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Studies on the turnover and messenger activity of rat-liver ribonucleic acids
BIOCHIMICA ET BIOPHYSICA ACTA
547
BBA 95406
S T U D I E S ON T H E T U R N O V E R AND M E S S E N G E R ACTIVITY, OF R A T - L I V E R R I B O N U C L E I C ACIDS
A. A. HADJIOLOV*
The Rocke/eller University, New York, N.Y. (U.S.A.) (Received September i7th , 1965)
SUMMARY
In studies with E14C]orotic acid, rat-liver ribosomes were found to exhibit a turnover with a half-life of about 40 h. This rate far exceeds the reported mean generation time of liver cells of at least 60 days. The two ribosomal subunits are degraded at essentially the same rate. After short-term labeling, initially the smaller ribosomal subunit and the derived I8-S RNA have a higher specific activity than the large ribosomal subunit and the derived 28-S RNA. The more highly labeled fraction could not be dissociated from the I8-S ribosomal RNA b y various treatments, including heating with 0. 4 M urea at 60 ° and subsequent centrifugation in urea density gradients. Of the nuclear RNA fractions extracted with sodium dodecyl sulfate/ phenol at 45 ° and 65 ° respectively, the RNA extracted at higher temperature had a 3-fold higher messenger activity in the Escherichia coli cell-free system. The synthesized product remained almost completely bound to the ribosomes, and no specific pattern in the stimulated incorporation of different amino acids could be detected.
INTRODUCTION
Experiments from a number of laboratoriesZ, 2, including recent reports from this laboratory ~, established that rat-liver microsomes and ribosomes differ markedly in their capacity to synthesize specific proteins, although the incorporation of amino acids is similar in both systems. Obviously, the understanding of the mechanism of specific protein synthesis requires a better knowledge of the manner in which messenger RNA interacts with ribosomes and probably with ribosomal RNA. In rat-liver cytoplasm a rapidly labeled RNA fraction has been found that sediments in sucrose gradients together with I8-S ribosomal RNA4, 5, but also lower and more dispersed sedimentation values are reported 6-s. These experiments have indicated, further* Present address: Biochemical Research Laboratory, Bulgarian Academy of Sciences, Sofia (Bulgaria).
Biochim. Biophys. Acta, 119 (1966) 547 556
548
h.a.
HADJIOLOV
more, that the ribosomal RNA of liver is labeled quite rapidly; its turnover, however, has not been investigated. During preparation of this manuscript, we learned of results by LOEB, HOWELLAND TOMKINS*on the decay of I14CJorotic acid-labeled RNA from "free ribosomes" of rat liver, with conclusions on the turnover of rat-liver ribosomes similar to those discussed here. Nuclear RNA stimulates polypeptide synthesis in cell-free extractsS,gJ ° to a much greater extent than the cytoplasmic RNA, which is associated mainly with the I8-S ribosomal RNAS, n. No characterization of the product synthesized as a result of addition of these RNA's has been reported.
METHODS
Experimental animals The experiments were carried out with male Sprague-Dawley albino rats with a body weight of 12o-16o g, fed ad libitum with the standard laboratory diet. In the labeling experiments in vivo, animals with a body weight of 12o-4-1o g were used. In control experiments, starvation of the animals did not affect the parameters studied. Ribosomes and ribosomal subunits were prepared according to the procedur e of TASHIRO AND SIEKEVITZ 12, except that intermediate cytoplasmic fractions were obtained by centrifuging the original homogenate at 15oo ×g for 5 min to sediment the nuclei, and at IO ooo ×g for IO min to sediment the mitochondria. Microsomes and ribosomes were then isolated from the postmitochondrial fractions. The "crude" ribosomal fraction was dissociated with 5/,moles EDTA/mg ribosomes to obtain the two ribosomal subunits. The quantity of ribosomes and ribosomal subunits was estimated on the basis of an extinction coefficient of 135 at 260 m/~ for a I °/o ribosome suspension. The average r a t i o A26om#/A235m# was 1.36 for crude ribosomes, 1.68 for the 47-S subunit, and 1.69 for the 32-S subunit.
Isolation o/ ribonucleic acids Liver RNA's were isolated and fractionated by the method of GEORGIEV AND MANTIEVA 13, modified as follows. The livers of lO-2O rats were removed, rinsed with
o.14 M NaC1, and homogenized directly in phenol (saturated with o.14 M NaC1 at pH 5.0). The homogenate was shaken for 30 min at 4 ° and centrifuged at IO ooo ×g for IO min. The water layer was aspirated and processed further to yield the fraction of cytoplasmic RNA. The interphase layer was extracted twice more at 4 ° with phenol-NaC1 for 15 rain and the water phases were discarded. After the last treatment the interphase layer was suspended in o.14 M NaC1; sodium dodecyl sulfate was then added to a final concentration of 0.25 %, followed by an equal volume of phenol. The mixture was extracted at 45 ° for 20 min, cooled, and centrifuged at IO ooo x g for IO min. The water phase was aspirated and used to prepare the nuclear "ribosomal-like" RNA. The resulting interphase layer was extracted once more for 5 rain at 45 °, and after centrifugation, the water layer was discarded and the interphase layer was suspended in o.14 M NaC1, made 0.25 % with respect to sodium " Personal communication. Biochim. Biophys. Acta, 119 (1966) 547-556
TURNOVER AND MESSENGER ACTIVITY OF LIVER
RNA
549
dodecyl sulfate, and an equal volume of phenol was again added. The mixture was heated at 65 ° for 20 min with continuous shaking, and was cooled, and centrifuged at IO ooo ×g for IO min. The water layer was used to prepare the nuclear " D N A like" RNA fraction. The cytoplasmic and the two nuclear fractions were deproteinized 3-5 times with cold phenol, and the RNA from the final water layers was precipitated with 2 volumes of 96 °/o ethanol at --20 °. The RNA precipitate was washed several times with ethanol, drained carefully, and dissolved in deionized water. The average yield of the different RNA fractions was: cytoplasmic RNA, 90-92 °/o; "ribosomallike" RNA, 4-6 °/o; and "DNA-like" RNA, 2-3 %. Ribosomes or ribosomal subunits were suspended in o.14 M NaC1 and RNA was extracted with an equal volume of cold phenol after the addition of 0.25 % sodium dodecyl sulfate. The different RNA preparations were purified from low molecular weight impurities by passage through a Sephadex G-25 column 14. The quantity of RNA was estimated by its absorbance at 26o m# (24 absorbance units = I mg RNA).
Density gradients Linear density gradients prepared according to BRITTEN AND ROBERTSls were used in all experiments. Centrifugation at 24 ooo rev./min for 9 or 12 h intervals was carried out in the Spinco, model L-2 ultracentrifuge using the SW 25 rotor at 3-4 °. Sucrose gradients (5-2o %) were used for the fractionation of ribosomal subunits 16 or cytoplasmic RNA's 4. Urea density gradients were prepared with solutions of urea (purified from ultraviolet absorbing material by Norit pretreatment) in deionized water, and adjusted to p H 7.0. The density of I M and 5 M urea corresponds approximately to that of 5 and 20 °/o sucrose. Fractions of about i ml were collected after puncturing the bottom of the tube. Aliquots of the appropriate volume were pipetted for absorbance and radioactivity determinations.
