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Effect of dansylation of ribosomal protein from Pinus lambertiana
Plant Science Letters, 2 (1974) 225--231 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Ne'herlands
EFFECT OF DANSYLATION OF RIBOSOMAL PROTEINS FROM P I N U S LAMBER TIANA *
THONG SUNG KO**, ROBERT E. ADAMS and LEWIS B. BARNE'~T
Departments of Biochemistry and Nutrition and Forestry and Forest Products, Virginia Polytechnic Institute and State University, Blacksburg, Va. 24061 (U.S.A.) (Received July 31st, 1973) (Revision received October 30th, 1973)
SUMMARY
Ribosomes from seeds of sugar pine, P i n u s lambertiana, were dansyhted (180 molecules of dansyl chloride per ribosome particle), derivatizing the protein and leaving the RNA unreacted. The dansylated ribosomes were more sensitive to pancreatic ribonuclease than normal ribosomes at 5 mM MgC12 and at 0.1 mM MgCl2. No difference was observed between the sedimentation patterns of normal ribosomes and dansylated ribosomes or between their dissociation and reassociation patterns. These observations are consistent with a model that envisions ribosomal proteins associated with non-helical portions of ribosomal RNA and protecting these sites from ribonuclease attack.
INTRODUCTION
Stepwise advancement of messenger RNA along the ribosome during protein synthesis may be associated with structural transitions of ribosomal RNA (r-RNA) helices [1]. Evidence indicates that the conformation of r-RNA within ribosomes is influenced by ribosomal protein (r-protein) [2] and Sypherd and Fansler [3], proposed that the primary role of r-proteins was to regulate conformation of the nucleic acid. The purpose of this work was to study the effect of r-proteins on r-RNA structure within ribosomes, using dansyl chloride dispersed on celite as a protein derivatizing reagent.
*Research supported by Hatch gra.~t No. 616161 and McIntire-Stennis Grant No. 636125. *~Present address: Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pa. 19104 (U.S.A.). Abbreviation: TCA, trichloroacetic acid. 225
MATERIALS AND METHODS
Preparation of ribosomes from Pinus lambertiana 50 g of pine seeds were washed thoroughly with tap water by shaking for 1 h, and then washed 4 times with distilled water. The washed seeds were soaked with distilled water overnight in the cold. All the following steps were done at 4 °. Seeds were homogenized for 2 rain in a SorvaU Omni mixer in 100 ml of homogenization medium containing 0.005 M MgCI2, 0.05 M Tris--HCl (pH 8.3), 0.02 M KCI, 0.006 M 2-mercaptoethanol, 0.4 M sucrose and 0.5% sodium deoxycholate. The deoxycholate was added just before homogenization. The homogenate was squeezed through cheesecloth and centrifuged for 30 min at 27 000 g and the semisolid lipid layer and precipitate were discarded. The supernate was centrifuged for 3 h at 105 000 g. The 105 000 g pellet was washed with 40 ml of 0.005 M MgCI:, 0.02 M KC1, 0.006 M 2-mercaptoethanol, 0.025 M Tris--HCl (pH 7.8). This buffer system will be referred to as TKM buffer. The pellet was washed again with 20 ml TKM buffer containing 0.35 M NH4CI and subsequently with TKM buffer.
Ribosomal analysis Protein content of the ribosome preparations was estimated by the method of Lowry et al. [4], using bovine serum albumin as the standard. Dische's orcinol method [5] was used for the RNA determination, using yeast RNA as the standard.
Analytical ultracentrifuge The Spinco Model E ultracentrifuge equipped with Schlieren optics was used for all sedimentation experiments. The centrifuge was run at 39 460 rev./ min with a phase angle af 50 ° and a temperature between 3 ° and 7 °. All patterns were taken about 15 rain after reaching speed and were of ribosome in 0.025 M Tris--HC1 (pH 7.8), 0.02 M KC1 and 0.006 M 2-mercaptoethanol. Sedimentation coefficients were corrected to water at 20 ° .
