Hoppe-Seyler's Z. Physiol. Chem. Bd. 356, S. 109 -118, Februar 1975
Studies on the Binding of /V-Bromoacetylpuromycin and /V-Bromoacetylaminonucleoside to Rat Liver Ribosomes Michael A. Minks, Mario Ariatti and Arthur O. Hawtrey
(Received 1 October 1974)
Summary: [ H^-Bromoacetylaminonucleoside and [3HyV-bromoacetylpuromycin have been synthesised as possible alkylating agents in order to study their interactions with rat liver ribosomes. Both compounds bind covalently to ribosomes to a considerable extent. The puromycin derivative binds to the extent of approximately 8 mol per ribosome, while the aminonucleoside derivative binds to the extent of approximately 13 mol per ribosome. Ammonium sulphate precipitation of ribosomes or treatment with puromycin, followed by washing of the ribosomes through NH4Cl-containing sucrose density gradients decreases the binding of both derivatives. Partial unfolding or de-
naturation of ribosomes by heating at 65 °C or through the action of various chemical reagents appears to expose more sites for binding. However,at 15 min of heating the binding of the puromycin derivative decreased by approximately 50% while the binding of the aminonucleoside derivative was almost zero. Binding of both labelled derivatives occurred only with the 5OS ribosomal subunit. The extent of binding to the smaller 30S subunit was approximately 4% that of the 50S subunit. Various other experiments are also described dealing with the binding of [3HyV-acetylphenylalanyl-tRNA to the A site of ribosomes following treatment with the TVfa romoacetyl derivatives.
Untersuchung des Bindungsvermögens von N-Bromacetylpuromycin und N-Bromacetylaminonucleosid an Rattenleber-Ribosomen Zusammenfassung: [3H]A^-Bromacetylpuromycin und [3H]Af-Bromacetylaminonucleosid wurden als mögliche Alkylierungsreagentien für das Studium von deren Bindungsvermögen an Rattenleberribosomen hergestellt. Beide Verbindungen bilden mit Ribosomen kovalente Verbindungen; das Puromycinderivat verbindet sich mit den Ribosomen im Verhältnis 8: l und das Aminonucleosidderivat im Verhältnis von ungefähr 13:1. Die Fällung der Ribosomen durch
Ammoniumsulfat oder deren Behandlung mit Puromycin und nachfolgendes Waschen mit Saccharosegradienten vermindert deren Bindungsvermögen. Die partielle Entfaltung oder Denaturierung der Ribosomen durch Erwärmen auf 65 °C oder Behandlung mit verschiedenen Reagentien legt offenbar weitere Bindungstellen frei. Nach 15minütigem Erwärmen vermindert sich jedoch das Bindungsvermögen des Puromycinderivates um ungefähr 50%, während das
Address: M. Minks, Department of Biochemistry, University of Rhodesia, P.O. Box MP. 167, Salisbury, Rhodesia. Abbreviations: Me 2 SO, dimethylsulphoxide; A^-BrAc[3HlPhe-tRNA, [3H]yV-bromoacetylphenylalanyl-tRNA; aminonucleoside (puromycin aminonucleoside), 6-dimethylamino-9-(3-deoxy-3-amino-j3-D-ribofuranosyl)-purine.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
110
M. Minks, M. Ariatti and A. O. Hawtrey
Bd. 356(1975)
Bindungsvermögen des Aminonucleosidderivates allmählich verschwindet. Die Bindung der beiden markierten Verbindungen findet hauptsächlich an der 50S-Ribosomenuntereinheit statt, während das Bindungsvermögen an die kleinere SOSUntereinheit nur 4% des Gesamtbindungsvermö-
gens beträgt. Verschiedene andere Versuche zur Bindung der [3H]-/V-Acetylphenylalanyl-tRNA an die A-Bindungsstelle der Ribosomen und die darauffolgende Behandlung mit TV-Bromacetylderivaten, werden außerdem beschrieben.
In an attempt to study the localisation of proteins in the area of the peptidyl synthetase in E. coli ribosomes, Cantor's group 1 1>2 1 synthesised the peptidyl analog, 7V-bromoacetylphenylalanyltRNA as an alkylating probe. These workers showed that7V-BrAc-[3H]Phe-tRNA reacts covalently with the 5OS ribosomal proteins L2 and L26 - L27, when the bromoacetylated tRNA was bound in the P site of the ribosome. Evidence for P site binding was based upon the ability of the covalently attached tRNA molecule to still participate in dipeptide formation. More recent work by this group^, however, indicates that the covalent attachment of-/V-BrAc-[3H]Phe-tRNA toE.coli ribosomes is in fact more complicated. In the presence of deacylated tRNA, binding occurs predominantly to the A site environment (LI6 protein). Sonnenberg^4! and co-workers have also observed covalent attachment of ./V-bromoacetylchoramphenicol to proteins L2 and L27 of E.coli ribosomes, while Pongs'5'6^ and associates have observed attachment of the same derivative as well as the monoiodo derivative toE.coli ribosomes. These latter alkylating reactions appear to involve mainly the A site of the ribosome. Puromycin is known to react at the A site of E. coli ribosomes and to bind to the 50S subunit^. Similar reactions appear to take place with rat-
liver ribosomesl8'9!. We therefore became interested in synthesising the [3H]./V-bromoacetyl derivatives of puromycin and of aminonucleoside (Scheme, compounds I and II, respectively) in order to study the interaction of those alkylating probes with a eukaryotic ribosomal system, such as rat-liver ribosomes. Experiments in this direction are reported in the present paper.
(III
HOCH,
C=0
CH30
-CH2CHNH-C CH2Br
I ^A/-
0 Scheme. W-Bromoacetylpuromycin (I) and TV-bromoacetylaminonucleoside (II). B stands for the base 6-dimethylaminopurine.
Materials and Methods Chemicals Puromycin dihydrochloride, aminonucleoside and GTP (disodium salt) were obtained from the Sigma Chemical Co., St. Louis, U.S.A. Dimethylsulphoxide, dimethylformamide, triethylamine and TLC plates (Silica gel 60F, 254, with fluorescent indicator) were supplied by E. Merck AG., Darmstadt, Germany. Sodium deoxycholate was purchased from General Biochemicals Inc., Chagrin Falls, Ohio, U.S.A. [ 3 H]Bromoacetic acid (190mCi/mmol), [ 3 H]puromycin-2 HCl(3.7Ci/mmol) and [ 3 H]L-phenylalanine (29Ci/mmol) were supplied by Amersham, England. Phenylalanyl-tRNA and ATP (disodium salt) were obtained from Boehringer Mannheim GmbH. Yeast tRNA was supplied by Schwarz Bio Research Inc., Orangeburg, N.Y., U.S.A. Dicyclohexylcarbodiimide was obtained from Koch Light Laboratories, Colnbrook, Bucks, England. All other reagents were of analytical grade. Preparation of poly somes Polysomes were prepared from rat liver using Medium A [containing (final concentrations); 0.25M sucrose, 5mM Mg(OAc) 2 , 25mM KC1, 50mM Tris/HCl, pH 7.6 and ImM dithiothreitol] according to the method of Wettstein, Staehelin and Nolll 101 with slight modification in that sodium deoxycholate was replaced by a mixture of sodium deoxycholate (final concentration, 0.5% w/v) and Triton X-100 (final concentration, 0.5% w/v). The polysomes were finally suspended in Medium A and stored at -15 °C. Ammonium sulphate precipitated polysomes Ammonium sulphate precipitation of polysomes was carried out by the addition of an equal volume of saturated (NH4)2SO4 (pH 7.6) to polysomes in Medium A. After standing for 10 min, the precipitated polysomes
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
Bd. 356 (1975)
Binding of ./V-Bromoacetylpuromycin to Rat Liver Ribosomes