Cell-[tee amino acid incorporation An Escherichia coli B cell-free system was prepared according to the procedure of NIRENBERGiv. In all experiments, the preincubated S-3o extract was used, dialysis of the preparation being replaced b y gel filtration through a Sephadex G-25 column equilibrated with the appropriate medium.
Radioactivity determinations Incorporation of amino acids was determined after heating the 5 % trichloroacetic acid precipitates for 20 min at 9 o°. The precipitates were plated on Millipore membrane filters, dried, and counted in a Nuclear Chicago gasf-low counter at about 22 ~o efficiency. Samples of ribosomes or RNA were precipitated with 5 % cold trichloroacetic acid after addition of 50 #g of bovine serum albumin to each sample.
MATERIALS
Analytical grade reagents were used. Uniformly labeled E14C]amino acids with Biochim. Biophys. Acta, 119 (1966) 547 556
55 °
A.A.
HADJIOLOV
specific activities of about 2oo mC/mmole were obtained from New England Nuclear Corp., Boston, Mass. E14C]orotic acid (30 mC/mmole) was a product of Nuclear Chicago Corp., Chicago, Ill. (U.S.A.).
RESULTS AND DISCUSSION
Turnover o[ rat-liver ribosomes and ribosomal subunits After dissociation of the "crude" ribosomal fraction, only 47-S and 32-S ribosomal subunits are detected on the sucrose gradient. In addition, varying amounts of undissociated ribosomes sediment to the bottom of the tube. After short term labeling in vivo with E6-14C]orotic acid, the radioactivity curve follows closely the A260 m~ curve (Fig. IA). Following such short term labeling, furthermore, a rapidly labeled fraction of low sedimentation value is also observed, probably reflecting the fast turnover of cytidylic acid in soluble RNA. The specific activity of the 32-S subunit is appreciably higher than that of the 47-S subunit; 9 ° min after injection of [6-14C]orotic acid, the ratio of the specific radioactivities 32 S: 47 S is 1.48. In experiments in which [12C]orotic acid is introduced I h after the [x4C3orotic acid, this ratio decreases rapidly, to 1.13 after 4 h and close to I.O after 8 h, remaining so in subsequent determinations (Table I, Fig. Ib). TABLE
I
SPECIFIC RADIOACTIVITY OF RAT-LIVER RIBOSOMAL SIJBUNITS AFTER LABELING [6-14C]OROTIC ACID AND [12C]OROTIC ACID CHASE
in vivo
WITH
T h e e x p e r i m e n t a l c o n d i t i o n s a r e t h e s a m e a s d e s c r i b e d f o r F i g s . I a n d 2. T h e s a m e e x t i n c t i o n c o e f f i c i e n t w a s u s e d f o r t h e 47-S a n d 32-S s u b u n i t .
Hours a/ter [l*C]orotic acid 1. 5 4 8 27
Hours a/ter [l~C]orotic acid I 3 7 26
Speci/ic radioactivity (Counts/rain/rag ribosomes) 47 S
32 S
21o 47 ° 126o 3720
31o 53 ° 124o 3720
Ratio 32 S/47 S
1.48 1-13 o.98 I.OO
In order to study the turnover of liver ribosomes and their subunits, a 6o-min pulse with E634C]orotic acid was followed b y the administration of an approx. 30o times higher dose of unlabeled orotic acid. The labeling of the two ribosomal subunits was followed up to 168 h and the specific activities of the free nucleotides and nuclear RNA (total nuclear RNA extracted at 65°with sodium dodecylsulfatephenol) were determined. The results of these experiments are presented in Fig. 2. As can be seen, a three-fold decrease in the labeling of the free nucleotides occurs in the first 4 h, and the label nearly disappears after 16 h. These results show that the pyrimidine nucleotide pool size in liver is relatively small, thus allowing efficient dilution of the label with added unlabeled orotic acid. The decrease in the specific activity of the nuclear RNA proceeds at a slower rate during the first 48 h. In contrast, the specific activity of the two ribosomal subunits increases almost Biochim. Biophys. Acta,
119 ( I 9 6 6 ) 547--556
TURNOVER
,oog
AND
MESSENGER
A C T I V I T Y OF L I V E R
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Fig. I. Sucrose density gradient sedimentation p a t t e r n of rat-liver ribosomal subunitsisolated from animals killed 90 miri (A) and 8 h (B) after administration in vivo of [6-1*C]orotic acid and subsequent chase with [l*C]orotic acid. [6-1*C]Orotic acid was injected intraperitoneally at zero time (2/iC/i2o g body weight), followed after 6o min by unlabeled orotic acid (20/~mole/I2O g body weight). Centrifugation was for 9 h at 24 ooo rev./min (Spinco SW 25 rotor). The radioactivity represents the counts precipitated with 5 % trichloroacetic acid at 4 °. 0 - 0 , A260m#; (2)---O, counts/min. The sedimentation coefficients are those reported by TASHIRO AND SIEKEVITZ
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Fig. 2. Specific radioactivity of rat-live~--ribosomal subunlts and free nucleotides after admiixistratiorl iii vivo of [6-x~0]orotic acid alxd subsequent chase with unlabeled orotic acid. Administration of [6-1*C]orotic acid (arrow-ziC] and of unlabeled orotic acid (arrow-12C) as described for Fig. I. The pooled livers from groups of four rats (body weight, 12O-/lO g) were used for the isolation of ribosomal subunits. During the whole period the animals were fed ad libitum with the standard laboratory diet. Abscissa, hours after the administration of [6-1¢C]orotic acid. The same extinction coefficient was used for the estimation of the q u a n t i t y of b o t h the 47-S and 32-S subunit (see METHODS). Biochim. Biophys. Acta, 119 (1966) 547-556
552
A . A . HADJIOLOV
linearly up to about 3 ° h, when it reaches a m a x i m u m that is about 15 times higher than the values obtained at 9 ° min. Analysis of this part of the curve suggests that ribosome formation in the nucleus precedes the release of these particles into the cytoplasm. These results are in agreement with those for H e L a cells reported by GIRARD, PENMAN AND DARNELL is who observed a release of ribosomes into the cytoplasm after actinomycin block of RNA synthesis (see review b y PRESCOTT19). Analysis of the curve in later periods shows a gradual and parallel decrease in the specific activity of both ribosomal subunits, almost reaching the base line at the 7th day. Since the body weight of the animals remained essentially unchanged, these results indicate that the turnover of rat-liver ribosomes is not directly correlated with the reported mitotic rate of rat-liver cells ~°. The half-life of rat liver ribosomes estimated from the rather accurate logarithmic decay curve was found to be about 4 ° h, whereas the half-life of liver cells in rats of this age group is estimated to be at least 60 days *°. The results obtained with rat liver differ from those with growing animals cells (see review b y GRAHAM AND RAKE21), and therefore we consider that more information is needed in order to explain the observed discrepancy. In addition, our results indicate that the total ribosomal population is essentially homogeneous with respect to its turnover rate. The fact that the RNA of the two ribosomal subunits is degraded at the same rate suggests that the release of these particles into the cytoplasm is synchronized.
