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Functional stability of messenger RNA in potato tuber slices
403
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95636
F U N C T I O N A L S T A B I L I T Y OF MESSENGER RNA IN POTATO T U B E R SLICES
R O B E R T E. CLICK* AND D A V I D P. H A C K E T T * *
Department o] Biochemistry, University o] Cali[ornia, Berkeley, Cali[. (U.S.A.) (Received D e c e m b e r I9th, 1966)
SUMMARY
I. The functional stability of messenger RNA (mRNA) was determined on potato tuber slices that had either an active or inactive metabolic rate. 2. Using the decline of amino acid incorporation in the presence of actinomycin as an index, the functional half-life during the inactive phase was 1.6 h. During the same period, the RNA required for the respiratory increase (4-fold in one day) had a half-life of 1. 7 h. 3. The functional stability during the active phase ( > 20 h incubation) was comprised of three types; one possessing a half-life of 1.6 h, one a half-life of over 2 h and a third that may have been pre-existing, but inactive.
INTRODUCTION
Since the original concept of messenger RNA (mRNA) I, many of the characteristic properties of this class of molecales have been investigated. One kind of characterization has involved determining the duration after cessation of RNA synthesis during which protein synthesis can still be maintained. Such characterizations have been interpreted in terms of the functional lifetime of m R N A and have been found to be on the order of a few minutes in bacteria 2-4 and a few hoursS, 6 or days 7-9 in animal cells. To date, however, very little evidence has accumulated concerning the characterization of m R N A in plant tissue 1°-12 and even less information is available concerning the m R N A stability 13-~5. A useful system for studying m R N A in plant tissue is based on the 4-fold increase in respiratory rate that develops in potato tuber slices incubated aerobically for 24 h; a process that depends on both RNA and RNAmediated protein synthesis 16. Furthermore, the stability can be compared at different rates of metabolism; namely an inactive (0-7 h) and an active ( > 18 h) state as measured by salt uptakO 7, protein synthesis le or carbohydrate metabolism as,19. The present communication is concerned with the determination of the functional stability of m R N A in these slices. * P r e s e n t address: D e p a r t m e n t of Zoology, U n i v e r s i t y of Wisconsin, Madison, Wisc., U.S.A. *~ Deceased: J a n u a r y 2I, 1965. Abbreviation: m R N A , m e s s e n g e r R N A .
Biochim. Biophys. Acta, 142 (1967) 403-409
404
R . E . CLICK, D. P. HACKETT
MATERIALS AND METHODS
Monthly shipments of potato tubers (Solanum tuberosurn, var. White Rose) were obtained monthly through the courtesy of Dr. H. TIMM, Department of Vegetable Crops, University of California at Davis and stored at 21 °. Cylinders of tissue were removed with a cork borer (I cm diameter) and I-ram-thick slices were cut therefrom using a hand microtome. The slices were washed for 25 rain in sterile, deionized water and then incubated in 0.02 M potassium phosphate (pH 6.7), I" Io -4 M CaSO~, I. Io -3 M MgC12, 50//g/ml dihydrostreptomycin and IOO units/ml penicillin at 21 ° in small petri dishes a6. Protein synthesis was determined by measuring the amount of !I-a4C]leucine incorporated into the slices. After appropriate intervals of incubation, the slices were removed from the radioactive solution and washed in 25 ml of non-radioactive leucine (0.2 mg/ml) for 30 rain to remove any readily exchangeable [aaClleucine. They were then extracted with 7° ~o ethanol for 8 h in a Soxhlet apparatus, after which they were rinsed briefly with absolute ethanol. The alcohol-insoluble residue was dried at 8o ° and counted directly in an automatic gas flow apparatus (NuclearChicago) with an efficiency of approx. 5 %. The alcohol-soluble material was concentrated and counted in a scintillation spectrometer with an efficiency of 55 %
RESULTS
The measurement of the metabolic stability of RNA can most easily be made in the absence of its synthesis. Therefore, actinomycin C1 (Merck, Sharp and Dohme), the classical inhibitor of RNA synthesis 2°, was employed in the experiments to be described. It will be assumed that actinomycin neither increased nor decreased the decay rate of m R N A 21-23 although there are reports challenging these assumptions 24,2~.