Dansylation of the ribosome The dansylation procedure has been reviewed by Gray [6] and the method using celite was reported by Rinderknecht [7]. Dansyl chloride dispersed on celite (dansyl-Cl-celite), colorimetric assay 11%, was purchased from Calbiochem and [Me-14C]dansyl chloride in acetone, specific activity 45 mCi/mmole, was from Schwarz Bioresearch Inc. To 5 ml of 0.5% ribosome, in a medium containing 0.025 M sodium cacodylate (pH 8.8}, 0.02 M KC1 and MgC12 at the desired level, was added 0.1 g of dansyl-Cl-celite which corresponded to 4 ;~moles of dansyl-C1, and the mixture was stirred gently for 2.5 h in the cold. The reaction mixture was then centrifuged 3 times at 12 000 g for 10 rain to eliminate dansyl-Cl-celite residues. The precipitate was discarded each time. The supernate was dialyzed overnight in the cold against a medium composed of 0.025 M Tris--HC1 (pH 7.8), 0.02 M KC1 with MgC12 at the appropriate 226
level. A system containing celite instead of dansyl-Cl-celite was used as a control. Time course of the dansylation of ribosomes and the extent of the dansylation were followed by measuring the radioactivity of the ribosomes dansylated with dansyl-Cl-celite containing [Me-Z4C]dar~syl-C1 (specific activity of the mixture was 35 pCi/mmole dansyl chloride) under the conditions described above. The labelled ribosomes, after separation from the dansyl-Cl-celite by centrifugation, were precipitated with 10% TCA and washed twice with 2 ml of 5% TCA and finally with an acetone--10% TCA mixture (1:9 v/v). The washed ribosomes were dissolved in formic acid and counted on planchets.
Effect of ribonuclease on ribosomes A 0.03-ml sample containing 0.5% ribosomes in TKM buffer was suspended in 3 ml of medium (preincubated to 30 ° ) containing 0.025 M Tris--HC1 (pH 7.8), 0.02 M KCI, and MgCI2 at the desired level and the resulting suspension was held in the spectrophotometer cuvette at 30 ° for 2 min. On the addition of 30 pg of ribonuclease (EC 2.7.7.16) (beef pancreatic ribonuclease purchased from Worthington Biochemical Corporation) dissolved in 0.03 ml of distilled water, the absorbance at 260 nm was observed at 1-min intervals until no change was observed. The zero time absorbance was between 0.4 and 0.6 at 260 nm.
Test ot the reactivity of ribosomal components with dansyl-Cl-celite Ribosomes, after treatment with dansyl-Cl-celite, were separated into the protein and the RNA moieties by a modification of LiCl-urea method of Leboy et al. [8]. A solution of 6 M LiC1 and 8 M urea was added to an equaJ volume of 1 to 3% suspension of ribosomes in TKM buffer. The solution was mixed and held for 24 h in the cold, following which the precipitated RNA was removed by centrifugation at 100 000 g for 20 rain. The supernatant, containing r-protein fractions, was dialyzed against 0.01 M HC1 for 48 h in the cold and then centrifuged at 13 000 g for 2 h and lyophilized. The 100 000 g pellet containing the r-RN ~ fraction, was washed with a medium containing equal volumes of TKM b'~ffi'er and the mixture of 6 M LiCl and 8 M urea. The RNA was collected by centrifugation and then dissolved in a buffer containing 0.1 M KC1, 0.1 M Tris--HC1 (pH 9) and 0.01 M EDTA, dialyzed against the same buffer, and lyophilized. RESULTS
Dansylation of ribosomes The site of dansylation of ribosomes was checked both by fluorescence and by radioac*Avity. Protein from dansylated ribosomes had the characteristic fluorescence and radioactivity both before and after lyophilization, whereas the RNA moiety from these particles did not exhibit either. The uptake of the dansyl group by the ribosome increased linearly with 227
time and reached a plateau after 2.5 h, with about 45 nmoles of dansyl-C1 bound per mg of ribosomes. This corresponds to 180 molecules of dansyl-Cl per ribosome particle, assuming the molecular weight of plant ribosome [9] to be 4-10 ~, and a specific activity of r-protein of 6.03 -103 cpm/mg. Dansylribosomes used in the following experiments were dansyhted to this extent unless otherwise specified.
