylenediaminetetraacetic acid.
622
MANUEL KRAUSKOPF, ALEJANDROARAYA AND SIMON LITVAK
1.6 12 08 04 0 24
o41 ~' 181 g
12
o
06 ~ .____.J
0 16 ~2
0-8 04 0 0
I
2
~
4
5
6
crn
FIc. 1. Disc-gel electrophoresis of (a) shark liver RNA obtained after elution of a bulk preparation through a DEAE--ceUulose column, (b) RNA separated from the previous fraction by means of a first isopropanol precipitation, (c) shark liver tRNA, product of a second isopropanol treatment and (d) E. coli tRNA. Each gel was loaded with 1 A2e0 unit of sample. The details of the electrophoretic run are described in Materials and Methods.
present in a considerable amount. In fact three peaks were separated from the tRNA. The major one migrated about 2 cm from the origin and it probably is 5 S ribosomal RNA. This peak will be referred in the text as 5 S RNA. This is in agreement with the results obtained for rat liver t R N A with a DEAE-cellulose batchwise adsorption elution process of bulk R N A (Rogg et al., 1969). Figure la also shows that this fraction presents some degradation products.
SHARKLIV~ tRNA
623
In order to free the shark liver tRNA fraction from their contaminants, an isopropanol precipitation step was introduced (Zubay, 1966; von Ehrenstein, 1967; Rogg et al., 1969). Isopropanol (0.56 vol.) was added to the DEAE-cellulose column tRNA fraction dissolved in 0.3 M sodium acetate, pH 7.0. The precipitate thus obtained was dissolved and subjected to polyacrylamide gel electrophoresis. The absorbancy scanning is shown in Fig. lb. Isopropanol (0.44 vol.) was added to the supernatant left by this procedure and a second precipitate was collected, dissolved and electrophoresed (Fig. lc). As seen in Fig. lb, the first precipitate contains most of the contaminants present in the previous fraction, although a considerable amount of tRNA is also present. The second precipitate is essentially pure tRNA, as shown in Fig. lc. The shape of the main peak (Fig. lc) suggests the presence of at least two electrophoretically distinctive groups of tRNA. This is perfectly understandable since this fraction, as a whole, must contain all the multiplicity of individual species that take part in the protein biosynthesis process. Analysis of rat liver tRNA in Sephadex G-200 columns indicates the presence of a small shoulder where the tRNA peak is detected (Rogg et al., 1969). Transfer ribonucleic acid multiple bands when total cytoplasmic rat liver, kidney and brain RNA was electrophoresed in polyacrylamide gels has also been reported (Peacock & Dingman, 1967). Cannon & Richards (1967) showed similar densitometric tracing for E. coli (strain B 163) tRNA. The mobility of shark liver tRNA was compared with E. coli tRNA, Figure ld shows the electrophoregram of a freshly prepared solution of commercial E. coli B tRNA. As seen, both tRNA have about equal electrophoretical behaviour. The latter is more contaminated with 5 S RNA but has less degradation products. The results presented here prove that polyacrylamide gel electrophoresis can be used as an excellent method in the analysis of the different steps involved in the tRNA fractionation procedure, as well as in the purity of the product, and offers significant advantages over other choices. The shark liver tRNA product, when studied in the analytical centrifuge (see Materials and Methods), sedimented as a single moving boundary. The calculated sedimentation coefficient gave S20.w= 4.01. This value is slightly lower with respect to other eukaryotic tRNAs. Values of 4.15 S and 4.2 S have been reported for rabbit liver and human placenta tRNA, respectively (Abadom & Elson, 1970; Igarashi & Dufresne, 1972). In order to obtain a more detailed characterization of the RNAs separated during the fractionation of shark liver tRNA, we are isolating each of the individual peaks obtained with the first isopropanol precipitation step with a suitable method of preparative electrophoresis (E. Rosenmann, A. Araya and M. Krauskopf, in preparation). Completeness of the t R N A The presence of the pCpCpA sequence at the Y end is a common feature for all the tRNAs species studies up to date (Jukes & Holmquist, 1972). The presence
624
MANUEL KRAUSKOPF, ALEJANDRO ARAYAAND SIMON LITVAK
or absence of these final residues can be used as a criterion in the analysis of the extent of the intactness of a tRNA preparation, tRNA molecules lacking this 3' terminal residues can be repaired by using a specific enzyme, the CTP (ATP)tRNA nucleotidyltransferase (Carre et al. 1970). When shark liver tRNA was tested for AMP or CMP incorporation into the 3' end using E. coli CCA-enzyme (see Materials & Methods) very little incorporation was observed (Fig. 2). These results may indicate that (a) the tRNA assayed has its 3' end practically intact; (b) the tRNA lacks more than the three terminal o