I. Actinomyein effects on respiration and RNA synthesis In light of the lethal action that actinomyein has on rats 9 and on mammalian tissue culture cells ~6, the toxicity of the antibiotic was examined by determining the TABLE I EFFECTS OF DINITROPHENOL ON FRESH SLICES AND SLICES AGED IN 25 p g / m l ACTINOMYCIN The r e s p i r a t o r y r a t e (#1 O2/h p e r g) w a s m e a s u r e d b y s t a n d a r d W a x b u r g m a n o m e t r i c t e c h n i q u e s a t p H 6.0 a f t e r a t o t a l i n c u b a t i o n t i m e of 24 h ( e x c e p t for t h e fre s h slices w h i c h w e r e a n a l y z e d imm e d i a t e l y ) . T h e c o n t r o l r a t e w a s d e t e r m i n e d p r i o r to t h e m e a s u r e m e n t in d i n i t r o p h e n o l (5" i o 5 M, p H 6.0).
Time o~ actionomycin addition
kd 02/h per g Control
DNP
A
F r e s h slices Initial 5 h I1 h Never
24 22 93 II 7 117
5° 46 122 143 145
26 24 29 26 28
Biochim. Biophys. Acta, 142 (1967) 403-409
MESSENGER RNA
4o7
described in other systems ~3-s7 or the activation of pre-existing non-translatable mRNA3S,39. It is rather unlikely that the first possibility is correct, since the percent inhibition of RNA synthesis increased directly with the length of incubation in the antibiotic. The pool size also is an unlikely factor since the control and treated tissue had equivalent isotopic material at the 48-h period, whereas the amount of isotope incorporated at this time is 2-fold higher in the control. Further experimental data will be required to understand this observation.
I I I . Actinomycin e/]ects on the increase in respiratory activity The final criterion used to measure the functional half-life is perhaps the most meaningful, namely the loss of the capacity to increase enzymatic activity. The enzyme(s) analyzed in this tissue are those involved in the development of the respiratory capacity. As discussed earlier, actinomycin has no effect on the respirat o r y rate of the tissue at the time of its addition (Table I); furthermore, puromycin, a protein synthesis inhibitor, also has no effect on the rate TM. These findings suggest that the enzyme(s) responsible for the increased respiratory rate were metabolically stable proteins. Thus analysis of the respiratory rate can be determined at a single time (all measurements were made after a total incubation of 24 h). The effect on the respiratory rate of adding actinomycin at different times of incubation is given in Fig. 3- If added at zero time, the increase in the respiratory i
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Fig. 3. The effect of delayed additions of a c t i n o m y c i n (to 25 ~g/ml) and p u r o m y c i n (to 5" lO-4 M) on the d e v e l o p m e n t of the r e s p i r a t o r y capacity. The inhibitors were added after o, 2.5 and 5 h of incubation. The r e s p i r a t o r y rate was m e a s u r e d at the end of 24 h. Q - O , control; [ ~ - - - [ ~ , a c t i n o m y c i n ; A- - - A, puromycin.
rate was completely suppressed. However, if added after 2.5 or 5 h of incubation, 31 9/0 and 61 °/o of the maximal increase occurred respectively. However, the minim u m time required for actinomycin to completely prevent the respiratory increase, when added at either 2.5 or 5 h, was constant, requiring 4-4-5 h (calculated from Fig. 3). These results suggest that the half-life of the RNA required for the synthesis of the respiratory enzyme(s) was approx. 1. 7 h, a value comparable to the functional life-time of m R N A utilized for bulk protein synthesis during this phase of metabolism. Biochim. Biophys. Acta, 142 (1967) 4o3-4o9
40~
R . E . CLICK, D. P. HACKETT
Recent evidence suggests that enzymes in both bacterial 21 and mammalian cells e° may have a time differential between their synthesis and the appearance of activity. Since any discernable lag between these two processes would lead to inaccurate estimates of the RNA half-life, they were analyzed in the absence of (net) protein synthesis. Addition of puromycin within 2.5 h after the initiation of the incubation period resulted in complete suppression of the development of respiration (Fig. 3) while actinomycin required 4 h for complete suppression. If enzyme activation itself does not require protein synthesis these results suggest that, at this time, enzyme activation (after polypeptide formation) cannot influence stability measurements. Thus the inhibition of the respiratory enhancement by actinomycin is a reliable measure of the mRNA required for this phenomenon. While addition of puromycin after 2.5 h did allow significant augmentation of the respiratory capacity, these results are most readily explained by the lack of total inhibition of protein synthesis (Fig. 2) and not by a time lag between the appearance of activity and synthesis de novo.