Effect o f the dansylation on the sedimentation patterns of ribosomes In the experiments of Fig. 1, in which only the variation of Mg2. concentra. tion is indicated, other buffer components were kept constant at: 0.025 M Tris-HC1 (pH 7.8); 0.02 M KCi; and 0.006 M 2-mercaptoethanol. Ribosomes dansylated at a MgC12 concentration of 5.10 -3 M showed the same sedimentation pattern as those of controls after dialysis at (a) 5.10 -3 M MgCl2 and (b) 6-10-4 M MgC12. Thus, dansylation had no apparent effect on the dissociation of ribosomes.
57S
0
77S
i
ts
I |59S
c
d
Fig. 1. Analytical ultracentrifuge patterns of ~ibosomes (6 mg/ml) dansylated at 5 "10-3M MgCI 2 and then dialyzed v s . (a) 5"10-3M MgC12 and (b) 6"10-4M MgCI2, and dansylated at 6 "10 -4 M MgCI2 and then dialyzed v s . (c) 6"10 -4 M MgC12 and (d) 5 "10 -3 M MgCI 2. Sedimentation is from right to left. Top pattern is dansyl ribosome. Bottom pattern is control. (See MATERIALS AND METHODS for further details.) 228
Dansylation of ribosomal subunits at 6 . 1 0 - 4 M MgC12 had no effect or, their sedimentation pattern (Fig. l c ) or on their reassociation at 5.10-3M MgCl2 (Fig. l d ) and the resulting sedimentation patterns were the same as controls. Controls were also run in the presence of celite alone and it had no apparent effect on dissociation, reassociation or sedimentation behavior of ribosomes.
The effect o f ribonuclease on dansyl-ribosomes Fig. 2 shows the effect of ribonuclease on dansyl-ribosomes and control ribosomes in the presence of 5-10-3M MgCl2 and 1.10 -4 MgC12. Although the original absorbance was about 0.5, all solutions were normalized to an absorbance value of 1.0 at zero time. It must be kept in mind t h a t the 80S ribosome is partially dissociated at 1-10-4M MgC12. 18 0 N
16
w z
in 12 w _>
I 2
1
I
1
4
6
8
I0
TIME (min)
Fig. 2. E f f e c t o f ribonuclease o n the absorbance o f r i b o s o m e and of dansyi-ribosomes at 5 "10 -3 M MgCI2 and at 1 o10 -4 M MgCI 2. Detailed e x p e r i m e n t a l conditions are described in M A T E R I A L S A N D M E T H O D S . o - - - - - - o , r i b o s o m e s 5 "10 -3 M MgCI 2 ; ~z % dansylr i b o s o m e s 5 . 1 0 -3 M MgCI2; $ - - - - - - $ , ribosomes 1 "10 -4 M MgCI2 ; ~ - - - - 4 , dansylr i b o s o m e s 1 "10 -4 M MgCI2.