Fractionation o/ rapidly labeled cytoplasmic RigA As shown, in short term labeling experiments with E6-x4C]orotic acid, the 32-S ribosomal subunit has a higher specific activity than the 47-S subunit. This would suggest that a rapidly labeled RNA is associated with the 32-S ribosomal subunit. Therefore, the relation of the rapidly labeled RNA fraction to the ribosomal RNA was further studied. When the RNA extracted with sodium dodecyl sulfatephenol from the postnuclear fraction after 9 ° rain labeling in vivo with [6-14C]orotic acid was fractionated by sucrose density gradient centrifugation, the patterns obtained were identical to those reported b y HIATT4. The same results were observed with cytoplasmic RNA extracted either from the postmitochondrial fraction or from microsomes or ribosomes. In all cases, the labeling of the I8-S RNA was higher than that of the 28-S RNA. The ratio of the specific activities of 18 S:28 S varies from 1.44 to 1.84. Fractionation of the two ribosomal subunits, followed by the separate extraction of their RNA's, yielded a single 28-S RNA peak from the 47-S subunit and a single I8-S peak from the 32-S subunit. These results indicate that the two types of RNA molecules (28 S and 18 S) correspond to the two types of ribosomal subunits (47 S and 32 S). A similar correlation is well documented for bacterial cells2~,~3. The radioactivity of the 28-S and I8-S RNA's followed the A260ma curve, and again, the specific activity of the I8-S RNA was always higher. It should be emphasized that under these conditions we have not been able to detect the rapidly labeled RNA fractions sedimenting between 18 S and 4 S described by others 6-s. The results of these experiments indicate that the rapidly labeled RNA fraction, presumably messenger RNA, is either intimately associated with the IS-S ribosomal RNA, or it has the same sedimentation Biochim. Biophys. Acta, 119 (1966) 547-556
TURNOVER AND MESSENGER ACTIVITY OF LIVER
RNA
553
coefficient. Therefore, an a t t e m p t was made to dissociate the obtained RNA fractions. For this purpose, a linear density gradient was run with I M to 5 M urea solutions as described under METHODS. The results of a typical fractionation of the postmitochondrial RNA are presented in Fig. 3. As can be seen, the fractionation obtained in an urea density gradient is similar to the pattern obtained in sucrose
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Fig. 3. U r e a d e n s i t y g r a d i e n t s e d i m e n t a t i o n p a t t e r n of r a t - l i v e r - p o s t m i t o c h o n d r i a l 1RNA isolated f r o m a n i m a l s killed 9o m i n a f t e r a d m i n i s t r a t i o n in vivo of [6-14C]orotic acid. E6-14C]Orotic acid (3.46/*C/Ioo g b o d y w e i g h t ) w a s i n j e c t e d i n t r a p e r i t o l t e a l l y a n d t h e a n i m a l s killed a f t e r 9o m i n . T h e livers were h o m o g e n i z e d i n m e d i u m A TM a n d t h e h o m o g e n a t e w a s cer~trifuged s u b s e q u e n t l y a t 15oo × g for 5 rain a n d a t io ooo x g for io mill. R N A w a s isolated f r o m t h e l a s t s u p e r n a t a n t as d e s c r i b e d u n d e r METHODS. T h e R N A s a m p l e in O.Ol 4 M NaC1 w a s applied on t o p of a 1-5 M u r e a g r a d i e n t . C e n t r i f u g a t i o u w a s for IO h a t 24 ooo r e v . / m i n . (Spinco, S W 25 rotor). O - O , A260 ms; (D- - - © , couixts/min. T h e s e d i m e n t a t i o n v a l u e s a r e t h o s e r e p o r t e d b y I-IIATT4.
density gradients. The radioactivity of the I8-S RNA is again higher than t h a t of the 28-S RNA. Evidently, the simple centrifugation in urea gradients does not affect the behavior of cytoplasmic RNA's. In further experiments, the 28-S and I8-S RNA's were extracted from the pooled corresponding fractions of several sucrose density gradients (average 18 S:28 S ratio of the specific a c t i v i t y - - 1 . 5 o ) . The two RNA fractions were then heated at 60 ° in 0.4 M urea for IO min, cooled rapidly in dry ice, and applied to more shallow urea gradients. The results are shown in Fig. 4. The Figure shows that the sedimentation rate of both 28-S and I8-S RNA's is decreased, suggesting a disruption of secondary structure, the reformation of helices being made unlikely b y the urea gradient. However, both the 28-S and the I8-S RNA's showed a single peak, the radioactivity coinciding with the A2~0ma curve. Again, the labeling of the I8-S RNA was higher than that of the 28-S RNA, with a 18 S :28 S ratio equal to 1.47. Centrifugation in urea gradients containing I . io -s M E D T A gave essentially the same results. The higher specific activities observed in the 32-S subunit and the corresponding I8-S RNA after short term [6-14C~orotic acid labeling could be interpreted in at least three ways: (a) rat-liver messenger RNA has a sedimentation coefficient close to 18 S, or, if it consists of shorter chains, they are extremely tightly bound to the I8-S ribosomal RNA. (b) The 32-S subunit is released from the nucleus into the Biochim. Biophys. Acta, 119 (1966) 5 4 7 - 5 5 6
554
A . A . HADJIOLOV A
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Fig. 4. U r e a d e n s i t y g r a d i e n t s e d i m e n t a t i o n p a t t e r n s of isolated 28-S (A) a n d I8-S (13) r a t - l i v e r p o s t m i t o c h o n d r i a l r i b o n u c l e i c acids. T h e R N A w a s isolated f r o m a n i m a l s killed 90 m i n after t h e a d m i n i s t r a t i o n of [6-a4Clorotic acid as d e s c r i b e d for Fig. 3. T h e 28-S a n d I8-S 1RNA's were isolated f r o m t h e pooled r e s p e c t i v e f r a c t i o n s o b t a i n e d b y s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . O n e or t w o i n t e r m e d i a t e t u b e s b e t w e e n 28-S a n d 18-S R N A ' s were o m i t t e d . T h e R N A w a s p r e c i p i t a t e d w i t h 2 v o l u m e s of cold 96 % e t h a n o l a n d k e p t o v e r n i g h t a t - - 2 o °. T h e 28-S a n d I8-S R N A ' s were d i s s o l v e d in 0. 4 M urea, h e a t e d a t 60 ° for io m i n , a n d cooled in d r y ice. T h e cooled s o l u t i o n s were a p p l i e d on t o p of linear u r e a d e n s i t y g r a d i e n t s w i t h t h e s h o w n c o n c e n t r a t i o n s (the arrow i n d i c a t e s t h e b o t t o m of t h e tube). C e n t r i f u g a t i o n w a s for 8 h a t 24 ooo r e v . / m i n . (Spinco, S W 25 rotor). O - Q , A260 in#; G - - -Q), c o u n t s / m i n . T h e a r r o w s i n d i c a t e t h e a v e r a g e specific a c t i v i t y in counts/min/mg RNA. Fig. 5. D i s t r i b u t i o n of [14C]lysine-labeled p o l y p e p t i d e p r o d u c t s y n t h e s i z e d in a n E. call cell-free s y s t e m s t i m u l a t e d b y r a t - l i v e r n u c l e a r r i b o n u c l e i c acids. T h e i n c u b a t i o n m i x t u r e is t h e s a m e as d e s c r i b e d for T a b l e II, e x c e p t t h a t t h e final c o n c e n t r a t i o n of [14C!lysine is o.o36/zmole/m]. A f t e r i n c u b a t i o n for 2o m i n a t 35 °, 15o ktg of b o v i n e s e r u m a l b u m i n / m l were a d d e d to each t u b e . T h e r i b o s o m e s were s e d i m e n t e d a t lO 5 ooo × g for 9o m i n . Clear areas, b a c k g r o u n d i n c o r p o r a t i o n ; s h a d e d areas, + 8 o o / t g of " r i b o s o m a l - l i k e " R N A / m l ; s t i p p l e d areas, + 2 5 o / z g of " D N A - l i k e " RNA/ml.
cytoplasm at a faster rate than the 47-S subunit, the higher labeling being inherent in the I8-S ribosomal RNA. (c) The time required to label the complete 32-S subunit (respectively the I8-S RNA) is shorter than the time needed to label the 47-S particle, the two subunits being released into the cytoplasm simultaneously. The results presented suggest that the third alternative is more likely, but do not rule out the first two possibilities.