CONCLUDING REMARKS
The present estimates of a short-lived (1.6 h half-life) functional mRNA that is required for protein synthesis and for the development of respiratory capacity during the metabolically inactive period of potato tuber slices are in accord with estimates made for pulse-labeled RNA in soybean hypocotyl tissuel4, ~6. However, a much shorter half-life has been reported for pea roots a5 (IO min) and a much longer one has been reported for germinating cotton embryos 13 ( > 16 h). The latter functional time, however, was estimated in the presence of RNA synthesis (37 °/1, of the control). In view of the differential inhibition of RNA synthesis by actinomycia (mRNA being the least affected), the value obtained by WATERS AND DURE13 may be over-estimated. Nevertheless, at the moment, insufficient evidence is available to make firm conclusions concerning the uniformity or variation in functional half-life of mRNA either between different plant species, different tissues within a single plant, or different cells within a single tissue. Ill addition, the possibilities of variation of mRNA stability in response to environmental shifts are yet to be explored with plant material.
ACKNOWLEDGEMENTS
We wish to thank Dr. D. SONNEBORN for critically reading the manuscript. This investigation was supported by National Science Foundation Grant GB-I75I. One of us (R.E.C.) was a Predoctoral Fellow of the Public Health Service. REFERENCES i 2 3 4
F. C. D. D.
JACOB AND J. ~IONOD, J. Mol. Biol., 3 (I96I) 318LEVINTHAL, A. KEYNAN AND A. HIGA, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1631. NAKADA AND B. MAGASANIK, J. Mob. Biol., 8 (1964) lO5. P. FAN, J. Mol. Biol., 16 (1966) 164 .
Biochim. Biophys. Acta, 142 (1967) 403-409
MESSENGER RNA
4o5
response of the potato slices to dinitrophenol, an agent that uncouples oxidative phosphorylation. Incubation of fresh slices in the antibiotic for 24 h resulted in tissue that retained the respiratory properties of freshly sliced tissue (Table I), namely the normal and uncoupled rates. Addition of the antibiotic after 5 or I I h of incubation, which normally permits some increase in the respiratory capacity 16, has no effect on the respiratory response to dinitrophenol. These results demonstrate that actinomyGin has no detrimental effect on the processes directly involved in oxygen consumption and, therefore, is presumed to be non-toxic to this tissue. RNA synthesis in potato tuber slices is effectively inhibited b y low levels of actinomycin 16. The action of the drug apparently is on the DNA-dependent RNA polymerase 27,2s since there is little effect of the inhibitor on the synthesis of nucleotide precursors in this tissue TM. The kinetics of inhibition (Fig. I) indicate that uracil 4-
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Fig. I. T h e effect of a d d i n g a c t i n o m y c i n (to 25 # g / m l ) , a f t e r 5.5 a n d 24 h of i n c u b a t i o n , on t h e i n c o r p o r a t i o n of [14C!uraciL I n c u b a t i o n m e d i u m c o n t a i n e d 1.25" lO s d i s i n t . / m i n p e r 8 m].
incorporation was quickly inhibited b y 25 #g/ml of actinomycin (the residual incorporation after 24 h m a y represent DNA labeling29). When the drug is added after 5 h of incubation, a loss of radioactive polymeric material occurred, followed, however, b y a subsequent increase. In this case, the loss of label is considered an unreliable measurement of m R N A half-life in view of both the low level of incorporation observed and the possible involvement of labeling other classes of RNA. Others, also have questioned the reliability of this technique 3°-32.