were centrifuged for 10 min at 2000 rpm. The polysome pellet was taken up in Medium A and dialysed against the same medium for 24 h. Ammonium chloride washed ribosomes Poly some s were incubated in a buffer containing 50mM Tris/HCl (pH 7.6), 25mM KC1, ImM dithiothreitol, 5mM MgCl 2 , 80mM NH4C1 and ImM puromycin,for 30 min at 37 °C (modification of the method of Siler and Moldavel8!)· Thereafter 10-m/ portions of the ribosome suspensions were layered over 25 ml of 0.5M sucrose in Medium A buffer containing 0.5M NH4C1 and lOmM MgCl2 and centrifuged at 99 000-g for 4 h in the Spinco no. 30 rotor. The polysemes were taken up in Medium A and stored at -15 °C. [ 3 H \N-Acetylphenylalanyl-tRNA [ 3 H]L-Phenylalanyl-tRNA was prepared from rat liver pH 5 enzyme as previously described! ll>i2]. (3H]N-Acetylphenylalanyl-tRNA was prepared from [ 3 H]L-phenylalanyl-tRNA by a modification of the method of Pellegrini, Oen and Cantori 11 (Ariatti and Hawtrey, manuscript in preparation) using ./V-hydroxysuccinimide activation of acetic acid. The final acetylated product gave a 260/280 ratio of 1.8 and 38· 104 cpm/100Mg of tRNA. It was stored in small portions at -15 °C. Translocation (G factor) This was prepared from a sample of rat liver pH 5 supernatant according to the method described by Felicetti and Limpanm 131. Purification was taken as far as the calci in phosphate gel stage. Binding of labelled N-bromoacetyl derivatives to ribosomes Binding of labelled TV-bromoacetyl derivatives to ribosomes was carried out in Medium A buffer containing 10% (v/v) dime thy Isulphoxide for various times at 37 °C. Reactions were stopped with cold 5% trichloroacetic acid. The reaction tubes were heated at 90 °C for 25 min (covalent binding to protein), cooled, and the precipitates collected on GF/C (Whatman) filters, followed by washing with 25 ml of 5 % trichloroacetic acid. After drying, samples were counted in a Scintillation Spectrometer. Labelled ribosomal RNA was extracted by the sodium dodecylsulphate/phenol method, followed by ethanol precipitation and exhaustive dialysis against distilled water. [ 3H\ N-Bromoacetylaminonucleoside To a solution of aminonucleoside (10 mg, 34 μπιοί) in 0.1 m/ of dry Me2SO was added a mixture of unlabelled bromoacetic acid (12 mg, 85 μπιοί), [3H]bromoacetic acid (1.05 mg, 8 μπιοί) and dicyclohexylcarbodiimide (10 mg, 49 μιτιοί) in 0.2 ml of dry Me 2 SO. After standing at room temperature for 30 min, dicyclohexylurea
111
was removed by filtration, and the solvent removed under vacuum at 37 °C. The residue was extracted twice with dry ether (2· 10 ml) to remove excess bromoacetic acid. Final traces of dicyclohexylurea were removed by dissolving the crude product in dioxane at 60 °C, from which the urea crystallized on cooling. Dioxane was removed under vacuum at 37 °C, and the residue subjected to thin-layer chromatography (Silica Gel 60F2S4, 10-20 cm plates) using methanol/chloroform 1:9 (v/v). The appropriate band was eluted with methanol/chloroform 1:1, (v/v) (3-10 m/). Recrystallisation from dry ethanol gave 12 mg of product (85% yield) in two crops, m.p., 202-204 °C. UV max (in dioxane), 276 nm (log e, 4.45). Thin-layer chromatography on Silica Gel 60 F 254 gave RF0.45, methanol/chloroform 1:9, (v/v). Radioactivity, 9.1· 106 cpm/μηιοΐ. C 14 H 19 04N 6 Br (415.3); mass spectrum, M®, 414, 416 (bromine isotopes). [3H\N-Bromoacetylpuromycin To a solution of puromycin dihydrochloride (12 mg, 22 μπιοί) in 0.2 ml Me2SO was added three drops of trie thy lamine. Triethylamine-HCl was removed by filtration and the filtrate co-evaporated twice with pyridine under vacuum. To the residue was added [3H]bromoacetic acid (3.5 mg, 25 μΐηοΐ), bromoacetic acid (3.5 mg, 25 μπιοί) and dicyclohexylcarbodiimide (12.5 mg, 60 μπιοί) in 0.45 ml of Me2SO. After standing at room temperature for 5 h, the desired product was isolated by the method described for the aminonucleoside (see above), m.p., 185-187 °C. UV max (ethanol), 275 nm (log e, 4.27). RF, 0.50, Silica Gel 60F 2S4 , solvent methanol/chloroform 1:9, (v/v). Radioactivity, 6·107 cpm/μιτιοΐ. C24H30N706Br (592.5) Calc. C 48.66 H 5.10 Ν 16.55 Found C 49.67 H 5.28 Ν 16.63 Mass spectrum, Me-HBr, 511. Counting of radioactive samples Ribosomal samples which had been precipitated with trichloroacetic acid and filtered on GF/C (Whatman) filters were counted with a solution containing 0.5% (w/v) 2,5-diphenyloxazole and 0.03% (w/v) 1,4-bis(5-phenyl-2-oxazolyl)benzene in chromatography-grade toluene. In experiments concerned with the interaction of [ 3 H]puromycin with unlabelled peptide chains on ribosomes, samples were counted in Bray's solution. Estimation of protein and RNA These were determined as previously described by Hawtrey, Schirren and Dijkstrai 141. ^4 2 6o units were determined by UV absorption measurement at 260 nm on a portion of the sample under test. One /4 26 Q unit is that amount of material in 1 ml of solution giving an optical density at 260 nm of 1.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
112
M. Minks, M. Ariatti and A. O. Hawtrey
Results 3
The covalent binding of [ HyV-bromoacetylpuromycin and [3H]7V-bromoacetylaminonucleoside to rat liver ribosomes was initially studied. Results presented in Fig. 1 illustrate the time course of binding of both derivatives to polysomes and (NH4)2S04-precipitated poly somes at different incubation temperatures. Fig. la illustrates the binding or attachment of [3H]7V-bromoacetylaminonucleoside and Fig. Ib the binding of [3H]^V-bromoacetylpuromycin. The number of moles ofAM>romoacetylpuromycin bound per mole of ribosome was found to be approximately 1, while with the aminonucleoside derivative the value was 9. Ammonium sulphate precipitation of polysomes appears to decrease to some extent their ability to bind theAM>romoacetyl derivati-
Bd. 356 (1975)
The results of the effect of increasing concentrations of [3HyV-bromoacetylaminonucleoside and [3H]7V-bromoacetylpuromycin on their binding to polysomes is shown in Fig. 2. The aminonucleoside derivative bound to the extent of 13 moles per ribosome, while the puromycin derivative bound to the extent of 8 moles per ribosome. Binding of the latter derivative shows a fairly linear increase with increasing concentration. It was of considerable interest to determine whether the binding of theTV-bromoacetyl derivatives to ribosomes was dependent upon the native structure of the ribosomal particles or not. For this purpose, the effects of heat treatment and fairly high concentrations of different chemicals on the subsequent ability of ribosomes to bind the./V-bromoacetyl derivatives was studied. Heat treatment of ribosomes at 65 °C for various times, followed by incubation of the heated ribo-
Fig. 1. Rate of binding of (a), [3H]W-bromoacetylaminonucleoside and (b), [ 3 H]7V-bromoacetylpuromycin to polysomes at different temperatures. Incubation tubes contained 5 /4260 units of polysomes plus 5 nmol of the respective labelled bromoderivative in 0.1 ml of Medium A containing 10% (v/v) Me2SO. Tubes were processed for counting as described in the Materials and Methods Section. (·), polysomes at 37 °C; (o) (NH4)2SO4-precipitated polysomes at 37 °C; («), polysomes at 20 °C; (±), (NH4)2SO4precipitated polysomes at 20 °C; (o), polysomes at 0 °C.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
113
Binding of 7V-Bromoacetylpuromy ein to Rat Liver Ribosomes
Bd. 356 (1975)
50
WO 150 cljumol/ll —*
200
250
Fig. 2. The effect of increasing concentrations of ^HJjV-bromoacetylaminonucleoside (·) and [ 3 H]./V-bromoacetylpuromycin (o) on thek covalent binding to polysomes. Incubations were as described for Fig. 1 (2 h at 37 °C).