Stimulation o/ cell-[ree polypeptide synthesis In preliminary experiments with [uC]leucine and [14C]lysine, it was found that after the addition of rat-liver RNA's, amino acid incorporation proceeds linearly with time up to 3o rain and with RNA concentration up to I mg/ml. When RNA is added, the Mg~+ concentration (i4-i6/,moles Mg2+/ml) needed for optimal activity is higher than the optimum for endogenous incorporation. The capacities of different 28-S and I8-S cytoplasmic RNA's to stimulate [14C]lysine incorporation were investigated. These two fractions were isolated from Biochim. Biophys. Acta, 119 (t966) 547-556
TURNOVER AND MESSENGER ACTIVITY OF LIVER TABLE
RNA
555
II
STIMULATION OF THE INCORPORATION ACIDS OF RAT LIVER
OF DIFFERENT
AMINO
ACIDS BY N U C L E A R
RIBONUCLEIC
The reaction mixtures contained, per ml: 160 ~tmoles Tris-HC1 buffer (pH 7.8), 16 ~moles MgCI2, io ~tmoles NH4CI, 4/,moles A T P , 2 ~tmoles G T P , io ~tmoles phosphoenolpyruvate sodium salt, 8o~tg of crystalline pyruvate kinase (EC 2.7.i.4o), 24~tmoles ~-mercaptoethanol, o.o 5 Fmole each of 2o L-amino acids minus the amino acid added as 14C. T h e concentration of the nuclear ribonucleic acids was 2oo ~tg/ml for "DNA-like" I R N A and 7oo ~g/ml for "ribosomal-like IRIWA". E. coli B cell-freesystem -- preincubated S-3 o. Total volume, o.25 ml. Incubation was for 2o rain a t 35 ° .
[14C]amino acid
Background Stimulated inPercent stimulation Final incorporation corporation conch. ~moles/ml) (/H~moles/ml) (izl, moles/ml per mg R N A ) " R i b o s o m a l - " D N A - l i k e " like" R N A RNA "Ribosomal- " D N A - l i k e " like" R N A RNA
Alanine Arginine Glutamic acid Isoleucine Leucine Lysine Phenylalanine Serine Valine
o.o33 o. o 17 o.o16 O.OLO o.o18 o.o 18 o.o12 o.o23 o.02o
4.64 2.oo 1.4o 0.64 7.95 1.35 16.o5 6.52 i. 15
8.47 5.63 2.43 1.68 16.55 4.4 ° 19.9o 16.oo 2.88
13.2o 15.6o 4.35 ii.io 51.7 ° 17.4 ° 36.70 31.6o 5.68
83 182 77 163 lO8 226 24 145 15 °
184 68o 21o 163 ° 55 ° I 19o 128 385 395
sucrose density gradients obtained from: (a) the fraction cytoplasmic RNA obtained as described under METHODS. (b) RNA isolated from the postmitochondrial fraction. (c) The 47-S and 32-S ribosomal subunits extracted with sodium dodecyl sulfatephenol. In all three groups, the stimulation of cell-free [14C]lysine incorporation was higher with I8-S RNA than with 28-S RNA (30-50 ~o with I8-S RNA and 5-1o °/o with 28-S RNA). These observations are in agreement with reported results with [14Clvaline5 and [14C]leucine n. Since large variations were observed and stimulation was relatively low, an accurate quantitative evaluation of the observed effects could not be made. The nuclear "ribosomal-like" RNA and "DNA-like" RNA are both 5-Io-fold more active than the cytoplasmic RNA's. Therefore the messenger activity of these two nuclear fractions was further investigated. The stimulation of the cell free incorporation of different amino acids is presented in Table II. Because of the complexity of the system, no definite conclusions on the amino acid composition of the product of stimulation can be derived. However, these results indicate: (a) that both "ribosomal-like" RNA and "DNA-like" RNA stimulate the incorporation of the different amino acids tested, the stimulation with"DNA-like" RNA being on the average three times higher than that of"ribosomal-like" RNA; and (b) that the estimated amino acid composition of the polypeptide product is similar for the two RNA preparations, despite the fact that they differ markedly in their mononucleotide composition 13. The high incorporation of E14Cllysine and [14Clarginine would suggest a product related to nuclear histones, but no preferential solubility of the stimulated product in 0.25 N HC1 could be detected. Furthermore, it could be shown that almost all of the product due to stimulation remained bound to the ribosomes, whereas the endogenous incorporation was located mainly on the lO5 ooo x g supernatant fraction (Fig. 5). Biochim. Biophys. Acta, 119 (1966) 547-556
556
A.A. HADJIOLOV
ACKNOWLEDGEMENTS
This work was carried out in the laboratory of Dr. F. LIPMANN,and was supported in part by a grant from the National Science Foundation and from the Inter-University Committee on Travel Grants. The author would like to thank Dr. LIPMANN for his generous counsel, and for stimulating discussions throughout the course of this work.
REFERENCES I 2 3 4 5 6 7 8 9 io II I2 13 14 15 16 17 18 19 20 21 22 23
A. VON DER DECKEN AND P. N. CAMPBELL, Biochem. J., 84 (1962) 449P. N. CAMPBELL, Progr. Biophys. Mol. Biol., 15 (1965) i. M. C. GANOZA, C. A. WILLIAMS AND F. LIPMANN, Proc. Natl. Acad. Sci. U.S., 53 (1965) 619. H. H. HIATT, J. Mol. Biol., 5 (1962) 217 . A. DIGIROLAMO, E. C. HENSHAW AND H. H. HIATT, J. Mol. Biol., 8 (1964) 479. T. STAEHELIN, F. O. WETTSTEIN, H. OURA AND H. NULL, Nature, 2Ol (1964) 264. A. J. MUNRO AND A. KORNER, Nature, 2Ol (1964) 1194. A. TRAKATELLIS, A. E. AXELROD AND M. MONTJAR, J. Biol. Chem., 239 (1964) 4237 • S. H. BARONDES, C. W. DINGMAN AND M. B. SPURN, Nature, 196 (1963) 145. N. LANG AND E. SEKERIS, Life Sci., 3 (1964) 161. G. BRAWERMAN, N. BIEZUNSKI AND J. EISENSTADT, Biochim. Biophys. Acta, lO 3 (1965) 2Ol. Y. TASHIRO AND P. SIEKEVITZ, J. Mol. Biol., I I (1965) I49. G. P. GEORGIEV AND V. P. MANTIEVA, Biokhimia, 27 (1962) 949. R. G. TSANEV, G. G. MARKOV AND G. DESSEV, Biochem. J., i n t h e press. R. J. BRITTEN AND R. B. ROBERTS, Science, 131 (196o) 32 . Y. TASHIRO AND P. SIEKEVITZ, J. Mol. Biol., I I (1965) 166. M. W. NIRENBERG, i n S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 6, A c a d e m i c Press, N e w Y o r k , 1963, p. 17. M. GIRARD, S. PENMAN AND J. E. DARNELL, Proc. Natl. Acad. Sci. U.S., 51 (1964) 205. D. M. PRESCOTT, Progr. Nucleic Acid Res. and Mol. Biol., 3 (1964) 33. R. A. McDONALD, Arch. Intern. Med., lO 7 (1961) 335. A. F. GRAHAM AND A. V. RAKE, Ann. Rev. Microbiol., 17 (1963) 139. A. S. SPIRIN, Macromolecular Structure o/Ribonucleic A cids, R e i n h o l d , N e w Y o r k , 1964, p. 21 o. W. M. STANLEY AND R. M. BUCK, Biochemistry, 4 (1965) 13°2.