8
(~J
{ X
0( 20
40
60 Time,
80
I00
120
rain
FIG. 2. Kinetics of °H-AMP or aH-CMP incorporation into shark liver tRNA by the E. coli CTP(ATP)-tRNA nucleofidyhransferase. The conditions were those given under Materials and Methods. Untreated shark liver tRNA as tested with aH-ATP ( 0 - - 0 ) and aH-CTP (•--A). Venom phosphodiesterase treated shark liver tRNA as tested with °H-ATP (O--©), CTP plus aH-ATP (V~--[~) and aH-CTP ( , - - A ) . XMP refers to either °H-AMP or aH-CMP. residues, hence the enzyme is not able to incorporate the nucleotides; and (c) shark liver tRNA is not a suitable substrate for the E. coli enzyme. To distinguish among these three possibilities, shark liver tRNA was partially digested with snake venom phosphodiesterase. As shown in Fig. 2, when the treated tRNA was incubated in the presence of 3H-ATP and the E. coli CCA-enzyme, some incorporation was attained. When cold CTP was added to the same mixture, the transfer of OH-AMP from 3H-ATP into the 3' end of the treated tRNA was greatly stimulated. Repair assays of the partially digested tRNA with 8H-CTP alone resulted in an important 8H-CMP incorporation (Fig. 2). When the digested tRNA was subjected to repair, and was subsequently assayed for 14C-valine acceptance with an aminoacyl-tRNA synthetase, a thirtyfold increase in the amino acid incorporation was obtained with respect to the unrepaired control.
STUDIES ON FISH LIVER PROTEIN SYNTHESIS--I. ISOLATION AND CHARACTERIZATION OF SHARK LIVER TRANSFER RIBONUCLEIC ACID* M A N U E L K R A U S K O P F , t 1 A L E J A N D R O ARAYA 1 and SIMON LITVAK 2 Instituto de Bioquimica, Facultad de Ciencias, Universidad Austral de Chile, Valdivia; a and Departamento de Qulmica, Facultad de Medicina, Universidad de Chile, Santiagofl Chile (Received 14 August 1973)
Abst~et--1. The isolation of tRNA from the liver of the shark Mustdus mento is described. The procedure involves phenol extraction, chromatography on DEAE-cellulose and isopropanol fractionation. The obtained fractions were analyzed by polyacrylamide gel electrophoresis. 2. The tRNA was shown to have an intact 3' end and to accept the four amino acids tested with homologous aminoacyl-tRNA synthetases. 3. Analytical centrifuge analysis gave a sedimentation coefficient of S2o,w = 4"01. 4. Thermal denaturation profiles were compared to other tRNAs. INTRODUCTION THE ~X~OWL~GE of biomoleeular mechanisms involved in protein synthesis has been gained mainly through the study of systems which have been isolated from bacteria, mammals and plants (Allende, 1969; Lengyel & Soil, 1969; Lucas-Lenard & Lipmann, 1971). Studies on fish systems have not been properly considered in spite of the fact that they are attractive models for the study of thermally directed changes in mechanisms of macromolecular synthesis. Cell-free incorporation of amino acids by a trout liver system has been reported. Rosen et al. (1967) showed an extremely heat-labile step in the aminoacyl transfer reaction with S a l m o gairdnerii liver ribosomes, postmitochondrial supernatant and rat liver aminoacyl-tRNA. We are initiating more extensive studies on the mechanisms of protein synthesis, in organisms other than the warm-blooded vertebrates, as a new approach to understand their regulation. In the present paper we deal mainly with the isolation and characterization of transfer ribonucleie acid from the liver of the shark, M u s t e l g s mento, as a first step in the fractionation of the components involved in the process of protein biosynthesis of this poikilotherm. * This work was supported by a grant from the Research Fund of the Universidad Austral de Chile (Project 72-72). ~"To whom inquiries should be addressed (Universidad Austral, Valdivia, Chile). 619
620
MANUEL KRAUSKOPF, ALEJANDRO ARAYA AND SIMON LITVAK
W e chose a r e p r e s e n t a t i v e of t h e m o s t a n c i e n t f o r m o f c a r t i l a g i n o u s fish available, since t h e s e s t u d i e s c o u l d give s o m e i n s i g h t in t h e v a r i a t i o n s p r o d u c e d t h r o u g h t h e e v o l u t i o n a r y scale. MATERIALS AND METHODS Collection of specimens Sharks, M. mento (de Buen, 1959), were collected by midwater trawling at depths down to 120 m in the cold waters of the Peril current, near the coast of Corral, Valdivia. Liver tissue was removed from the freshly caught sharks and transported at 0°C to the laboratory where it was processed immediately, or stored at - 18°C.