somes with the ^-bromoacetyl derivatives for l h at 37 °C indicates (Fig. 3) that initially as the ribosomes unfold more sites become available for covalent binding, which, however, is then followed by a decrease in binding at the longer times of heating. Treatment of ribosomes for 16 min at 65 °C completely destroys their ability to interact with the TV-bromoacetyl aminonucleoside.Table 1 shows the results of preincubating ribosomes with methanol, urea and EDTA on their subsequent binding of the labelled A^bromoacetyl derivatives. All three reagents bring about an increase in the binding of [3H]/V-bromoacetylpuromycin suggesting that unfolding or partial denaturation of the ribosome exposes or makes available more sites for binding. These results are thus similar to those obtained in the heating experiment (Fig. 3). All incubations contained 10% (v/v) dimethylsulphoxide (Me2 SO) in order to keep the bromo derivatives in solution. A check was made therefore on the possible effects of this chemical on ribosome structure and function. Dimethylsulphoxide (10% v/v) in incubation mixtures had no effect on polysome profiles as evidenced by sucrose density-gradient centrifugation (Figs. 4 and 5). Further, no inhibitory effects were observed in the reaction of [3H]W-
Table 1. The effect of different chemical reagents on the binding of [3H]A^-bromoacetylpuromycin to ribosomes. Reaction mixtures contained: polysomes (5 A2(>o units), Medium A buffer, 10% (v/v) Me2SO, [3H]Nbromoacetylpuromycin (5 nmol) in a final volume of 0.1 ml, with or without reagents shown in the table. Incubations were carried out for 60 min at 37 °C. Samples were cooled in ice and processed as described in the Materials and Methods Section. System
[3H)^-bromoacetylpuromycin bound [cpm/5^260 units]
Polysomes (control) Polysomes + 20% (v/v) MeOH Polysomes + IM urea Polysomes + 2M urea Polysomes + 20mM Mg(OAc)2 Polysomes + lOmM EDTA
1630 2110 2290 5671 1738 9599
11 mini —»·Fig. 3. Effect of heat treatment of polysomes at 65 °C for various times on their subsequent binding of the bromoacetyl derivatives. Polysomes were heated at 65 °C for various times, cooled in ice, and then incubated for l h at 37 °C with the labelled compounds as described for Fig. 1. (·), Binding of [ 3 H]A^-bromoacetylpuromycin; (o), binding of [3H],/V-bromoacetylaminonucleoside.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
114
M. Minks, M. Ariatti and A. O. Hawtrey
acetylphenylalanyl-tRNA bound to washed ribosomes with puromycin. Protein synthesis in a complete system using 14C-labelled yeast protein hydrolysis was also not affected (unpublished results of M. Ariatti and A. 0.Hawtrey). Having shown that [3H]jY-bromoacetylpuromycin and [3HyV-bromoacetylaminonucleoside bind to rat liver ribosomes, it was of considerable interest to determine if these substances bound to either the 5OS or 3OS ribosomal subunit or to both subunits. Ribosomes were therefore labelled with theJV-bromoacetyl derivatives for l h at
Bd. 356(1975)
0.8 0.6 O.I. 02
A
0.6·
10
15
20
25
Fract. no. —^-
Fig. 4. Effect of dimethylsulphoxide on polysome profiles. Polysemes were preincubated with and without 10% (v/v) Me2SO for 2 h at 0 °C. They were then subjected to separation by means of centrifugation through a 15 - 35% (w/v) linear sucrose gradient (S.W. 25 rotor, 90 000·,§· for 4 h at 4 °C). (a), Control poly somes; (b), Polysomes plus 10% (v/v) Me2SO.
Fig. 5. The effect of preincubating (NH4)2SO4-precipitated polysomes with ./V-bromoacetyl derivatives as evidenced by centrifugation through linear sucrose gradients. Polysomes with or without the ./V-bromoacetyl derivatives but containing 10% Me 2 SO were preincubated for 2 h at 37 °C. They were then subjected to centrifugation through 15 - 35% (w/v) linear sucrose gradients (S.W. 25 rotor, 90000·^ for 5 h at 4 °C). (a), Control polysomes; (b) polysomes preincubated with ./V-bromoacetylpuromycin (5·10~5Μ); (c) polysomes preincubated with 7V-bromoacetylaminonucleoside (5-10~ 5 M).
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
Bd. 356 (1975)
Binding of W-Bromoacetylpuromycin to Rat Liver Ribosomes
5
W
15
20
25
5
W
15
20
115
25
Fig. 6. Distribution of (a), [3H]JV-bromoacetylaminonucleoside and (b), [3H]./V-bromoacetylpuromycin on rat liver ribosomal subunits following sucrose density-gradient centrifugation. Polysomes (25 Λ 2 60 units) were incubated with the labelled compounds (25 nmol) for l h at 37 °C. After cooling in ice, the labelled poly somes were washed by means of discontinuous sucrose gradient centrifugation. They were then dissociated into subunits by treatment with 5mM EDTA. Separation of subunits was carried out using a linear 10 - 30% (w/v) sucrose gradient containing lOmM Tris/HCl; pH 7.6, 25mM KC1 and ImM EDTA in the S.W. 25 -t rotor (22000 rpm for 16 h at 5 °C). AI^Q was determined and samples counted in Bray's solution. (—), (·), cpm.
37 °C, and then washed by centrifugation through a discontinuous sucrose gradient. The labelledwashed ribosomes were dissociated into subunits with EDTA and separated from each other by sucrose density-gradient centrifugation. Results presented in Figs.oa and 6b, respectively, show that binding of both labelled TV-bromoacetyl derivatives occurs mainly on the 50S ribosomal subunit. The extent of binding of both compounds to the 30S subunit was negligible, amounting to approximately 4% of that bound to the 50S subunit. Theoretically, binding of the labelled W-bromoacetyl derivatives to ribosomes can occur through attachment to protein or to RNA or both. Experiments (results not shown) have indicated that considerable binding occurs with the 50S subunit proteins. However, it was also found that binding of the labelled derivatives took place with ribosomal RNA. Careful extraction of RNA from labelled ribosomes by the phenol/sodium dodecylsulphate method at different times of incubation indicated a rapid rate of binding up to l h followed by a decrease in binding up to 2 h (Fig.7).
Fig. 7. Time course of binding of [3H]7V-bromoacetylpuromycin to rat liver ribosomal RNA. Polysomes (48 ^260 units) were incubated in Medium A containing 10% (v/v) Me2SO with 40 nmol of the labelled compound for various times at 37 °C. RNA was extracted as described in the Materials and Methods Section. Counting in Bray's solution. (·), cpm/5 A26Q units of RNA.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
116
M. Minks, M. Ariatti and A. O. Hawtrey
Reasons for the subsequent decrease in binding are not known. In vitro experiments with various radioactively labelled puromycin and aminonucleoside derivatives incubated with purified yeast tRNA gave no evidence of binding to the tRNA (Table 2). It is generally assumed that puromycin interacts with peptidyl-tRNA already in the P (peptidyl) site on the ribosome. One would assume therefore that the puromycin molecule could interact or bind possibly in an area of the ribosome around or close to the A (aminoacyl) site for its subsequent reaction with peptidyl-tRNA. One would also expect theTV-bromoacetylated puromycin and aminonucleoside derivatives possibly to interact with ribosomes in the same area (A site). If this were so, prior incubation of washed ribosomes with theTV-bromoacetylated derivatives might possibly interfere with the subsequent binding of [3H]W-acetylphenylalanyl-tRNA to the A site of the ribosome. Experiments to test this idea were therefore carried out.