Biochim. Biophys. Acta, 119 (1966) 547-556
547
BBA 95406
S T U D I E S ON T H E T U R N O V E R AND M E S S E N G E R ACTIVITY, OF R A T - L I V E R R I B O N U C L E I C ACIDS
A. A. HADJIOLOV*
The Rocke/eller University, New York, N.Y. (U.S.A.) (Received September i7th , 1965)
SUMMARY
In studies with E14C]orotic acid, rat-liver ribosomes were found to exhibit a turnover with a half-life of about 40 h. This rate far exceeds the reported mean generation time of liver cells of at least 60 days. The two ribosomal subunits are degraded at essentially the same rate. After short-term labeling, initially the smaller ribosomal subunit and the derived I8-S RNA have a higher specific activity than the large ribosomal subunit and the derived 28-S RNA. The more highly labeled fraction could not be dissociated from the I8-S ribosomal RNA b y various treatments, including heating with 0. 4 M urea at 60 ° and subsequent centrifugation in urea density gradients. Of the nuclear RNA fractions extracted with sodium dodecyl sulfate/ phenol at 45 ° and 65 ° respectively, the RNA extracted at higher temperature had a 3-fold higher messenger activity in the Escherichia coli cell-free system. The synthesized product remained almost completely bound to the ribosomes, and no specific pattern in the stimulated incorporation of different amino acids could be detected.
INTRODUCTION
Experiments from a number of laboratoriesZ, 2, including recent reports from this laboratory ~, established that rat-liver microsomes and ribosomes differ markedly in their capacity to synthesize specific proteins, although the incorporation of amino acids is similar in both systems. Obviously, the understanding of the mechanism of specific protein synthesis requires a better knowledge of the manner in which messenger RNA interacts with ribosomes and probably with ribosomal RNA. In rat-liver cytoplasm a rapidly labeled RNA fraction has been found that sediments in sucrose gradients together with I8-S ribosomal RNA4, 5, but also lower and more dispersed sedimentation values are reported 6-s. These experiments have indicated, further* Present address: Biochemical Research Laboratory, Bulgarian Academy of Sciences, Sofia (Bulgaria).
Biochim. Biophys. Acta, 119 (1966) 547 556
548
h.a.
HADJIOLOV
more, that the ribosomal RNA of liver is labeled quite rapidly; its turnover, however, has not been investigated. During preparation of this manuscript, we learned of results by LOEB, HOWELLAND TOMKINS*on the decay of I14CJorotic acid-labeled RNA from "free ribosomes" of rat liver, with conclusions on the turnover of rat-liver ribosomes similar to those discussed here. Nuclear RNA stimulates polypeptide synthesis in cell-free extractsS,gJ ° to a much greater extent than the cytoplasmic RNA, which is associated mainly with the I8-S ribosomal RNAS, n. No characterization of the product synthesized as a result of addition of these RNA's has been reported.
METHODS
Experimental animals The experiments were carried out with male Sprague-Dawley albino rats with a body weight of 12o-16o g, fed ad libitum with the standard laboratory diet. In the labeling experiments in vivo, animals with a body weight of 12o-4-1o g were used. In control experiments, starvation of the animals did not affect the parameters studied. Ribosomes and ribosomal subunits were prepared according to the procedur e of TASHIRO AND SIEKEVITZ 12, except that intermediate cytoplasmic fractions were obtained by centrifuging the original homogenate at 15oo ×g for 5 min to sediment the nuclei, and at IO ooo ×g for IO min to sediment the mitochondria. Microsomes and ribosomes were then isolated from the postmitochondrial fractions. The "crude" ribosomal fraction was dissociated with 5/,moles EDTA/mg ribosomes to obtain the two ribosomal subunits. The quantity of ribosomes and ribosomal subunits was estimated on the basis of an extinction coefficient of 135 at 260 m/~ for a I °/o ribosome suspension. The average r a t i o A26om#/A235m# was 1.36 for crude ribosomes, 1.68 for the 47-S subunit, and 1.69 for the 32-S subunit.
Isolation o/ ribonucleic acids Liver RNA's were isolated and fractionated by the method of GEORGIEV AND MANTIEVA 13, modified as follows. The livers of lO-2O rats were removed, rinsed with
o.14 M NaC1, and homogenized directly in phenol (saturated with o.14 M NaC1 at pH 5.0). The homogenate was shaken for 30 min at 4 ° and centrifuged at IO ooo ×g for IO min. The water layer was aspirated and processed further to yield the fraction of cytoplasmic RNA. The interphase layer was extracted twice more at 4 ° with phenol-NaC1 for 15 rain and the water phases were discarded. After the last treatment the interphase layer was suspended in o.14 M NaC1; sodium dodecyl sulfate was then added to a final concentration of 0.25 %, followed by an equal volume of phenol. The mixture was extracted at 45 ° for 20 min, cooled, and centrifuged at IO ooo x g for IO min. The water phase was aspirated and used to prepare the nuclear "ribosomal-like" RNA. The resulting interphase layer was extracted once more for 5 rain at 45 °, and after centrifugation, the water layer was discarded and the interphase layer was suspended in o.14 M NaC1, made 0.25 % with respect to sodium " Personal communication. Biochim. Biophys. Acta, 119 (1966) 547-556
TURNOVER AND MESSENGER ACTIVITY OF LIVER
RNA
549
dodecyl sulfate, and an equal volume of phenol was again added. The mixture was heated at 65 ° for 20 min with continuous shaking, and was cooled, and centrifuged at IO ooo ×g for IO min. The water layer was used to prepare the nuclear " D N A like" RNA fraction. The cytoplasmic and the two nuclear fractions were deproteinized 3-5 times with cold phenol, and the RNA from the final water layers was precipitated with 2 volumes of 96 °/o ethanol at --20 °. The RNA precipitate was washed several times with ethanol, drained carefully, and dissolved in deionized water. The average yield of the different RNA fractions was: cytoplasmic RNA, 90-92 °/o; "ribosomallike" RNA, 4-6 °/o; and "DNA-like" RNA, 2-3 %. Ribosomes or ribosomal subunits were suspended in o.14 M NaC1 and RNA was extracted with an equal volume of cold phenol after the addition of 0.25 % sodium dodecyl sulfate. The different RNA preparations were purified from low molecular weight impurities by passage through a Sephadex G-25 column 14. The quantity of RNA was estimated by its absorbance at 26o m# (24 absorbance units = I mg RNA).