Isolation of shark liver t R N A Unless otherwise specified, all procedures were carried out at 4°C. Six hundred g of frozen shark liver were minced, and 900 ml of 0.1 M Tris-HC1 buffer, p H 7"2, containing 0"1% bentonite and 0-25 M sucrose were added and homogenized batchwise, with a glassTeflon motor-driven Potter homogenizer. T h e homogenate was centrifuged for 10 min at 16,000 g and fihered through glass-wool into a separatory funnel. One vol. of phenol saturated with 0"1 M potassium acetate, p H 5'6, containing 0"1% of 8-hydroxyquinoline was added to the oil-free post-mitochondrial supernatant. It was then stirred vigorously for 1 hr at room temperature. After centrifugation at 4000 g for 1 hr the aqueous layer was collected by aspiration and precipitated by the addition of 0"1 vol. of 20% potassium acetate, p H 5"6, and 2 vol. of cold ethanol and left overnight at - 1 8 ° C . T h e precipitate was collected by centrifugation and washed twice with cold ethanol and twice with ether. T h e dried pellet was suspended in 0"1 M potassium acetate buffer, p H 5"6, centrifuged at 27,000 g for 10 min and the supernatant loaded into a DEAE-cellulose column (2 x 20 cm) previously equilibrated with the same buffer. Contaminants were eluted by use of a salt gradient, from 0"1 to 0"5 M NaC1, in 0"1 M potassium acetate buffer, p H 5.6. The t R N A fraction was eluted by changing the buffer to 1 M NaC1. T h e A260 absorbing peak was pooled and precipitated with 2 vol. of cold ethanol. Finally, the precipitate collected by centrifugation was dissolved in 0"3 M sodium acetate, p H 7"0, and subjected to isopropanol precipitation at room temperature, as described by Rogg et al. (1969). Hydrolysis of the aminoacyl residues from the t R N A thus isolated was accomplished by incubation in 0"5 M Tris-HC1, p H 8"7, for 1 hr at 37°C. T h e discharged t R N A was recovered by ethanol precipitation. Assay of amino acid acceptor activity Crude shark liver aminoacyl-tRNA synthetase was prepared as described by Nishimura & Weinstein (1969) for the rat liver system. T h e enzyme was stored in 0"1 M Tris-HC1, p H 7'2, 5 m M MgCI~ and 50% glycerol at - 1 5 ° C . Amino acid acceptance assays were performed essentially as previously described (Henes et al., 1969). Mixtures (0'1 mi) containing 0.1 M 3,3-dimethylglutaric acid buffer, p H 7"0, 10 m M magnesium acetate, 10 m M KC1, 12 m M A T P , 0"02 m M l~C-amino acid, shark liver t R N A and enzyme, were incubated for 30 rain at 30°C. The amounts of t R N A and enzyme were adjusted to give complete charging of the tRNA. Reactions were stopped with cold 5 % trichloroacetic acid, and the precipitate was collected on Millipore filters (HA, 0"45/~m), washed three times with cold trichloroacetic acid and dissolved in Bray's solution (Bray, 1960) for counting at 77 per cent efficiency. Gel electrophoresis Polyacrylamide gel electrophoresis was performed according to the method of Peacock & Dingman (1967). Runs of 10% disc gel were made at 4 mA/gel either at 4°C or at room
SHARK LIV-~ tRNA
621
temperature. The stained gels (Peacock & Dingman, 1967) were scanned at 615 nm in a Gilford Model 2400 spectrophotometer using the Model 2410-S linear transport system.