Table 2. Binding of [ 3 H] puromycin and related compounds to tRNA in vitro. Each incubation tube contained yeast tRNA (1 mg), 50 mM Tris/HCl pH 7.6, 25mM KC1, 5mM MgCl2, 10% Me 2 SO (v/v) and the appropriate labelled compound in a final volume of 1.0 m/. Solutions were incubated for 3 h at 37 °C, cooled in ice, and then mixed with 0.1 ml of 20% (w/v) potassium acetate. Cold ethanol (2.5 m/) was added and the mixture kept at 4 °C overnight. The resulting RNA precipitate was recovered by low-speed centrifugation (2000 rpm/5 min), dissolved in 1 ml water and reprecipitated as described above. The final precipitate was washed twice with ethanol by low-speed centrifugation and dissolved in water (1 ml). Portions were taken for ^260 measurements and radioactive counting (Instagel). System
Bromoacetic acid Puromycin ./V-Acetylaminonucleoside Λ^-Acetylpuromycin Af-Bromoacetylaminonucleoside ./V-Bromoacetylpuromycin
nmol of 3H-labelled compound bound/nmol of tRNA 5.28-10-1 3.28-10-4 1.11·10~2
i.oo-io-
2
2.03-10-2 4.05-10-3
Bd. 356 (1975)
In the first experiments, washed ribosomes were incubated with the unlabelledAf-bromoacetyl derivatives (concentration, 5· 10~ 5 M) for 2 h at 37 °C. Thereafter, the reaction mixtures were directly interacted with [3H]./V-acetyrphenylalanyl-tRNA in a complete binding system. Results presented in Table 3 show that bothN-bromoacetyl derivatives had little effect on the non-enzymatic binding of the labelled aminoacyltRNA to the A site of the ribosome. In a further series of experiments, washed ribosomes were incubated with unlabelled N-bromoacetylpuromycin at three different concentrations for 2 h at 37 °C, and then purified by washing through discontinuous sucrose gradients. The washed ribosomes were then incubated with
Table 3. Binding of [3H]^V-acetylphenylalanyl-tRNA to washed rat liver ribosomes previously treated with jV-bromoacetylpuromycin and ./V-bromoacetylaminonucleoside. Polysomes were previously treated with puromycin and washed through discontinuous sucrose gradients containing 0.5M NH4C1 (washed ribosomes); see Materials and Methods Section. Washed ribosomes (5 Λ2 60 units) in 0.1 ml of Medium A containing 10% (w/v) Me2SO were incubated with the jV-bromoacetylated derivatives (final concentration 0.05mM) for 2 h at 37 °C. To the incubation tubes various reagents were then (directly) added (final concentrations), 50mM Tris/HCl, pH 8.0, 20mM Mg(OAc) 2 , 60mM KC1, 20mM NH4C1, ImM mercaptoethanol, 30 Mg poly(U) and 15 Mg [3H]JV-acetylphenylalanyl-tRNA (5.3Ί04 cpm). Final volume, 0.25 ml. All reaction tubes were incubated for 30 min at 37 °C and then cooled in ice. Samples were filtered through Millipore filters and washed with 20 ml of cold Nirenberg buffer (lOOmM Tris/HCl, pH 7.6, 20mM Mg(OAc) 2 and 50mM KC1). After drying, samples were counted as previously described. System
Ribosomes (control) Ribosomes C/V-BrAc-puromycin) Ribosomes C/V-BrAc-aminonucleoside)
[3H]^-Acetylphenylalanyl tRNA bound [cpm/5^260 u n i t s l 8068
6321 7853
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
Bd. 356 (1975)
Binding of W-Bromoacetylpuromycin to Rat Liver Ribosomes
Table 4. Binding of [3H]7V-acetylphenylalanyl-tRNA to washed rat liver ribosomes following previous treatment with W-bromoacetylpuromycin and centrifugation through discontinuous sucrose gradients. Washed ribosomes after incubation with JV-bromoacetylpuromycin for 2 h at 37 °C were centrifuged through a discontinuous sucrose gradient containing 0.5M sucrose in Medium A at 215 000 ·£ for 90 min (No. 50 rotor). The pellets were taken up in Medium A and treated exactly as described for Table 3 for the binding of labelled tRNA. System
Ribosomes (control) Ribosomes (JV-BrAc-puromycin O.OSmM) Ribosomes C/V-Br-Ac-puromycin 0.1 5mM) Ribosomes (W-BrAc-puromycin 0.25mM)
[3H]^-Acetylphenylalanyl-tRNA bound [cpm/1.0 ^260 units l
117
indicate that the process of binding is not specific with regard to the number of moles of compound bound per ribosome. The puromycin derivative binds to the extent of 8 moles per ribosome, while the aminonucleoside derivative binds to the extent of approximately 13 moles per ribosome. Partial unfolding or denaturation of ribosomes by heating at 65 °C for short periods of time or through the action of urea, methanol and EDTA bring about a considerable increase in binding of both derivatives, suggesting that as the ribosome structure unfolds, more sites become available for interaction and attach-
1163 1150 1143
I 6·
1350
[3H]^Vr-acetylphenylalanyl-tRNA in a complete binding system. Results in Table 4 again show that prior incubation of washed ribosomes with JV-bromoacetylated derivatives had little effect on their subsequent non-enzymatic binding of the labelled tRNA to the A site of the ribosome. The third type of experiment involved the interaction of polysomes (not washed) with the unlabelled TV-bromoacetylated derivatives for 2 h at 37 °C, followed by washing of the polysomes through discontinuous sucrose gradients. The washed polysomes were then incubated with [3H]puromycin in order to measure the degree of reaction with unlabelled peptide chains in the P site on the ribosome. Results presented in Fig. 8 indicate very little difference in the interaction of [3H]puromycin with peptide chains, following treatment with the TV-bromoacetylated derivatives. Discussion
The present experiments on the binding of [ 3 H]-/V-bromoacetylpuromycin and [ HJ/V-bromoacetylaminonucleoside to rat liver ribosomes
Fig. 8. Time course of the interaction of [3H]puromycin with peptide chains on rat liver polysomes after previous treatment of polysomes with ./V-bromoacetylated derivatives. Polysomes in Medium A containing 10% (w/v) Me2SO were incubated with the W-bromoacetylated derivatives (0.05mM) for 2 h at 37 °C. The reacted polysomes were then washed by discontinuous sucrose-gradient centrifugation as described for Table 4. Pellets were resuspended in Medium A (1 m/, 40/4260 units), and incubated with [3H]puromycin (1.25 μΟ) at 37 °C. Samples (0.2 m/) were withdrawn at various times and injected into a solution (0.2 ml of 20% trichloroacetic acid) containing unlabelled puromycin (0.27 mg). After standing in ice for 15 min, the precipitates were collected on Whatman GF/C filters and washed with 20 ml of cold 5% trichloroacetic acid by 20 ml of 96% ethanol. After drying, samples were counted as described in the Materials and Methods Section. (A), Control polysomes; (o), ./V-bromoacetylpuromycintreated polysomes; (·), Λ^-bromoacetylaminonucleoside-treated polysomes.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
118
M. Minks, M. Ariatti and A. O. Hawtrey
ment. The only specific process in the alkylation reactions of both derivatives appears to be their interaction with the 50S ribosomal subunit. In this respect the 5 OS subunit involvement is similar to that observed by other workers using #-BrAc-[3H]-Phe-tRNA11-31 and JV-bromoacetylchloramphenicol^ 4 ~ 6 ',even though the binding interactions of these substances are obviously different. Puromycin, because of its properties as a donor substrate in the peptidyl transferase re action I 7 " 9 ', presumably interacts and binds (weakly) to the A site of ribosomes. It was of interest therefore to observe that prior incubation of washed ribosomes with theTV-bromoacetyl derivatives had little or no effect on the subsequent binding of [3HyV-acetylphenylalanyl-tRNA to the A site. This result may support the ideas of Raacke1151, who on the basis of model building suggested that puromycin interacts with peptidyl-tRNA in the P site through a backside attack on the activated ester grouping of the peptidyl-tRNA, and therefore does not interact or bind directly to the A site. However, binding or attachment of puromycin presumably occurs in close vicinity to both the A and P sites of the ribosome. The finding that [3H]7V-bromoacetylpuromycin also binds to rat liver ribosomal RNA is of interest. Recently, Symons and co-workers^16' have reported on the interaction of the puromycin analogue, 5'-0-(7V-bromoacetyl-p -aminophenylphosphono)-3'^-L-phenylalanylaminonucleoside withJS'.co// ribosomes. These workers showed that this affinity label specifically interacts with ribosomal RNA of the 50S subunit. However, the binding of alkylating probes to RNA should be viewed with caution, in that proteins still firmly attached to RNA might be
Bd. 356 (1975)
responsible for the binding observed. Further experiments are in progress studying the 50S subunit proteins involved in binding and also the nature of the binding occurring with ribosomal RNA. Literature 1 Pellegrini, M., Oen, H. & Cantor, C. (1972) Proc. Nat. Acad. Sei. U.S.A. 69, 837 - 841. 2 Oen, H., Pellegrini, M., Eilat, D. & Cantor, C. (1973) Proc. Nat. Acad. Sei. U.S.A. 70, 2799 - 2803. 3 Eilat, D., Pellegrini, M., Oen, H., De Groot, N., Lapidot, Y. & Cantor, C. (1974) Nature (London) 250,514-516. 4 Sonnenberg, N., Wüchek, M. & Zamir, A. (1973) Proc. Nat. Acad. Sei. U.S.A. 70, 1423 - 1426. 5 Bald, R., Erdmann, V. A. & Pongs, O. (1972) FEBS Lett. 28, 149 -152. 6 Pongs, 0., Bald, R. & Erdmann, V. A. (1973) Proc. Nat. Acad. Sei. U.S.A. 70, 2229 - 2233. 7 Pestka, S. (1970) Arch. Biochem. Biophys. 136, 80-88. 8 Siler, J. & Moldave, K. (1969) Biochim. Biophys. Acta 195, 123 -129. 9 Siler, J. & Moldave, K. (1969) Biochim. Biophys. Acta 195, 130-137. 10 Wettstein, F. O., Staehelin, T. & Noll, . (1963) Nature (London) 197,430 - 435. 11 Hawtrey, A. O. (1965) 5. Afr. J. Med. Sei. 30, 100- 104. 12 Herrington, M. D. & Hawtrey, A. O. (1971) Biochem. J. 121,279-285. 13 Felicetti, L. & Lipmann, F. (1968) Arch. Biochem. Biophys. 125, 548 - 557. 14 Hawtrey, A. 0., Schirren, V. & Dijkstra, J. (1963) Biochem. J. 88, 106-114. 15 Raacke, I. D. (1971) Biochem. Biophys. Res. Commun. 43,168 - 173. 16 Harris, R.J., Greenwell, P. & Symons, R. H. (1973) Biochem. Biophys. Res. Commun. 55, 117 -124.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
Studies on the Binding of /V-Bromoacetylpuromycin and /V-Bromoacetylaminonucleoside to Rat Liver Ribosomes Michael A. Minks, Mario Ariatti and Arthur O. Hawtrey
(Received 1 October 1974)
Summary: [ H^-Bromoacetylaminonucleoside and [3HyV-bromoacetylpuromycin have been synthesised as possible alkylating agents in order to study their interactions with rat liver ribosomes. Both compounds bind covalently to ribosomes to a considerable extent. The puromycin derivative binds to the extent of approximately 8 mol per ribosome, while the aminonucleoside derivative binds to the extent of approximately 13 mol per ribosome. Ammonium sulphate precipitation of ribosomes or treatment with puromycin, followed by washing of the ribosomes through NH4Cl-containing sucrose density gradients decreases the binding of both derivatives. Partial unfolding or de-
naturation of ribosomes by heating at 65 °C or through the action of various chemical reagents appears to expose more sites for binding. However,at 15 min of heating the binding of the puromycin derivative decreased by approximately 50% while the binding of the aminonucleoside derivative was almost zero. Binding of both labelled derivatives occurred only with the 5OS ribosomal subunit. The extent of binding to the smaller 30S subunit was approximately 4% that of the 50S subunit. Various other experiments are also described dealing with the binding of [3HyV-acetylphenylalanyl-tRNA to the A site of ribosomes following treatment with the TVfa romoacetyl derivatives.