II. A ctinomycin el/ects on bulk protein synthesis Estimates of the functional life-time of the messengers can presumably be made b y studying the kinetics of protein synthesis after the addition of actinomycin*. Fig. 2 shows the effect of the antibiotic on the incorporation of El*C]leucine into protein during the metabolically inactive phase. As shown, 4 h were required to completely inhibit the incorporation, suggesting that the RNA required for protein synthesis at this stage of incubation had a half-life of about 1.6 h (assuming an exponential decay). Biochim. Biophys. Acta, 142 (1967) 4 o 3 - 4 o 9
400
R . E . CLICK, D. P. HACKETT 34 ~
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Fig. 2. The effect of a d d i n g a c t i n o m y c i n (to 25/*g/ml) a n d p u r o m y c i n (to 5" IO 4 M), a f t e r 5 h of i n c u b a t i o n , on t h e i n c o r p o r a t i o n of ~14Clleucine. I n c u b a t i o n m e d i u m c o n t a i n e d o.41. lO s d i s i n t . / m i n per 8 ml.
The stability of mRNA was also determined during the metabolically active period following the completion of the respiratory rise (24 h). Evaluated from pulselabeling experiments (compared with the previous continuous-labeling experiments) protein synthesis decreased to 50 °o of the control 4 h after addition of actinomycin (Table I1L a result similar to that found for the inactive period. In the subsequent TABLE II CAPACITY FOR PROTEIN SYNTHESIS AFTER ACTINOMYCIN ADDITION T w e l v e slices ( a b o u t 0.83 g) were a g e d p e r e x p e r i m e n t for 18 h. A t t h i s t i m e , a c t i n o m y c i n D (to 25 u g / m l ) w a s a d d e d to o n e - h a l f of t h e s a m p l e s (6 dishes). A t t h e d e s i g n a t e d t i m e s one c o n t r o l s a m p l e a n d one a c t i n o m y c i n - t r e a t e d s a m p l e were t r a n s f e r r e d t o s t e ri l e s o l u t i o n s c o n t a i n i n g [14C]leucine (5.6- i o 5 d i s i n t . / m i n per 8 ml) a n d p u l s e d for 2 h.
Incubation time prior to pulse labeling
Counts~rain per g in protein
Disint./min per g in soluble pool*
(h)
Control
A ctinomycin
Control
A ctinomycin
18 22 26 32 4° 48
2966 3612 4557 5776 9344 12 600
3900 2o75 19o7 3790 5025 6989
52 49 43 34 15 io
48 44 ° 24 75 ° 22 27 ° 12 61o 797 ° 8630
980 59o ioo 640 040 74 °
* C o r r e c t e d to i o o °/o c o u n t i n g efficiency.
4 h, no further change was observed. However, after a further 8 h incubation in the antibiotic, protein synthesis increased to a level comparable to that occurring after 18 h incubation. These changes are not readily explainable at present, but may be due to the "apparently incomplete" inhibition of RNA synthesis by actinomycin (Fig. I), the differences in the transfer of radioactive amino acid from the medium into the soluble pool (Table II), selectivity of RNA inhibition by actinomycin Biochim. Biophys. Acta, 142 (1967) 403-409
MESSENGER R N A
409
5 E. REICH, R. M. FRANKLIN, A. J. SHATKIN AND E. L. TATUM, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1238 . 6 S. PENMAN, K. SCHERRER, Y. BECKER AND J. E. DARNELL, Proc. Natl. Acad. Sci. U.S., 49 (1963) 654. 7 p. R. GRoss AND G. H. COUSINEAU, Biochem. Biophys. Res. Commun., io (1963) 321. 8 T. HUMPHREYS, S. PENMAN AND E. BELL, Biochem. Biophys. Res. Commun., 17 (1964) 618. 9 M. REVEL AND ~-I. H. HIATT, Proc. Natl. Acad. Sci. U.S., 51 (1964) 81o. IO U. E. LOENING, Nature, 195 (1962) 467 . i i R. ]3. VAN HUYSTEE AND J. H. CHERRY, Biochem. Biophys. Res. Commun., 23 (1966) 835. 12 C. Y. LIN, J. L. KEY AND C. E. ]3RACKER, Plant Physiol., 41 (1966) 976. 