Dansylation of the ribosomes increased the sensitivity to ribonuclease, as measured by the chromicity change at 260 nm, at both 5 . 1 0 - 3 M MgCl: and 1 o10-4M MgC12. At 5.10 -3 M MgC12 there was an increased rate of change in absorbance although the total hyperchromicity was about the same for the dansyl-ribosomes and the control ribosomes. At 1 . 1 0 - 4 M MgC12 there was an increase in both the rate and degree of absorbance change. DISCUSSION
Dansylation of the ribosomes of seeds of Pinus lambertiana was restricted to the r-proteins and did n o t occur to any detectable extent on the r-RNA. This conclusion is based b o t h on the fluorescence properties and the radioactivity of the r-protein and r-RNA which was isolated after dansylation with labeled dansyl-chloride. 229
The failure to observe any difference between the normal ribosomes and the dansyl-ribosomes in their dissociation, reassociation, or sedimentation patte.ms (Fig. 1), as examined by sedimentation analysis, may indicate that dansylation of the ribosome under these conditions did not appreciably affect intra-subunit interactions which are responsible for the conformation of the ribosome. This supports observations which have shown that modification (a) of the r-protein moiety with 2,4-dinitrofluorobenzene, (b) of the r-RN_~, moiety with ribonuclease, or (c) of both r-proteins and r-RNA with formaldehyde was not accompanied by alteration in sedimentation pattern or the electron microscopic appearance of the ribosomes [10--12]. However, dansylation did increase susceptibility of the ribosome to ribonuclease attack, as measured by absorbance changes at 260 nm (Fig. 2). Therefore, dansylation of the protein in these ribosomes probably did affect the protein--RNA interaction. This is to be expected since electrostatic attractions between the positively charged groups on the proteins and the negatively charged groups on the RNA are considered to be a significant force in nucleoprotein stability, and dansylation of amino groups removes the positive charge from amino groups. Since ribonuclea~e attack occurs primarily at single-stranded regions of RNA and not at double-stranded regions, it is likely that the affected interactions involve single-stranded regions of RNA. The removal of the positive charge in the amino groups through dansylation does n o t break down ribonucleoprotein structure to such an extent that sedimentation profiles are altered. The effect of magnesium ions on ribonuclease attack on ribosomes is also consistent with many studies that have shown that the dissociation of the ribosome into subunits results in particles which are more susceptible to ribonuclease digestion (el. ref. 13). The effect of dansylation was not simply the removal of large, amounts of protein from the ribosomes since both undansylated and dansylated ribosomes contained the same gross composition of 42% RNA and 58% protein. Supporting evidence for increased instability of ribosomes after dansylation was obtained through the comparison of melting curves of control and dansylated ribosomes. In both instances the melting curves were biphasic and the melting temperature was 3--5 ° lower after dansylation at each phase. Cotter and Gratzer [14] have summarized evidence for the location of a sigmficant amount of RNA at the surface of the ribosome. Several workers [15--17] have presented evidence that r-proteins are associated with nonhelical portions of the r-RNA and protect these sites from attack by ribonuclease. Our observations in this paper that dansylation of r-protein increases the rate of r-RNA attack by ribonuclease are consistent with this model. The dansylation technique may be useful for studies on biochemical function of r-proteins because r-RNA does not become dansylated. The dansylation does not involve any solvent that might affect the ribosome and can be carTied out under conditions (buffer, temperature, reaction periods, etc.) suitable for the maintenance of ribosome integrity without side effects. 230
F u r t h e r m o r e , t h e strong f l u o r e s c e n c e o f t h e d a n s y l g r o u p can be u s e d as a m a r k e r a n d c a n be useful f o r p h y s i c a l - c h e m i c a l studies o f r i b o s o m e s . REFERENCES
1 S. Arnott, W. Fuller, A. Hodgson and I. Prutton, Nature, 220 (1968) 561. 2 P.S. Sypherd, J. Mol. Biol., 56 (1971) 311. 3 P.S. Sypherd and B.S. Fansler, J. Bacteriol., 93 (1967) 920. 4 0 . H . Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. 5 Z. Dische, Color reactions of nucleic acid components, in E. Chargaff and J.N. Davidson (Eds.), The Nucleic Acids, Vol. I, Academic Press, New York, 1955, p. 300. 6 W.R. Gray, .Dansyl chloride procedure, in S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology, Vol. XI, Academic Press, New York, 1967, p. 139. 7 H. Rinderknecht, Nature, 193 (1962) 167. 8 P.S. Leboy, E.C. Cox and J.G. Flaks, Proc. Natl. Acad. Sci. (U.S.), 52 (1964) 1367. 9 M.L. Petermann, The Physical and Chemical Properties of Ribosomes, Elsevier, New York, 1964, p. 121. 10 P.B. Moore, J. Mol. Biol., 22 (1966) 145. 11 R.A. Cox, Biochem. J., 114 (1969) 753. 12 A.S. Spirin, N.V. Belitsina and M.I. Terman, J. Mol. Biol., 14 (1965) 611. 13 A. Worcel, D.S. Goldman and I.B. Sachs, Proc. Natl. Acad. Sci. (U.S.), 61 (1968) 122. 14 I.R, Cotter and W.B. Gratzer, European J. Biochem., 23 (19~ 1) 468. 15 G.N. Dessev and K. Grantcharon, Life Sci., 9 (Pt. II) (1970) 1181. 16 P. McPhie, J. Hounsell and W.B. Gratzer, Biochemistry, 5 (1966) 988. 17 W. Moller, R. Amons, J.C.L. Groene, R.A. Garrett and C.P. Lerhorst, Biochim. Biophys. Acta, 190 (1969) 381.