Untersuchung des Bindungsvermögens von N-Bromacetylpuromycin und N-Bromacetylaminonucleosid an Rattenleber-Ribosomen Zusammenfassung: [3H]A^-Bromacetylpuromycin und [3H]Af-Bromacetylaminonucleosid wurden als mögliche Alkylierungsreagentien für das Studium von deren Bindungsvermögen an Rattenleberribosomen hergestellt. Beide Verbindungen bilden mit Ribosomen kovalente Verbindungen; das Puromycinderivat verbindet sich mit den Ribosomen im Verhältnis 8: l und das Aminonucleosidderivat im Verhältnis von ungefähr 13:1. Die Fällung der Ribosomen durch
Ammoniumsulfat oder deren Behandlung mit Puromycin und nachfolgendes Waschen mit Saccharosegradienten vermindert deren Bindungsvermögen. Die partielle Entfaltung oder Denaturierung der Ribosomen durch Erwärmen auf 65 °C oder Behandlung mit verschiedenen Reagentien legt offenbar weitere Bindungstellen frei. Nach 15minütigem Erwärmen vermindert sich jedoch das Bindungsvermögen des Puromycinderivates um ungefähr 50%, während das
Address: M. Minks, Department of Biochemistry, University of Rhodesia, P.O. Box MP. 167, Salisbury, Rhodesia. Abbreviations: Me 2 SO, dimethylsulphoxide; A^-BrAc[3HlPhe-tRNA, [3H]yV-bromoacetylphenylalanyl-tRNA; aminonucleoside (puromycin aminonucleoside), 6-dimethylamino-9-(3-deoxy-3-amino-j3-D-ribofuranosyl)-purine.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
110
M. Minks, M. Ariatti and A. O. Hawtrey
Bd. 356(1975)
Bindungsvermögen des Aminonucleosidderivates allmählich verschwindet. Die Bindung der beiden markierten Verbindungen findet hauptsächlich an der 50S-Ribosomenuntereinheit statt, während das Bindungsvermögen an die kleinere SOSUntereinheit nur 4% des Gesamtbindungsvermö-
gens beträgt. Verschiedene andere Versuche zur Bindung der [3H]-/V-Acetylphenylalanyl-tRNA an die A-Bindungsstelle der Ribosomen und die darauffolgende Behandlung mit TV-Bromacetylderivaten, werden außerdem beschrieben.
In an attempt to study the localisation of proteins in the area of the peptidyl synthetase in E. coli ribosomes, Cantor's group 1 1>2 1 synthesised the peptidyl analog, 7V-bromoacetylphenylalanyltRNA as an alkylating probe. These workers showed that7V-BrAc-[3H]Phe-tRNA reacts covalently with the 5OS ribosomal proteins L2 and L26 - L27, when the bromoacetylated tRNA was bound in the P site of the ribosome. Evidence for P site binding was based upon the ability of the covalently attached tRNA molecule to still participate in dipeptide formation. More recent work by this group^, however, indicates that the covalent attachment of-/V-BrAc-[3H]Phe-tRNA toE.coli ribosomes is in fact more complicated. In the presence of deacylated tRNA, binding occurs predominantly to the A site environment (LI6 protein). Sonnenberg^4! and co-workers have also observed covalent attachment of ./V-bromoacetylchoramphenicol to proteins L2 and L27 of E.coli ribosomes, while Pongs'5'6^ and associates have observed attachment of the same derivative as well as the monoiodo derivative toE.coli ribosomes. These latter alkylating reactions appear to involve mainly the A site of the ribosome. Puromycin is known to react at the A site of E. coli ribosomes and to bind to the 50S subunit^. Similar reactions appear to take place with rat-
liver ribosomesl8'9!. We therefore became interested in synthesising the [3H]./V-bromoacetyl derivatives of puromycin and of aminonucleoside (Scheme, compounds I and II, respectively) in order to study the interaction of those alkylating probes with a eukaryotic ribosomal system, such as rat-liver ribosomes. Experiments in this direction are reported in the present paper.
(III
HOCH,
C=0
CH30
-CH2CHNH-C CH2Br
I ^A/-
0 Scheme. W-Bromoacetylpuromycin (I) and TV-bromoacetylaminonucleoside (II). B stands for the base 6-dimethylaminopurine.
Materials and Methods Chemicals Puromycin dihydrochloride, aminonucleoside and GTP (disodium salt) were obtained from the Sigma Chemical Co., St. Louis, U.S.A. Dimethylsulphoxide, dimethylformamide, triethylamine and TLC plates (Silica gel 60F, 254, with fluorescent indicator) were supplied by E. Merck AG., Darmstadt, Germany. Sodium deoxycholate was purchased from General Biochemicals Inc., Chagrin Falls, Ohio, U.S.A. [ 3 H]Bromoacetic acid (190mCi/mmol), [ 3 H]puromycin-2 HCl(3.7Ci/mmol) and [ 3 H]L-phenylalanine (29Ci/mmol) were supplied by Amersham, England. Phenylalanyl-tRNA and ATP (disodium salt) were obtained from Boehringer Mannheim GmbH. Yeast tRNA was supplied by Schwarz Bio Research Inc., Orangeburg, N.Y., U.S.A. Dicyclohexylcarbodiimide was obtained from Koch Light Laboratories, Colnbrook, Bucks, England. All other reagents were of analytical grade. Preparation of poly somes Polysomes were prepared from rat liver using Medium A [containing (final concentrations); 0.25M sucrose, 5mM Mg(OAc) 2 , 25mM KC1, 50mM Tris/HCl, pH 7.6 and ImM dithiothreitol] according to the method of Wettstein, Staehelin and Nolll 101 with slight modification in that sodium deoxycholate was replaced by a mixture of sodium deoxycholate (final concentration, 0.5% w/v) and Triton X-100 (final concentration, 0.5% w/v). The polysomes were finally suspended in Medium A and stored at -15 °C. Ammonium sulphate precipitated polysomes Ammonium sulphate precipitation of polysomes was carried out by the addition of an equal volume of saturated (NH4)2SO4 (pH 7.6) to polysomes in Medium A. After standing for 10 min, the precipitated polysomes
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
Bd. 356 (1975)
Binding of ./V-Bromoacetylpuromycin to Rat Liver Ribosomes
were centrifuged for 10 min at 2000 rpm. The polysome pellet was taken up in Medium A and dialysed against the same medium for 24 h. Ammonium chloride washed ribosomes Poly some s were incubated in a buffer containing 50mM Tris/HCl (pH 7.6), 25mM KC1, ImM dithiothreitol, 5mM MgCl 2 , 80mM NH4C1 and ImM puromycin,for 30 min at 37 °C (modification of the method of Siler and Moldavel8!)