Density gradients Linear density gradients prepared according to BRITTEN AND ROBERTSls were used in all experiments. Centrifugation at 24 ooo rev./min for 9 or 12 h intervals was carried out in the Spinco, model L-2 ultracentrifuge using the SW 25 rotor at 3-4 °. Sucrose gradients (5-2o %) were used for the fractionation of ribosomal subunits 16 or cytoplasmic RNA's 4. Urea density gradients were prepared with solutions of urea (purified from ultraviolet absorbing material by Norit pretreatment) in deionized water, and adjusted to p H 7.0. The density of I M and 5 M urea corresponds approximately to that of 5 and 20 °/o sucrose. Fractions of about i ml were collected after puncturing the bottom of the tube. Aliquots of the appropriate volume were pipetted for absorbance and radioactivity determinations.
Cell-[tee amino acid incorporation An Escherichia coli B cell-free system was prepared according to the procedure of NIRENBERGiv. In all experiments, the preincubated S-3o extract was used, dialysis of the preparation being replaced b y gel filtration through a Sephadex G-25 column equilibrated with the appropriate medium.
Radioactivity determinations Incorporation of amino acids was determined after heating the 5 % trichloroacetic acid precipitates for 20 min at 9 o°. The precipitates were plated on Millipore membrane filters, dried, and counted in a Nuclear Chicago gasf-low counter at about 22 ~o efficiency. Samples of ribosomes or RNA were precipitated with 5 % cold trichloroacetic acid after addition of 50 #g of bovine serum albumin to each sample.
MATERIALS
Analytical grade reagents were used. Uniformly labeled E14C]amino acids with Biochim. Biophys. Acta, 119 (1966) 547 556
55 °
A.A.
HADJIOLOV
specific activities of about 2oo mC/mmole were obtained from New England Nuclear Corp., Boston, Mass. E14C]orotic acid (30 mC/mmole) was a product of Nuclear Chicago Corp., Chicago, Ill. (U.S.A.).
RESULTS AND DISCUSSION
Turnover o[ rat-liver ribosomes and ribosomal subunits After dissociation of the "crude" ribosomal fraction, only 47-S and 32-S ribosomal subunits are detected on the sucrose gradient. In addition, varying amounts of undissociated ribosomes sediment to the bottom of the tube. After short term labeling in vivo with E6-14C]orotic acid, the radioactivity curve follows closely the A260 m~ curve (Fig. IA). Following such short term labeling, furthermore, a rapidly labeled fraction of low sedimentation value is also observed, probably reflecting the fast turnover of cytidylic acid in soluble RNA. The specific activity of the 32-S subunit is appreciably higher than that of the 47-S subunit; 9 ° min after injection of [6-14C]orotic acid, the ratio of the specific radioactivities 32 S: 47 S is 1.48. In experiments in which [12C]orotic acid is introduced I h after the [x4C3orotic acid, this ratio decreases rapidly, to 1.13 after 4 h and close to I.O after 8 h, remaining so in subsequent determinations (Table I, Fig. Ib). TABLE
I
SPECIFIC RADIOACTIVITY OF RAT-LIVER RIBOSOMAL SIJBUNITS AFTER LABELING [6-14C]OROTIC ACID AND [12C]OROTIC ACID CHASE
in vivo
WITH
T h e e x p e r i m e n t a l c o n d i t i o n s a r e t h e s a m e a s d e s c r i b e d f o r F i g s . I a n d 2. T h e s a m e e x t i n c t i o n c o e f f i c i e n t w a s u s e d f o r t h e 47-S a n d 32-S s u b u n i t .
Hours a/ter [l*C]orotic acid 1. 5 4 8 27
Hours a/ter [l~C]orotic acid I 3 7 26
Speci/ic radioactivity (Counts/rain/rag ribosomes) 47 S
32 S
21o 47 ° 126o 3720
31o 53 ° 124o 3720
Ratio 32 S/47 S
1.48 1-13 o.98 I.OO
In order to study the turnover of liver ribosomes and their subunits, a 6o-min pulse with E634C]orotic acid was followed b y the administration of an approx. 30o times higher dose of unlabeled orotic acid. The labeling of the two ribosomal subunits was followed up to 168 h and the specific activities of the free nucleotides and nuclear RNA (total nuclear RNA extracted at 65°with sodium dodecylsulfatephenol) were determined. The results of these experiments are presented in Fig. 2. As can be seen, a three-fold decrease in the labeling of the free nucleotides occurs in the first 4 h, and the label nearly disappears after 16 h. These results show that the pyrimidine nucleotide pool size in liver is relatively small, thus allowing efficient dilution of the label with added unlabeled orotic acid. The decrease in the specific activity of the nuclear RNA proceeds at a slower rate during the first 48 h. In contrast, the specific activity of the two ribosomal subunits increases almost Biochim. Biophys. Acta,
119 ( I 9 6 6 ) 547--556
TURNOVER
,oog
AND
MESSENGER
A C T I V I T Y OF L I V E R
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A
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55~
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Fig. I. Sucrose density gradient sedimentation p a t t e r n of rat-liver ribosomal subunitsisolated from animals killed 90 miri (A) and 8 h (B) after administration in vivo of [6-1*C]orotic acid and subsequent chase with [l*C]orotic acid. [6-1*C]Orotic acid was injected intraperitoneally at zero time (2/iC/i2o g body weight), followed after 6o min by unlabeled orotic acid (20/~mole/I2O g body weight). Centrifugation was for 9 h at 24 ooo rev./min (Spinco SW 25 rotor). The radioactivity represents the counts precipitated with 5 % trichloroacetic acid at 4 °. 0 - 0 , A260m#; (2)---O, counts/min. The sedimentation coefficients are those reported by TASHIRO AND SIEKEVITZ
12.