231
EFFECT OF DANSYLATION OF RIBOSOMAL PROTEINS FROM P I N U S LAMBER TIANA *
THONG SUNG KO**, ROBERT E. ADAMS and LEWIS B. BARNE'~T
Departments of Biochemistry and Nutrition and Forestry and Forest Products, Virginia Polytechnic Institute and State University, Blacksburg, Va. 24061 (U.S.A.) (Received July 31st, 1973) (Revision received October 30th, 1973)
SUMMARY
Ribosomes from seeds of sugar pine, P i n u s lambertiana, were dansyhted (180 molecules of dansyl chloride per ribosome particle), derivatizing the protein and leaving the RNA unreacted. The dansylated ribosomes were more sensitive to pancreatic ribonuclease than normal ribosomes at 5 mM MgC12 and at 0.1 mM MgCl2. No difference was observed between the sedimentation patterns of normal ribosomes and dansylated ribosomes or between their dissociation and reassociation patterns. These observations are consistent with a model that envisions ribosomal proteins associated with non-helical portions of ribosomal RNA and protecting these sites from ribonuclease attack.
INTRODUCTION
Stepwise advancement of messenger RNA along the ribosome during protein synthesis may be associated with structural transitions of ribosomal RNA (r-RNA) helices [1]. Evidence indicates that the conformation of r-RNA within ribosomes is influenced by ribosomal protein (r-protein) [2] and Sypherd and Fansler [3], proposed that the primary role of r-proteins was to regulate conformation of the nucleic acid. The purpose of this work was to study the effect of r-proteins on r-RNA structure within ribosomes, using dansyl chloride dispersed on celite as a protein derivatizing reagent.
*Research supported by Hatch gra.~t No. 616161 and McIntire-Stennis Grant No. 636125. *~Present address: Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pa. 19104 (U.S.A.). Abbreviation: TCA, trichloroacetic acid. 225
MATERIALS AND METHODS
Preparation of ribosomes from Pinus lambertiana 50 g of pine seeds were washed thoroughly with tap water by shaking for 1 h, and then washed 4 times with distilled water. The washed seeds were soaked with distilled water overnight in the cold. All the following steps were done at 4 °. Seeds were homogenized for 2 rain in a SorvaU Omni mixer in 100 ml of homogenization medium containing 0.005 M MgCI2, 0.05 M Tris--HCl (pH 8.3), 0.02 M KCI, 0.006 M 2-mercaptoethanol, 0.4 M sucrose and 0.5% sodium deoxycholate. The deoxycholate was added just before homogenization. The homogenate was squeezed through cheesecloth and centrifuged for 30 min at 27 000 g and the semisolid lipid layer and precipitate were discarded. The supernate was centrifuged for 3 h at 105 000 g. The 105 000 g pellet was washed with 40 ml of 0.005 M MgCI:, 0.02 M KC1, 0.006 M 2-mercaptoethanol, 0.025 M Tris--HCl (pH 7.8). This buffer system will be referred to as TKM buffer. The pellet was washed again with 20 ml TKM buffer containing 0.35 M NH4CI and subsequently with TKM buffer.