· Thereafter 10-m/ portions of the ribosome suspensions were layered over 25 ml of 0.5M sucrose in Medium A buffer containing 0.5M NH4C1 and lOmM MgCl2 and centrifuged at 99 000-g for 4 h in the Spinco no. 30 rotor. The polysemes were taken up in Medium A and stored at -15 °C. [ 3 H \N-Acetylphenylalanyl-tRNA [ 3 H]L-Phenylalanyl-tRNA was prepared from rat liver pH 5 enzyme as previously described! ll>i2]. (3H]N-Acetylphenylalanyl-tRNA was prepared from [ 3 H]L-phenylalanyl-tRNA by a modification of the method of Pellegrini, Oen and Cantori 11 (Ariatti and Hawtrey, manuscript in preparation) using ./V-hydroxysuccinimide activation of acetic acid. The final acetylated product gave a 260/280 ratio of 1.8 and 38· 104 cpm/100Mg of tRNA. It was stored in small portions at -15 °C. Translocation (G factor) This was prepared from a sample of rat liver pH 5 supernatant according to the method described by Felicetti and Limpanm 131. Purification was taken as far as the calci in phosphate gel stage. Binding of labelled N-bromoacetyl derivatives to ribosomes Binding of labelled TV-bromoacetyl derivatives to ribosomes was carried out in Medium A buffer containing 10% (v/v) dime thy Isulphoxide for various times at 37 °C. Reactions were stopped with cold 5% trichloroacetic acid. The reaction tubes were heated at 90 °C for 25 min (covalent binding to protein), cooled, and the precipitates collected on GF/C (Whatman) filters, followed by washing with 25 ml of 5 % trichloroacetic acid. After drying, samples were counted in a Scintillation Spectrometer. Labelled ribosomal RNA was extracted by the sodium dodecylsulphate/phenol method, followed by ethanol precipitation and exhaustive dialysis against distilled water. [ 3H\ N-Bromoacetylaminonucleoside To a solution of aminonucleoside (10 mg, 34 μπιοί) in 0.1 m/ of dry Me2SO was added a mixture of unlabelled bromoacetic acid (12 mg, 85 μπιοί), [3H]bromoacetic acid (1.05 mg, 8 μπιοί) and dicyclohexylcarbodiimide (10 mg, 49 μιτιοί) in 0.2 ml of dry Me 2 SO. After standing at room temperature for 30 min, dicyclohexylurea
111
was removed by filtration, and the solvent removed under vacuum at 37 °C. The residue was extracted twice with dry ether (2· 10 ml) to remove excess bromoacetic acid. Final traces of dicyclohexylurea were removed by dissolving the crude product in dioxane at 60 °C, from which the urea crystallized on cooling. Dioxane was removed under vacuum at 37 °C, and the residue subjected to thin-layer chromatography (Silica Gel 60F2S4, 10-20 cm plates) using methanol/chloroform 1:9 (v/v). The appropriate band was eluted with methanol/chloroform 1:1, (v/v) (3-10 m/). Recrystallisation from dry ethanol gave 12 mg of product (85% yield) in two crops, m.p., 202-204 °C. UV max (in dioxane), 276 nm (log e, 4.45). Thin-layer chromatography on Silica Gel 60 F 254 gave RF0.45, methanol/chloroform 1:9, (v/v). Radioactivity, 9.1· 106 cpm/μηιοΐ. C 14 H 19 04N 6 Br (415.3); mass spectrum, M®, 414, 416 (bromine isotopes). [3H\N-Bromoacetylpuromycin To a solution of puromycin dihydrochloride (12 mg, 22 μπιοί) in 0.2 ml Me2SO was added three drops of trie thy lamine. Triethylamine-HCl was removed by filtration and the filtrate co-evaporated twice with pyridine under vacuum. To the residue was added [3H]bromoacetic acid (3.5 mg, 25 μΐηοΐ), bromoacetic acid (3.5 mg, 25 μπιοί) and dicyclohexylcarbodiimide (12.5 mg, 60 μπιοί) in 0.45 ml of Me2SO. After standing at room temperature for 5 h, the desired product was isolated by the method described for the aminonucleoside (see above), m.p., 185-187 °C. UV max (ethanol), 275 nm (log e, 4.27). RF, 0.50, Silica Gel 60F 2S4 , solvent methanol/chloroform 1:9, (v/v). Radioactivity, 6·107 cpm/μιτιοΐ. C24H30N706Br (592.5) Calc. C 48.66 H 5.10 Ν 16.55 Found C 49.67 H 5.28 Ν 16.63 Mass spectrum, Me-HBr, 511. Counting of radioactive samples Ribosomal samples which had been precipitated with trichloroacetic acid and filtered on GF/C (Whatman) filters were counted with a solution containing 0.5% (w/v) 2,5-diphenyloxazole and 0.03% (w/v) 1,4-bis(5-phenyl-2-oxazolyl)benzene in chromatography-grade toluene. In experiments concerned with the interaction of [ 3 H]puromycin with unlabelled peptide chains on ribosomes, samples were counted in Bray's solution. Estimation of protein and RNA These were determined as previously described by Hawtrey, Schirren and Dijkstrai 141. ^4 2 6o units were determined by UV absorption measurement at 260 nm on a portion of the sample under test. One /4 26 Q unit is that amount of material in 1 ml of solution giving an optical density at 260 nm of 1.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
112
M. Minks, M. Ariatti and A. O. Hawtrey
Results 3
The covalent binding of [ HyV-bromoacetylpuromycin and [3H]7V-bromoacetylaminonucleoside to rat liver ribosomes was initially studied. Results presented in Fig. 1 illustrate the time course of binding of both derivatives to polysomes and (NH4)2S04-precipitated poly somes at different incubation temperatures. Fig. la illustrates the binding or attachment of [3H]7V-bromoacetylaminonucleoside and Fig. Ib the binding of [3H]^V-bromoacetylpuromycin. The number of moles ofAM>romoacetylpuromycin bound per mole of ribosome was found to be approximately 1, while with the aminonucleoside derivative the value was 9. Ammonium sulphate precipitation of polysomes appears to decrease to some extent their ability to bind theAM>romoacetyl derivati-
Bd. 356 (1975)
The results of the effect of increasing concentrations of [3HyV-bromoacetylaminonucleoside and [3H]7V-bromoacetylpuromycin on their binding to polysomes is shown in Fig. 2. The aminonucleoside derivative bound to the extent of 13 moles per ribosome, while the puromycin derivative bound to the extent of 8 moles per ribosome. Binding of the latter derivative shows a fairly linear increase with increasing concentration. It was of considerable interest to determine whether the binding of theTV-bromoacetyl derivatives to ribosomes was dependent upon the native structure of the ribosomal particles or not. For this purpose, the effects of heat treatment and fairly high concentrations of different chemicals on the subsequent ability of ribosomes to bind the./V-bromoacetyl derivatives was studied. Heat treatment of ribosomes at 65 °C for various times, followed by incubation of the heated ribo-
Fig. 1. Rate of binding of (a), [3H]W-bromoacetylaminonucleoside and (b), [ 3 H]7V-bromoacetylpuromycin to polysomes at different temperatures. Incubation tubes contained 5 /4260 units of polysomes plus 5 nmol of the respective labelled bromoderivative in 0.1 ml of Medium A containing 10% (v/v) Me2SO. Tubes were processed for counting as described in the Materials and Methods Section. (·), polysomes at 37 °C; (o) (NH4)2SO4-precipitated polysomes at 37 °C; («), polysomes at 20 °C; (±), (NH4)2SO4precipitated polysomes at 20 °C; (o), polysomes at 0 °C.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
113
Binding of 7V-Bromoacetylpuromy ein to Rat Liver Ribosomes
Bd. 356 (1975)
50
WO 150 cljumol/ll —*
200
250
Fig. 2. The effect of increasing concentrations of ^HJjV-bromoacetylaminonucleoside (·) and [ 3 H]./V-bromoacetylpuromycin (o) on thek covalent binding to polysomes. Incubations were as described for Fig. 1 (2 h at 37 °C).