'~
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Fig. 2. Specific radioactivity of rat-live~--ribosomal subunlts and free nucleotides after admiixistratiorl iii vivo of [6-x~0]orotic acid alxd subsequent chase with unlabeled orotic acid. Administration of [6-1*C]orotic acid (arrow-ziC] and of unlabeled orotic acid (arrow-12C) as described for Fig. I. The pooled livers from groups of four rats (body weight, 12O-/lO g) were used for the isolation of ribosomal subunits. During the whole period the animals were fed ad libitum with the standard laboratory diet. Abscissa, hours after the administration of [6-1¢C]orotic acid. The same extinction coefficient was used for the estimation of the q u a n t i t y of b o t h the 47-S and 32-S subunit (see METHODS). Biochim. Biophys. Acta, 119 (1966) 547-556
552
A . A . HADJIOLOV
linearly up to about 3 ° h, when it reaches a m a x i m u m that is about 15 times higher than the values obtained at 9 ° min. Analysis of this part of the curve suggests that ribosome formation in the nucleus precedes the release of these particles into the cytoplasm. These results are in agreement with those for H e L a cells reported by GIRARD, PENMAN AND DARNELL is who observed a release of ribosomes into the cytoplasm after actinomycin block of RNA synthesis (see review b y PRESCOTT19). Analysis of the curve in later periods shows a gradual and parallel decrease in the specific activity of both ribosomal subunits, almost reaching the base line at the 7th day. Since the body weight of the animals remained essentially unchanged, these results indicate that the turnover of rat-liver ribosomes is not directly correlated with the reported mitotic rate of rat-liver cells ~°. The half-life of rat liver ribosomes estimated from the rather accurate logarithmic decay curve was found to be about 4 ° h, whereas the half-life of liver cells in rats of this age group is estimated to be at least 60 days *°. The results obtained with rat liver differ from those with growing animals cells (see review b y GRAHAM AND RAKE21), and therefore we consider that more information is needed in order to explain the observed discrepancy. In addition, our results indicate that the total ribosomal population is essentially homogeneous with respect to its turnover rate. The fact that the RNA of the two ribosomal subunits is degraded at the same rate suggests that the release of these particles into the cytoplasm is synchronized.
Fractionation o/ rapidly labeled cytoplasmic RigA As shown, in short term labeling experiments with E6-x4C]orotic acid, the 32-S ribosomal subunit has a higher specific activity than the 47-S subunit. This would suggest that a rapidly labeled RNA is associated with the 32-S ribosomal subunit. Therefore, the relation of the rapidly labeled RNA fraction to the ribosomal RNA was further studied. When the RNA extracted with sodium dodecyl sulfatephenol from the postnuclear fraction after 9 ° rain labeling in vivo with [6-14C]orotic acid was fractionated by sucrose density gradient centrifugation, the patterns obtained were identical to those reported b y HIATT4. The same results were observed with cytoplasmic RNA extracted either from the postmitochondrial fraction or from microsomes or ribosomes. In all cases, the labeling of the I8-S RNA was higher than that of the 28-S RNA. The ratio of the specific activities of 18 S:28 S varies from 1.44 to 1.84. Fractionation of the two ribosomal subunits, followed by the separate extraction of their RNA's, yielded a single 28-S RNA peak from the 47-S subunit and a single I8-S peak from the 32-S subunit. These results indicate that the two types of RNA molecules (28 S and 18 S) correspond to the two types of ribosomal subunits (47 S and 32 S). A similar correlation is well documented for bacterial cells2~,~3. The radioactivity of the 28-S and I8-S RNA's followed the A260ma curve, and again, the specific activity of the I8-S RNA was always higher. It should be emphasized that under these conditions we have not been able to detect the rapidly labeled RNA fractions sedimenting between 18 S and 4 S described by others 6-s. The results of these experiments indicate that the rapidly labeled RNA fraction, presumably messenger RNA, is either intimately associated with the IS-S ribosomal RNA, or it has the same sedimentation Biochim. Biophys. Acta, 119 (1966) 547-556
TURNOVER AND MESSENGER ACTIVITY OF LIVER
RNA
553
coefficient. Therefore, an a t t e m p t was made to dissociate the obtained RNA fractions. For this purpose, a linear density gradient was run with I M to 5 M urea solutions as described under METHODS. The results of a typical fractionation of the postmitochondrial RNA are presented in Fig. 3. As can be seen, the fractionation obtained in an urea density gradient is similar to the pattern obtained in sucrose
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Fig. 3. U r e a d e n s i t y g r a d i e n t s e d i m e n t a t i o n p a t t e r n of r a t - l i v e r - p o s t m i t o c h o n d r i a l 1RNA isolated f r o m a n i m a l s killed 9o m i n a f t e r a d m i n i s t r a t i o n in vivo of [6-14C]orotic acid. E6-14C]Orotic acid (3.46/*C/Ioo g b o d y w e i g h t ) w a s i n j e c t e d i n t r a p e r i t o l t e a l l y a n d t h e a n i m a l s killed a f t e r 9o m i n . T h e livers were h o m o g e n i z e d i n m e d i u m A TM a n d t h e h o m o g e n a t e w a s cer~trifuged s u b s e q u e n t l y a t 15oo × g for 5 rain a n d a t io ooo x g for io mill. R N A w a s isolated f r o m t h e l a s t s u p e r n a t a n t as d e s c r i b e d u n d e r METHODS. T h e R N A s a m p l e in O.Ol 4 M NaC1 w a s applied on t o p of a 1-5 M u r e a g r a d i e n t . C e n t r i f u g a t i o u w a s for IO h a t 24 ooo r e v . / m i n . (Spinco, S W 25 rotor). O - O , A260 ms; (D- - - © , couixts/min. T h e s e d i m e n t a t i o n v a l u e s a r e t h o s e r e p o r t e d b y I-IIATT4.
density gradients. The radioactivity of the I8-S RNA is again higher than t h a t of the 28-S RNA. Evidently, the simple centrifugation in urea gradients does not affect the behavior of cytoplasmic RNA's. In further experiments, the 28-S and I8-S RNA's were extracted from the pooled corresponding fractions of several sucrose density gradients (average 18 S:28 S ratio of the specific a c t i v i t y - - 1 . 5 o ) . The two RNA fractions were then heated at 60 ° in 0.4 M urea for IO min, cooled rapidly in dry ice, and applied to more shallow urea gradients. The results are shown in Fig. 4. The Figure shows that the sedimentation rate of both 28-S and I8-S RNA's is decreased, suggesting a disruption of secondary structure, the reformation of helices being made unlikely b y the urea gradient. However, both the 28-S and the I8-S RNA's showed a single peak, the radioactivity coinciding with the A2~0ma curve. Again, the labeling of the I8-S RNA was higher than that of the 28-S RNA, with a 18 S :28 S ratio equal to 1.47. Centrifugation in urea gradients containing I . io -s M E D T A gave essentially the same results. The higher specific activities observed in the 32-S subunit and the corresponding I8-S RNA after short term [6-14C~orotic acid labeling could be interpreted in at least three ways: (a) rat-liver messenger RNA has a sedimentation coefficient close to 18 S, or, if it consists of shorter chains, they are extremely tightly bound to the I8-S ribosomal RNA. (b) The 32-S subunit is released from the nucleus into the Biochim. Biophys. Acta, 119 (1966) 5 4 7 - 5 5 6
554
A . A . HADJIOLOV A
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Fig. 4. U r e a d e n s i t y g r a d i e n t s e d i m e n t a t i o n p a t t e r n s of isolated 28-S (A) a n d I8-S (13) r a t - l i v e r p o s t m i t o c h o n d r i a l r i b o n u c l e i c acids. T h e R N A w a s isolated f r o m a n i m a l s killed 90 m i n after t h e a d m i n i s t r a t i o n of [6-a4Clorotic acid as d e s c r i b e d for Fig. 3. T h e 28-S a n d I8-S 1RNA's were isolated f r o m t h e pooled r e s p e c t i v e f r a c t i o n s o b t a i n e d b y s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . O n e or t w o i n t e r m e d i a t e t u b e s b e t w e e n 28-S a n d 18-S R N A ' s were o m i t t e d . T h e R N A w a s p r e c i p i t a t e d w i t h 2 v o l u m e s of cold 96 % e t h a n o l a n d k e p t o v e r n i g h t a t - - 2 o °. T h e 28-S a n d I8-S R N A ' s were d i s s o l v e d in 0. 4 M urea, h e a t e d a t 60 ° for io m i n , a n d cooled in d r y ice. T h e cooled s o l u t i o n s were a p p l i e d on t o p of linear u r e a d e n s i t y g r a d i e n t s w i t h t h e s h o w n c o n c e n t r a t i o n s (the arrow i n d i c a t e s t h e b o t t o m of t h e tube). C e n t r i f u g a t i o n w a s for 8 h a t 24 ooo r e v . / m i n . (Spinco, S W 25 rotor). O - Q , A260 in#; G - - -Q), c o u n t s / m i n . T h e a r r o w s i n d i c a t e t h e a v e r a g e specific a c t i v i t y in counts/min/mg RNA. Fig. 5. D i s t r i b u t i o n of [14C]lysine-labeled p o l y p e p t i d e p r o d u c t s y n t h e s i z e d in a n E. call cell-free s y s t e m s t i m u l a t e d b y r a t - l i v e r n u c l e a r r i b o n u c l e i c acids. T h e i n c u b a t i o n m i x t u r e is t h e s a m e as d e s c r i b e d for T a b l e II, e x c e p t t h a t t h e final c o n c e n t r a t i o n of [14C!lysine is o.o36/zmole/m]. A f t e r i n c u b a t i o n for 2o m i n a t 35 °, 15o ktg of b o v i n e s e r u m a l b u m i n / m l were a d d e d to each t u b e . T h e r i b o s o m e s were s e d i m e n t e d a t lO 5 ooo × g for 9o m i n . Clear areas, b a c k g r o u n d i n c o r p o r a t i o n ; s h a d e d areas, + 8 o o / t g of " r i b o s o m a l - l i k e " R N A / m l ; s t i p p l e d areas, + 2 5 o / z g of " D N A - l i k e " RNA/ml.