Ribosomal analysis Protein content of the ribosome preparations was estimated by the method of Lowry et al. [4], using bovine serum albumin as the standard. Dische's orcinol method [5] was used for the RNA determination, using yeast RNA as the standard.
Analytical ultracentrifuge The Spinco Model E ultracentrifuge equipped with Schlieren optics was used for all sedimentation experiments. The centrifuge was run at 39 460 rev./ min with a phase angle af 50 ° and a temperature between 3 ° and 7 °. All patterns were taken about 15 rain after reaching speed and were of ribosome in 0.025 M Tris--HC1 (pH 7.8), 0.02 M KC1 and 0.006 M 2-mercaptoethanol. Sedimentation coefficients were corrected to water at 20 ° .
Dansylation of the ribosome The dansylation procedure has been reviewed by Gray [6] and the method using celite was reported by Rinderknecht [7]. Dansyl chloride dispersed on celite (dansyl-Cl-celite), colorimetric assay 11%, was purchased from Calbiochem and [Me-14C]dansyl chloride in acetone, specific activity 45 mCi/mmole, was from Schwarz Bioresearch Inc. To 5 ml of 0.5% ribosome, in a medium containing 0.025 M sodium cacodylate (pH 8.8}, 0.02 M KC1 and MgC12 at the desired level, was added 0.1 g of dansyl-Cl-celite which corresponded to 4 ;~moles of dansyl-C1, and the mixture was stirred gently for 2.5 h in the cold. The reaction mixture was then centrifuged 3 times at 12 000 g for 10 rain to eliminate dansyl-Cl-celite residues. The precipitate was discarded each time. The supernate was dialyzed overnight in the cold against a medium composed of 0.025 M Tris--HC1 (pH 7.8), 0.02 M KC1 with MgC12 at the appropriate 226
level. A system containing celite instead of dansyl-Cl-celite was used as a control. Time course of the dansylation of ribosomes and the extent of the dansylation were followed by measuring the radioactivity of the ribosomes dansylated with dansyl-Cl-celite containing [Me-Z4C]dar~syl-C1 (specific activity of the mixture was 35 pCi/mmole dansyl chloride) under the conditions described above. The labelled ribosomes, after separation from the dansyl-Cl-celite by centrifugation, were precipitated with 10% TCA and washed twice with 2 ml of 5% TCA and finally with an acetone--10% TCA mixture (1:9 v/v). The washed ribosomes were dissolved in formic acid and counted on planchets.
Effect of ribonuclease on ribosomes A 0.03-ml sample containing 0.5% ribosomes in TKM buffer was suspended in 3 ml of medium (preincubated to 30 ° ) containing 0.025 M Tris--HC1 (pH 7.8), 0.02 M KCI, and MgCI2 at the desired level and the resulting suspension was held in the spectrophotometer cuvette at 30 ° for 2 min. On the addition of 30 pg of ribonuclease (EC 2.7.7.16) (beef pancreatic ribonuclease purchased from Worthington Biochemical Corporation) dissolved in 0.03 ml of distilled water, the absorbance at 260 nm was observed at 1-min intervals until no change was observed. The zero time absorbance was between 0.4 and 0.6 at 260 nm.