somes with the ^-bromoacetyl derivatives for l h at 37 °C indicates (Fig. 3) that initially as the ribosomes unfold more sites become available for covalent binding, which, however, is then followed by a decrease in binding at the longer times of heating. Treatment of ribosomes for 16 min at 65 °C completely destroys their ability to interact with the TV-bromoacetyl aminonucleoside.Table 1 shows the results of preincubating ribosomes with methanol, urea and EDTA on their subsequent binding of the labelled A^bromoacetyl derivatives. All three reagents bring about an increase in the binding of [3H]/V-bromoacetylpuromycin suggesting that unfolding or partial denaturation of the ribosome exposes or makes available more sites for binding. These results are thus similar to those obtained in the heating experiment (Fig. 3). All incubations contained 10% (v/v) dimethylsulphoxide (Me2 SO) in order to keep the bromo derivatives in solution. A check was made therefore on the possible effects of this chemical on ribosome structure and function. Dimethylsulphoxide (10% v/v) in incubation mixtures had no effect on polysome profiles as evidenced by sucrose density-gradient centrifugation (Figs. 4 and 5). Further, no inhibitory effects were observed in the reaction of [3H]W-
Table 1. The effect of different chemical reagents on the binding of [3H]A^-bromoacetylpuromycin to ribosomes. Reaction mixtures contained: polysomes (5 A2(>o units), Medium A buffer, 10% (v/v) Me2SO, [3H]Nbromoacetylpuromycin (5 nmol) in a final volume of 0.1 ml, with or without reagents shown in the table. Incubations were carried out for 60 min at 37 °C. Samples were cooled in ice and processed as described in the Materials and Methods Section. System
[3H)^-bromoacetylpuromycin bound [cpm/5^260 units]
Polysomes (control) Polysomes + 20% (v/v) MeOH Polysomes + IM urea Polysomes + 2M urea Polysomes + 20mM Mg(OAc)2 Polysomes + lOmM EDTA
1630 2110 2290 5671 1738 9599
11 mini —»·Fig. 3. Effect of heat treatment of polysomes at 65 °C for various times on their subsequent binding of the bromoacetyl derivatives. Polysomes were heated at 65 °C for various times, cooled in ice, and then incubated for l h at 37 °C with the labelled compounds as described for Fig. 1. (·), Binding of [ 3 H]A^-bromoacetylpuromycin; (o), binding of [3H],/V-bromoacetylaminonucleoside.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
114
M. Minks, M. Ariatti and A. O. Hawtrey
acetylphenylalanyl-tRNA bound to washed ribosomes with puromycin. Protein synthesis in a complete system using 14C-labelled yeast protein hydrolysis was also not affected (unpublished results of M. Ariatti and A. 0.Hawtrey). Having shown that [3H]jY-bromoacetylpuromycin and [3HyV-bromoacetylaminonucleoside bind to rat liver ribosomes, it was of considerable interest to determine if these substances bound to either the 5OS or 3OS ribosomal subunit or to both subunits. Ribosomes were therefore labelled with theJV-bromoacetyl derivatives for l h at
Bd. 356(1975)
0.8 0.6 O.I. 02
A
0.6·
10
15
20
25
Fract. no. —^-
Fig. 4. Effect of dimethylsulphoxide on polysome profiles. Polysemes were preincubated with and without 10% (v/v) Me2SO for 2 h at 0 °C. They were then subjected to separation by means of centrifugation through a 15 - 35% (w/v) linear sucrose gradient (S.W. 25 rotor, 90 000·,§· for 4 h at 4 °C). (a), Control poly somes; (b), Polysomes plus 10% (v/v) Me2SO.
Fig. 5. The effect of preincubating (NH4)2SO4-precipitated polysomes with ./V-bromoacetyl derivatives as evidenced by centrifugation through linear sucrose gradients. Polysomes with or without the ./V-bromoacetyl derivatives but containing 10% Me 2 SO were preincubated for 2 h at 37 °C. They were then subjected to centrifugation through 15 - 35% (w/v) linear sucrose gradients (S.W. 25 rotor, 90000·^ for 5 h at 4 °C). (a), Control polysomes; (b) polysomes preincubated with ./V-bromoacetylpuromycin (5·10~5Μ); (c) polysomes preincubated with 7V-bromoacetylaminonucleoside (5-10~ 5 M).
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
Bd. 356 (1975)
Binding of W-Bromoacetylpuromycin to Rat Liver Ribosomes
5
W
15
20
25
5
W
15
20
115
25
Fig. 6. Distribution of (a), [3H]JV-bromoacetylaminonucleoside and (b), [3H]./V-bromoacetylpuromycin on rat liver ribosomal subunits following sucrose density-gradient centrifugation. Polysomes (25 Λ 2 60 units) were incubated with the labelled compounds (25 nmol) for l h at 37 °C. After cooling in ice, the labelled poly somes were washed by means of discontinuous sucrose gradient centrifugation. They were then dissociated into subunits by treatment with 5mM EDTA. Separation of subunits was carried out using a linear 10 - 30% (w/v) sucrose gradient containing lOmM Tris/HCl; pH 7.6, 25mM KC1 and ImM EDTA in the S.W. 25 -t rotor (22000 rpm for 16 h at 5 °C). AI^Q was determined and samples counted in Bray's solution. (—), (·), cpm.
37 °C, and then washed by centrifugation through a discontinuous sucrose gradient. The labelledwashed ribosomes were dissociated into subunits with EDTA and separated from each other by sucrose density-gradient centrifugation. Results presented in Figs.oa and 6b, respectively, show that binding of both labelled TV-bromoacetyl derivatives occurs mainly on the 50S ribosomal subunit. The extent of binding of both compounds to the 30S subunit was negligible, amounting to approximately 4% of that bound to the 50S subunit. Theoretically, binding of the labelled W-bromoacetyl derivatives to ribosomes can occur through attachment to protein or to RNA or both. Experiments (results not shown) have indicated that considerable binding occurs with the 50S subunit proteins. However, it was also found that binding of the labelled derivatives took place with ribosomal RNA. Careful extraction of RNA from labelled ribosomes by the phenol/sodium dodecylsulphate method at different times of incubation indicated a rapid rate of binding up to l h followed by a decrease in binding up to 2 h (Fig.7).
Fig. 7. Time course of binding of [3H]7V-bromoacetylpuromycin to rat liver ribosomal RNA. Polysomes (48 ^260 units) were incubated in Medium A containing 10% (v/v) Me2SO with 40 nmol of the labelled compound for various times at 37 °C. RNA was extracted as described in the Materials and Methods Section. Counting in Bray's solution. (·), cpm/5 A26Q units of RNA.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
116
M. Minks, M. Ariatti and A. O. Hawtrey
Reasons for the subsequent decrease in binding are not known. In vitro experiments with various radioactively labelled puromycin and aminonucleoside derivatives incubated with purified yeast tRNA gave no evidence of binding to the tRNA (Table 2). It is generally assumed that puromycin interacts with peptidyl-tRNA already in the P (peptidyl) site on the ribosome. One would assume therefore that the puromycin molecule could interact or bind possibly in an area of the ribosome around or close to the A (aminoacyl) site for its subsequent reaction with peptidyl-tRNA. One would also expect theTV-bromoacetylated puromycin and aminonucleoside derivatives possibly to interact with ribosomes in the same area (A site). If this were so, prior incubation of washed ribosomes with theTV-bromoacetylated derivatives might possibly interfere with the subsequent binding of [3H]W-acetylphenylalanyl-tRNA to the A site of the ribosome. Experiments to test this idea were therefore carried out.