Assay of A M P and CMP incorporating activity of tRNA A 1-ml reaction mixture containing 50 mM Tris-HCl buffer, pH 8"5, 10 mM MgC12, 2 mM fl-mercaptoethanol, 5 mM 8H-ATP or 3H-CTP (1.7 x 10 ~ counts/rain), 13 A~6o units of tRNA (M. mento) and 5-15/zg of purified Escherichia coil CTP (ATP)-tRNA nucleotidyltransferase (CCA-enzyme) was incubated at 37°C (Carre et al., 1970). At different lengths of time 100-/~1 samples were withdrawn and the reaction stopped by the addition of 0.1 mg of bovine serum albumin and 0"2 ml of 50% triehloroaeetie acid containing 3 % of nucleosides triphosphate. The precipitates were filtered through glass fiber discs, washed with 5% trichloroacetic acid and counted in a toluene PPO,* POPOP scintillation mixture. Venom phosphodiesterase digestion of tRNA was accomplished essentially as described byMiller et aL (1970). The partially digested tRNA was recovered in the void volume from a Sephadex G-25 column (1 x 45 cm) with a 1-cm layer of acid-washed silicic acid on top, by elution with 0"1 M potassium acetate, pH 4"5. The A,e0 peak was precipitated overnight with 2 vol. of ethanol at - 20°C. The precipitate was collected by centrifugation, washed twice with cold ethanol, dried and dissolved in distilled water. Thermal denaturation studies The variation of absorbancy at 260 nm as a function of temperature was measured in a Beckmann DU spectrophotometer equipped with a thermostatically controlled cuvette block. Heating was produced by means of a circulating water-bath which was manually controlled. Sedimentation coefficient The sedimentation velocity boundary profile of the shark liver tRNA diluted in 0"1 M KCI, was measured in a Beckmann Model E analytical ultracentrifuge, at 56,000 rev/min at 20°C. The time between scans was 16 min. This work was kindly performed by Mr. Daniel Luk, from the Roche Institute of Molecular Biology in Nutley, N.J. Materials 14C-Valine (41 mCi/m-mole), 14C-methionine (60 mCi/m-mole) and 14C-phenylalanine (10 mCi/m-mole) were obtained from Amersham/Searle. t*C-Alanine (63 mCi/m-mole) was from CEA--France and aH-ATP and aH-CTP from Schwarz BioResearch. Snake venom phosphodiesterase (E.C. 3.1.4.1), B grade, was from Calbiochem. E. coli (strain B) tRNA was from Schwarz BioResearch. RESULTS AND D I S C U S S I O N Isolation of the shark liver t R N A T h e different R N A families obtained through the main fractionation steps during the isolation of M . mento liver t R N A were studied by gel electrophoresis. Figure la shows the absorbancy tracing of the fraction obtained by elution with 1 M NaC1 of the bulk R N A f r o m a DEAE--cellulose column as detailed under Materials and Methods. T h e major peak which migrates 3 cm f r o m the origin under the given conditions is the t R N A . Figure la shows that this step in the purification procedure is not enough, since higher molecular weight R N A s are * Abbreviations used: PPO, 2,5-diphenyloxazole; POPOP, 4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene; EDTA, ethylenediaminetetraacetic acid.
622
MANUEL KRAUSKOPF, ALEJANDROARAYA AND SIMON LITVAK
1.6 12 08 04 0 24
o41 ~' 181 g
12
o
06 ~ .____.J
0 16 ~2
0-8 04 0 0
I
2
~
4
5
6
crn
FIc. 1. Disc-gel electrophoresis of (a) shark liver RNA obtained after elution of a bulk preparation through a DEAE--ceUulose column, (b) RNA separated from the previous fraction by means of a first isopropanol precipitation, (c) shark liver tRNA, product of a second isopropanol treatment and (d) E. coli tRNA. Each gel was loaded with 1 A2e0 unit of sample. The details of the electrophoretic run are described in Materials and Methods.
present in a considerable amount. In fact three peaks were separated from the tRNA. The major one migrated about 2 cm from the origin and it probably is 5 S ribosomal RNA. This peak will be referred in the text as 5 S RNA. This is in agreement with the results obtained for rat liver t R N A with a DEAE-cellulose batchwise adsorption elution process of bulk R N A (Rogg et al., 1969). Figure la also shows that this fraction presents some degradation products.