Test ot the reactivity of ribosomal components with dansyl-Cl-celite Ribosomes, after treatment with dansyl-Cl-celite, were separated into the protein and the RNA moieties by a modification of LiCl-urea method of Leboy et al. [8]. A solution of 6 M LiC1 and 8 M urea was added to an equaJ volume of 1 to 3% suspension of ribosomes in TKM buffer. The solution was mixed and held for 24 h in the cold, following which the precipitated RNA was removed by centrifugation at 100 000 g for 20 rain. The supernatant, containing r-protein fractions, was dialyzed against 0.01 M HC1 for 48 h in the cold and then centrifuged at 13 000 g for 2 h and lyophilized. The 100 000 g pellet containing the r-RN ~ fraction, was washed with a medium containing equal volumes of TKM b'~ffi'er and the mixture of 6 M LiCl and 8 M urea. The RNA was collected by centrifugation and then dissolved in a buffer containing 0.1 M KC1, 0.1 M Tris--HC1 (pH 9) and 0.01 M EDTA, dialyzed against the same buffer, and lyophilized. RESULTS
Dansylation of ribosomes The site of dansylation of ribosomes was checked both by fluorescence and by radioac*Avity. Protein from dansylated ribosomes had the characteristic fluorescence and radioactivity both before and after lyophilization, whereas the RNA moiety from these particles did not exhibit either. The uptake of the dansyl group by the ribosome increased linearly with 227
time and reached a plateau after 2.5 h, with about 45 nmoles of dansyl-C1 bound per mg of ribosomes. This corresponds to 180 molecules of dansyl-Cl per ribosome particle, assuming the molecular weight of plant ribosome [9] to be 4-10 ~, and a specific activity of r-protein of 6.03 -103 cpm/mg. Dansylribosomes used in the following experiments were dansyhted to this extent unless otherwise specified.
Effect o f the dansylation on the sedimentation patterns of ribosomes In the experiments of Fig. 1, in which only the variation of Mg2. concentra. tion is indicated, other buffer components were kept constant at: 0.025 M Tris-HC1 (pH 7.8); 0.02 M KCi; and 0.006 M 2-mercaptoethanol. Ribosomes dansylated at a MgC12 concentration of 5.10 -3 M showed the same sedimentation pattern as those of controls after dialysis at (a) 5.10 -3 M MgCl2 and (b) 6-10-4 M MgC12. Thus, dansylation had no apparent effect on the dissociation of ribosomes.
57S
0
77S
i
ts
I |59S
c
d
Fig. 1. Analytical ultracentrifuge patterns of ~ibosomes (6 mg/ml) dansylated at 5 "10-3M MgCI 2 and then dialyzed v s . (a) 5"10-3M MgC12 and (b) 6"10-4M MgCI2, and dansylated at 6 "10 -4 M MgCI2 and then dialyzed v s . (c) 6"10 -4 M MgC12 and (d) 5 "10 -3 M MgCI 2. Sedimentation is from right to left. Top pattern is dansyl ribosome. Bottom pattern is control. (See MATERIALS AND METHODS for further details.) 228
Dansylation of ribosomal subunits at 6 . 1 0 - 4 M MgC12 had no effect or, their sedimentation pattern (Fig. l c ) or on their reassociation at 5.10-3M MgCl2 (Fig. l d ) and the resulting sedimentation patterns were the same as controls. Controls were also run in the presence of celite alone and it had no apparent effect on dissociation, reassociation or sedimentation behavior of ribosomes.
The effect o f ribonuclease on dansyl-ribosomes Fig. 2 shows the effect of ribonuclease on dansyl-ribosomes and control ribosomes in the presence of 5-10-3M MgCl2 and 1.10 -4 MgC12. Although the original absorbance was about 0.5, all solutions were normalized to an absorbance value of 1.0 at zero time. It must be kept in mind t h a t the 80S ribosome is partially dissociated at 1-10-4M MgC12. 18 0 N
16
w z
in 12 w _>
I 2
1
I
1
4
6
8
I0
TIME (min)
Fig. 2. E f f e c t o f ribonuclease o n the absorbance o f r i b o s o m e and of dansyi-ribosomes at 5 "10 -3 M MgCI2 and at 1 o10 -4 M MgCI 2. Detailed e x p e r i m e n t a l conditions are described in M A T E R I A L S A N D M E T H O D S . o - - - - - - o , r i b o s o m e s 5 "10 -3 M MgCI 2 ; ~z % dansylr i b o s o m e s 5 . 1 0 -3 M MgCI2; $ - - - - - - $ , ribosomes 1 "10 -4 M MgCI2 ; ~ - - - - 4 , dansylr i b o s o m e s 1 "10 -4 M MgCI2.