cytoplasm at a faster rate than the 47-S subunit, the higher labeling being inherent in the I8-S ribosomal RNA. (c) The time required to label the complete 32-S subunit (respectively the I8-S RNA) is shorter than the time needed to label the 47-S particle, the two subunits being released into the cytoplasm simultaneously. The results presented suggest that the third alternative is more likely, but do not rule out the first two possibilities.
Stimulation o/ cell-[ree polypeptide synthesis In preliminary experiments with [uC]leucine and [14C]lysine, it was found that after the addition of rat-liver RNA's, amino acid incorporation proceeds linearly with time up to 3o rain and with RNA concentration up to I mg/ml. When RNA is added, the Mg~+ concentration (i4-i6/,moles Mg2+/ml) needed for optimal activity is higher than the optimum for endogenous incorporation. The capacities of different 28-S and I8-S cytoplasmic RNA's to stimulate [14C]lysine incorporation were investigated. These two fractions were isolated from Biochim. Biophys. Acta, 119 (t966) 547-556
TURNOVER AND MESSENGER ACTIVITY OF LIVER TABLE
RNA
555
II
STIMULATION OF THE INCORPORATION ACIDS OF RAT LIVER
OF DIFFERENT
AMINO
ACIDS BY N U C L E A R
RIBONUCLEIC
The reaction mixtures contained, per ml: 160 ~tmoles Tris-HC1 buffer (pH 7.8), 16 ~moles MgCI2, io ~tmoles NH4CI, 4/,moles A T P , 2 ~tmoles G T P , io ~tmoles phosphoenolpyruvate sodium salt, 8o~tg of crystalline pyruvate kinase (EC 2.7.i.4o), 24~tmoles ~-mercaptoethanol, o.o 5 Fmole each of 2o L-amino acids minus the amino acid added as 14C. T h e concentration of the nuclear ribonucleic acids was 2oo ~tg/ml for "DNA-like" I R N A and 7oo ~g/ml for "ribosomal-like IRIWA". E. coli B cell-freesystem -- preincubated S-3 o. Total volume, o.25 ml. Incubation was for 2o rain a t 35 ° .
[14C]amino acid
Background Stimulated inPercent stimulation Final incorporation corporation conch. ~moles/ml) (/H~moles/ml) (izl, moles/ml per mg R N A ) " R i b o s o m a l - " D N A - l i k e " like" R N A RNA "Ribosomal- " D N A - l i k e " like" R N A RNA
Alanine Arginine Glutamic acid Isoleucine Leucine Lysine Phenylalanine Serine Valine
o.o33 o. o 17 o.o16 O.OLO o.o18 o.o 18 o.o12 o.o23 o.02o
4.64 2.oo 1.4o 0.64 7.95 1.35 16.o5 6.52 i. 15
8.47 5.63 2.43 1.68 16.55 4.4 ° 19.9o 16.oo 2.88
13.2o 15.6o 4.35 ii.io 51.7 ° 17.4 ° 36.70 31.6o 5.68
83 182 77 163 lO8 226 24 145 15 °
184 68o 21o 163 ° 55 ° I 19o 128 385 395
sucrose density gradients obtained from: (a) the fraction cytoplasmic RNA obtained as described under METHODS. (b) RNA isolated from the postmitochondrial fraction. (c) The 47-S and 32-S ribosomal subunits extracted with sodium dodecyl sulfatephenol. In all three groups, the stimulation of cell-free [14C]lysine incorporation was higher with I8-S RNA than with 28-S RNA (30-50 ~o with I8-S RNA and 5-1o °/o with 28-S RNA). These observations are in agreement with reported results with [14Clvaline5 and [14C]leucine n. Since large variations were observed and stimulation was relatively low, an accurate quantitative evaluation of the observed effects could not be made. The nuclear "ribosomal-like" RNA and "DNA-like" RNA are both 5-Io-fold more active than the cytoplasmic RNA's. Therefore the messenger activity of these two nuclear fractions was further investigated. The stimulation of the cell free incorporation of different amino acids is presented in Table II. Because of the complexity of the system, no definite conclusions on the amino acid composition of the product of stimulation can be derived. However, these results indicate: (a) that both "ribosomal-like" RNA and "DNA-like" RNA stimulate the incorporation of the different amino acids tested, the stimulation with"DNA-like" RNA being on the average three times higher than that of"ribosomal-like" RNA; and (b) that the estimated amino acid composition of the polypeptide product is similar for the two RNA preparations, despite the fact that they differ markedly in their mononucleotide composition 13. The high incorporation of E14Cllysine and [14Clarginine would suggest a product related to nuclear histones, but no preferential solubility of the stimulated product in 0.25 N HC1 could be detected. Furthermore, it could be shown that almost all of the product due to stimulation remained bound to the ribosomes, whereas the endogenous incorporation was located mainly on the lO5 ooo x g supernatant fraction (Fig. 5). Biochim. Biophys. Acta, 119 (1966) 547-556
556
A.A. HADJIOLOV
ACKNOWLEDGEMENTS
This work was carried out in the laboratory of Dr. F. LIPMANN,and was supported in part by a grant from the National Science Foundation and from the Inter-University Committee on Travel Grants. The author would like to thank Dr. LIPMANN for his generous counsel, and for stimulating discussions throughout the course of this work.
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Biochim. Biophys. Acta, 119 (1966) 547-556