Table 2. Binding of [ 3 H] puromycin and related compounds to tRNA in vitro. Each incubation tube contained yeast tRNA (1 mg), 50 mM Tris/HCl pH 7.6, 25mM KC1, 5mM MgCl2, 10% Me 2 SO (v/v) and the appropriate labelled compound in a final volume of 1.0 m/. Solutions were incubated for 3 h at 37 °C, cooled in ice, and then mixed with 0.1 ml of 20% (w/v) potassium acetate. Cold ethanol (2.5 m/) was added and the mixture kept at 4 °C overnight. The resulting RNA precipitate was recovered by low-speed centrifugation (2000 rpm/5 min), dissolved in 1 ml water and reprecipitated as described above. The final precipitate was washed twice with ethanol by low-speed centrifugation and dissolved in water (1 ml). Portions were taken for ^260 measurements and radioactive counting (Instagel). System
Bromoacetic acid Puromycin ./V-Acetylaminonucleoside Λ^-Acetylpuromycin Af-Bromoacetylaminonucleoside ./V-Bromoacetylpuromycin
nmol of 3H-labelled compound bound/nmol of tRNA 5.28-10-1 3.28-10-4 1.11·10~2
i.oo-io-
2
2.03-10-2 4.05-10-3
Bd. 356 (1975)
In the first experiments, washed ribosomes were incubated with the unlabelledAf-bromoacetyl derivatives (concentration, 5· 10~ 5 M) for 2 h at 37 °C. Thereafter, the reaction mixtures were directly interacted with [3H]./V-acetyrphenylalanyl-tRNA in a complete binding system. Results presented in Table 3 show that bothN-bromoacetyl derivatives had little effect on the non-enzymatic binding of the labelled aminoacyltRNA to the A site of the ribosome. In a further series of experiments, washed ribosomes were incubated with unlabelled N-bromoacetylpuromycin at three different concentrations for 2 h at 37 °C, and then purified by washing through discontinuous sucrose gradients. The washed ribosomes were then incubated with
Table 3. Binding of [3H]^V-acetylphenylalanyl-tRNA to washed rat liver ribosomes previously treated with jV-bromoacetylpuromycin and ./V-bromoacetylaminonucleoside. Polysomes were previously treated with puromycin and washed through discontinuous sucrose gradients containing 0.5M NH4C1 (washed ribosomes); see Materials and Methods Section. Washed ribosomes (5 Λ2 60 units) in 0.1 ml of Medium A containing 10% (w/v) Me2SO were incubated with the jV-bromoacetylated derivatives (final concentration 0.05mM) for 2 h at 37 °C. To the incubation tubes various reagents were then (directly) added (final concentrations), 50mM Tris/HCl, pH 8.0, 20mM Mg(OAc) 2 , 60mM KC1, 20mM NH4C1, ImM mercaptoethanol, 30 Mg poly(U) and 15 Mg [3H]JV-acetylphenylalanyl-tRNA (5.3Ί04 cpm). Final volume, 0.25 ml. All reaction tubes were incubated for 30 min at 37 °C and then cooled in ice. Samples were filtered through Millipore filters and washed with 20 ml of cold Nirenberg buffer (lOOmM Tris/HCl, pH 7.6, 20mM Mg(OAc) 2 and 50mM KC1). After drying, samples were counted as previously described. System
Ribosomes (control) Ribosomes C/V-BrAc-puromycin) Ribosomes C/V-BrAc-aminonucleoside)
[3H]^-Acetylphenylalanyl tRNA bound [cpm/5^260 u n i t s l 8068
6321 7853
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
Bd. 356 (1975)
Binding of W-Bromoacetylpuromycin to Rat Liver Ribosomes
Table 4. Binding of [3H]7V-acetylphenylalanyl-tRNA to washed rat liver ribosomes following previous treatment with W-bromoacetylpuromycin and centrifugation through discontinuous sucrose gradients. Washed ribosomes after incubation with JV-bromoacetylpuromycin for 2 h at 37 °C were centrifuged through a discontinuous sucrose gradient containing 0.5M sucrose in Medium A at 215 000 ·£ for 90 min (No. 50 rotor). The pellets were taken up in Medium A and treated exactly as described for Table 3 for the binding of labelled tRNA. System
Ribosomes (control) Ribosomes (JV-BrAc-puromycin O.OSmM) Ribosomes C/V-Br-Ac-puromycin 0.1 5mM) Ribosomes (W-BrAc-puromycin 0.25mM)
[3H]^-Acetylphenylalanyl-tRNA bound [cpm/1.0 ^260 units l
117
indicate that the process of binding is not specific with regard to the number of moles of compound bound per ribosome. The puromycin derivative binds to the extent of 8 moles per ribosome, while the aminonucleoside derivative binds to the extent of approximately 13 moles per ribosome. Partial unfolding or denaturation of ribosomes by heating at 65 °C for short periods of time or through the action of urea, methanol and EDTA bring about a considerable increase in binding of both derivatives, suggesting that as the ribosome structure unfolds, more sites become available for interaction and attach-
1163 1150 1143
I 6·
1350
[3H]^Vr-acetylphenylalanyl-tRNA in a complete binding system. Results in Table 4 again show that prior incubation of washed ribosomes with JV-bromoacetylated derivatives had little effect on their subsequent non-enzymatic binding of the labelled tRNA to the A site of the ribosome. The third type of experiment involved the interaction of polysomes (not washed) with the unlabelled TV-bromoacetylated derivatives for 2 h at 37 °C, followed by washing of the polysomes through discontinuous sucrose gradients. The washed polysomes were then incubated with [3H]puromycin in order to measure the degree of reaction with unlabelled peptide chains in the P site on the ribosome. Results presented in Fig. 8 indicate very little difference in the interaction of [3H]puromycin with peptide chains, following treatment with the TV-bromoacetylated derivatives. Discussion
The present experiments on the binding of [ 3 H]-/V-bromoacetylpuromycin and [ HJ/V-bromoacetylaminonucleoside to rat liver ribosomes
Fig. 8. Time course of the interaction of [3H]puromycin with peptide chains on rat liver polysomes after previous treatment of polysomes with ./V-bromoacetylated derivatives. Polysomes in Medium A containing 10% (w/v) Me2SO were incubated with the W-bromoacetylated derivatives (0.05mM) for 2 h at 37 °C. The reacted polysomes were then washed by discontinuous sucrose-gradient centrifugation as described for Table 4. Pellets were resuspended in Medium A (1 m/, 40/4260 units), and incubated with [3H]puromycin (1.25 μΟ) at 37 °C. Samples (0.2 m/) were withdrawn at various times and injected into a solution (0.2 ml of 20% trichloroacetic acid) containing unlabelled puromycin (0.27 mg). After standing in ice for 15 min, the precipitates were collected on Whatman GF/C filters and washed with 20 ml of cold 5% trichloroacetic acid by 20 ml of 96% ethanol. After drying, samples were counted as described in the Materials and Methods Section. (A), Control polysomes; (o), ./V-bromoacetylpuromycintreated polysomes; (·), Λ^-bromoacetylaminonucleoside-treated polysomes.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
118
M. Minks, M. Ariatti and A. O. Hawtrey
ment. The only specific process in the alkylation reactions of both derivatives appears to be their interaction with the 50S ribosomal subunit. In this respect the 5 OS subunit involvement is similar to that observed by other workers using #-BrAc-[3H]-Phe-tRNA11-31 and JV-bromoacetylchloramphenicol^ 4 ~ 6 ',even though the binding interactions of these substances are obviously different. Puromycin, because of its properties as a donor substrate in the peptidyl transferase re action I 7 " 9 ', presumably interacts and binds (weakly) to the A site of ribosomes. It was of interest therefore to observe that prior incubation of washed ribosomes with theTV-bromoacetyl derivatives had little or no effect on the subsequent binding of [3HyV-acetylphenylalanyl-tRNA to the A site. This result may support the ideas of Raacke1151, who on the basis of model building suggested that puromycin interacts with peptidyl-tRNA in the P site through a backside attack on the activated ester grouping of the peptidyl-tRNA, and therefore does not interact or bind directly to the A site. However, binding or attachment of puromycin presumably occurs in close vicinity to both the A and P sites of the ribosome. The finding that [3H]7V-bromoacetylpuromycin also binds to rat liver ribosomal RNA is of interest. Recently, Symons and co-workers^16' have reported on the interaction of the puromycin analogue, 5'-0-(7V-bromoacetyl-p -aminophenylphosphono)-3'^-L-phenylalanylaminonucleoside withJS'.co// ribosomes. These workers showed that this affinity label specifically interacts with ribosomal RNA of the 50S subunit. However, the binding of alkylating probes to RNA should be viewed with caution, in that proteins still firmly attached to RNA might be
Bd. 356 (1975)
responsible for the binding observed. Further experiments are in progress studying the 50S subunit proteins involved in binding and also the nature of the binding occurring with ribosomal RNA. Literature 1 Pellegrini, M., Oen, H. & Cantor, C. (1972) Proc. Nat. Acad. Sei. U.S.A. 69, 837 - 841. 2 Oen, H., Pellegrini, M., Eilat, D. & Cantor, C. (1973) Proc. Nat. Acad. Sei. U.S.A. 70, 2799 - 2803. 3 Eilat, D., Pellegrini, M., Oen, H., De Groot, N., Lapidot, Y. & Cantor, C. (1974) Nature (London) 250,514-516. 4 Sonnenberg, N., Wüchek, M. & Zamir, A. (1973) Proc. Nat. Acad. Sei. U.S.A. 70, 1423 - 1426. 5 Bald, R., Erdmann, V. A. & Pongs, O. (1972) FEBS Lett. 28, 149 -152. 6 Pongs, 0., Bald, R. & Erdmann, V. A. (1973) Proc. Nat. Acad. Sei. U.S.A. 70, 2229 - 2233. 7 Pestka, S. (1970) Arch. Biochem. Biophys. 136, 80-88. 8 Siler, J. & Moldave, K. (1969) Biochim. Biophys. Acta 195, 123 -129. 9 Siler, J. & Moldave, K. (1969) Biochim. Biophys. Acta 195, 130-137. 10 Wettstein, F. O., Staehelin, T. & Noll, . (1963) Nature (London) 197,430 - 435. 11 Hawtrey, A. O. (1965) 5. Afr. J. Med. Sei. 30, 100- 104. 12 Herrington, M. D. & Hawtrey, A. O. (1971) Biochem. J. 121,279-285. 13 Felicetti, L. & Lipmann, F. (1968) Arch. Biochem. Biophys. 125, 548 - 557. 14 Hawtrey, A. 0., Schirren, V. & Dijkstra, J. (1963) Biochem. J. 88, 106-114. 15 Raacke, I. D. (1971) Biochem. Biophys. Res. Commun. 43,168 - 173. 16 Harris, R.J., Greenwell, P. & Symons, R. H. (1973) Biochem. Biophys. Res. Commun. 55, 117 -124.
Brought to you by | University of Arizo Authenticated Download Date | 5/30/15 1:24 PM