Dansylation of the ribosomes increased the sensitivity to ribonuclease, as measured by the chromicity change at 260 nm, at both 5 . 1 0 - 3 M MgCl: and 1 o10-4M MgC12. At 5.10 -3 M MgC12 there was an increased rate of change in absorbance although the total hyperchromicity was about the same for the dansyl-ribosomes and the control ribosomes. At 1 . 1 0 - 4 M MgC12 there was an increase in both the rate and degree of absorbance change. DISCUSSION
Dansylation of the ribosomes of seeds of Pinus lambertiana was restricted to the r-proteins and did n o t occur to any detectable extent on the r-RNA. This conclusion is based b o t h on the fluorescence properties and the radioactivity of the r-protein and r-RNA which was isolated after dansylation with labeled dansyl-chloride. 229
The failure to observe any difference between the normal ribosomes and the dansyl-ribosomes in their dissociation, reassociation, or sedimentation patte.ms (Fig. 1), as examined by sedimentation analysis, may indicate that dansylation of the ribosome under these conditions did not appreciably affect intra-subunit interactions which are responsible for the conformation of the ribosome. This supports observations which have shown that modification (a) of the r-protein moiety with 2,4-dinitrofluorobenzene, (b) of the r-RN_~, moiety with ribonuclease, or (c) of both r-proteins and r-RNA with formaldehyde was not accompanied by alteration in sedimentation pattern or the electron microscopic appearance of the ribosomes [10--12]. However, dansylation did increase susceptibility of the ribosome to ribonuclease attack, as measured by absorbance changes at 260 nm (Fig. 2). Therefore, dansylation of the protein in these ribosomes probably did affect the protein--RNA interaction. This is to be expected since electrostatic attractions between the positively charged groups on the proteins and the negatively charged groups on the RNA are considered to be a significant force in nucleoprotein stability, and dansylation of amino groups removes the positive charge from amino groups. Since ribonuclea~e attack occurs primarily at single-stranded regions of RNA and not at double-stranded regions, it is likely that the affected interactions involve single-stranded regions of RNA. The removal of the positive charge in the amino groups through dansylation does n o t break down ribonucleoprotein structure to such an extent that sedimentation profiles are altered. The effect of magnesium ions on ribonuclease attack on ribosomes is also consistent with many studies that have shown that the dissociation of the ribosome into subunits results in particles which are more susceptible to ribonuclease digestion (el. ref. 13). The effect of dansylation was not simply the removal of large, amounts of protein from the ribosomes since both undansylated and dansylated ribosomes contained the same gross composition of 42% RNA and 58% protein. Supporting evidence for increased instability of ribosomes after dansylation was obtained through the comparison of melting curves of control and dansylated ribosomes. In both instances the melting curves were biphasic and the melting temperature was 3--5 ° lower after dansylation at each phase. Cotter and Gratzer [14] have summarized evidence for the location of a sigmficant amount of RNA at the surface of the ribosome. Several workers [15--17] have presented evidence that r-proteins are associated with nonhelical portions of the r-RNA and protect these sites from attack by ribonuclease. Our observations in this paper that dansylation of r-protein increases the rate of r-RNA attack by ribonuclease are consistent with this model. The dansylation technique may be useful for studies on biochemical function of r-proteins because r-RNA does not become dansylated. The dansylation does not involve any solvent that might affect the ribosome and can be carTied out under conditions (buffer, temperature, reaction periods, etc.) suitable for the maintenance of ribosome integrity without side effects. 230
F u r t h e r m o r e , t h e strong f l u o r e s c e n c e o f t h e d a n s y l g r o u p can be u s e d as a m a r k e r a n d c a n be useful f o r p h y s i c a l - c h e m i c a l studies o f r i b o s o m e s . REFERENCES
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