SHARKLIV~ tRNA
623
In order to free the shark liver tRNA fraction from their contaminants, an isopropanol precipitation step was introduced (Zubay, 1966; von Ehrenstein, 1967; Rogg et al., 1969). Isopropanol (0.56 vol.) was added to the DEAE-cellulose column tRNA fraction dissolved in 0.3 M sodium acetate, pH 7.0. The precipitate thus obtained was dissolved and subjected to polyacrylamide gel electrophoresis. The absorbancy scanning is shown in Fig. lb. Isopropanol (0.44 vol.) was added to the supernatant left by this procedure and a second precipitate was collected, dissolved and electrophoresed (Fig. lc). As seen in Fig. lb, the first precipitate contains most of the contaminants present in the previous fraction, although a considerable amount of tRNA is also present. The second precipitate is essentially pure tRNA, as shown in Fig. lc. The shape of the main peak (Fig. lc) suggests the presence of at least two electrophoretically distinctive groups of tRNA. This is perfectly understandable since this fraction, as a whole, must contain all the multiplicity of individual species that take part in the protein biosynthesis process. Analysis of rat liver tRNA in Sephadex G-200 columns indicates the presence of a small shoulder where the tRNA peak is detected (Rogg et al., 1969). Transfer ribonucleic acid multiple bands when total cytoplasmic rat liver, kidney and brain RNA was electrophoresed in polyacrylamide gels has also been reported (Peacock & Dingman, 1967). Cannon & Richards (1967) showed similar densitometric tracing for E. coli (strain B 163) tRNA. The mobility of shark liver tRNA was compared with E. coli tRNA, Figure ld shows the electrophoregram of a freshly prepared solution of commercial E. coli B tRNA. As seen, both tRNA have about equal electrophoretical behaviour. The latter is more contaminated with 5 S RNA but has less degradation products. The results presented here prove that polyacrylamide gel electrophoresis can be used as an excellent method in the analysis of the different steps involved in the tRNA fractionation procedure, as well as in the purity of the product, and offers significant advantages over other choices. The shark liver tRNA product, when studied in the analytical centrifuge (see Materials and Methods), sedimented as a single moving boundary. The calculated sedimentation coefficient gave S20.w= 4.01. This value is slightly lower with respect to other eukaryotic tRNAs. Values of 4.15 S and 4.2 S have been reported for rabbit liver and human placenta tRNA, respectively (Abadom & Elson, 1970; Igarashi & Dufresne, 1972). In order to obtain a more detailed characterization of the RNAs separated during the fractionation of shark liver tRNA, we are isolating each of the individual peaks obtained with the first isopropanol precipitation step with a suitable method of preparative electrophoresis (E. Rosenmann, A. Araya and M. Krauskopf, in preparation). Completeness of the t R N A The presence of the pCpCpA sequence at the Y end is a common feature for all the tRNAs species studies up to date (Jukes & Holmquist, 1972). The presence
624
MANUEL KRAUSKOPF, ALEJANDRO ARAYAAND SIMON LITVAK
or absence of these final residues can be used as a criterion in the analysis of the extent of the intactness of a tRNA preparation, tRNA molecules lacking this 3' terminal residues can be repaired by using a specific enzyme, the CTP (ATP)tRNA nucleotidyltransferase (Carre et al. 1970). When shark liver tRNA was tested for AMP or CMP incorporation into the 3' end using E. coli CCA-enzyme (see Materials & Methods) very little incorporation was observed (Fig. 2). These results may indicate that (a) the tRNA assayed has its 3' end practically intact; (b) the tRNA lacks more than the three terminal o
8
(~J
{ X
0( 20
40
60 Time,
80
I00
120
rain
FIG. 2. Kinetics of °H-AMP or aH-CMP incorporation into shark liver tRNA by the E. coli CTP(ATP)-tRNA nucleofidyhransferase. The conditions were those given under Materials and Methods. Untreated shark liver tRNA as tested with aH-ATP ( 0 - - 0 ) and aH-CTP (•--A). Venom phosphodiesterase treated shark liver tRNA as tested with °H-ATP (O--©), CTP plus aH-ATP (V~--[~) and aH-CTP ( , - - A ) . XMP refers to either °H-AMP or aH-CMP. residues, hence the enzyme is not able to incorporate the nucleotides; and (c) shark liver tRNA is not a suitable substrate for the E. coli enzyme. To distinguish among these three possibilities, shark liver tRNA was partially digested with snake venom phosphodiesterase. As shown in Fig. 2, when the treated tRNA was incubated in the presence of 3H-ATP and the E. coli CCA-enzyme, some incorporation was attained. When cold CTP was added to the same mixture, the transfer of OH-AMP from 3H-ATP into the 3' end of the treated tRNA was greatly stimulated. Repair assays of the partially digested tRNA with 8H-CTP alone resulted in an important 8H-CMP incorporation (Fig. 2). When the digested tRNA was subjected to repair, and was subsequently assayed for 14C-valine acceptance with an aminoacyl-tRNA synthetase, a thirtyfold increase in the amino acid incorporation was obtained with respect to the unrepaired control.