13 L. C. WATERS AND L. S. OURE, J. Mol. Biol., 19 (1966) i. 14 J. INGLE, J. L. KEY AND R. E. HOLM, J. Mcl. Biol., I I (1965) 73 ° . 15 U. E. LOENING, Proc. Roy. Soc., London Ser. B, 162 (1965) 121. 16 R. E. CLICK AND D. P. HACKETT, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 243. 17 F. C. STEWARD AND J. F. SUTCLIFFE, in F. C. STEWARD, Plant Physiology, Academic Press, N e w York, 1959, p. 25318 T. Ap F.EES AND H. BEEVERS, Plant Physiol., 35 (196o) 839. 19 J. A. ROMBERGER AND G. NORTON, Plant Physiol., 36 (1961) 20. 20 J. M. KIRK, Biochim. Biophys. Acta, 42 (196o) 167. 21 L. I-I. HARTWELL AND B. MAGASANIK, J. Mol. Biol., 7 (1963) 4 °122 A. C. TRAKATELLIS, ]~. HEINLE, M. MONTJAR, i . E. AXELROD AND W . N. JENSEN, Arch. Biochem. Biophys., 112 (1965) 89. 23 H. CHANTRENNE, Biochim. Biophys. Acta, 95 (1965) 351. 24 G. Acs, E. REICH AND S. VALANJU, Biochim. Biophys. Acta, 76 (1963) 68. 25 i . C. TRAKATELLIS, A. E. AXELROD AND M. MONTJAR, Nature, 203 (1964) 1134. 26 R. WII~2SNER, G. ACS, E. REICH AND A. SHAFIQ, J. Cell. Biol., 27 (1965) 47. 27 I. H. GOLDBERG AND M. RABINOWITZ, Science, 136 (1962) 315 • 28 J. HURWlTZ, J. J. FURTH, M. MALAMY AND M. ALEXANDER, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1222. 29 R. E. CLICK AND D. P. HACKETT, J. Mol. Biol., 17 (1966) 279. 3 ° F. GROS, J. M. DUBERT, A. TISSlI~RES, S. BOURGEOIS, M. MICHELSON, R. SOFFER AND L. LEGAULT, Cold Spring Harbor Syrup. Quant. Biol., 28 (1963) 299. 31 D. KENNELL, J. Mol. Biol., 9 (1964) 789 . 32 N. S. GIRIJA AND A. SREENIVASAN, Biochem. Biophys. Res. Commun., 21 (1965) 404 . 33 G. P. GEORGIEV, Q). P. SAMARINA, M. I. LERMAN, M. N. SMIRNOV AND A. N. SEVERTZOV, Nature, 200 (1963) 1291. 34 M. R. POLLOCK, Biochim. Biophys. Acta, 76 (1963) 80. 35 l{. P. PERRY, P. R. SRINIVASAN AND D. E. KELLEY, Science, 145 (1964) 504 . 36 J. L. KEY AND J. INGLE, Proc. Natl. Acad. Sci. U.S., 52 (1964) 1382. 37 D. P. TSCHUDY, H. S. MARVER AND A. COLLINS, Biochem. Biophys. Res. Commun., 2i (1065) 480. 38 J. BRACHET, H. DENIS AND F. DE VITY, Develop. Biol., 9 (1964) 398. 39 P. R. GROSS, L. I. MALKIN AND VV'. A. MOYER, Prec. Natl. Acad. Sci. U.S., 51 (1964) 407 • 4 ° G. GUIDICE, F. T. KENNEY AND G. n . NOVELLI, Biochim. Biophys. Acta, 87 (1964) 171.
Biochim. Biophys. Acta, 142 (1967) 403 409
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95636
F U N C T I O N A L S T A B I L I T Y OF MESSENGER RNA IN POTATO T U B E R SLICES
R O B E R T E. CLICK* AND D A V I D P. H A C K E T T * *
Department o] Biochemistry, University o] Cali[ornia, Berkeley, Cali[. (U.S.A.) (Received D e c e m b e r I9th, 1966)
SUMMARY
I. The functional stability of messenger RNA (mRNA) was determined on potato tuber slices that had either an active or inactive metabolic rate. 2. Using the decline of amino acid incorporation in the presence of actinomycin as an index, the functional half-life during the inactive phase was 1.6 h. During the same period, the RNA required for the respiratory increase (4-fold in one day) had a half-life of 1. 7 h. 3. The functional stability during the active phase ( > 20 h incubation) was comprised of three types; one possessing a half-life of 1.6 h, one a half-life of over 2 h and a third that may have been pre-existing, but inactive.
INTRODUCTION
Since the original concept of messenger RNA (mRNA) I, many of the characteristic properties of this class of molecales have been investigated. One kind of characterization has involved determining the duration after cessation of RNA synthesis during which protein synthesis can still be maintained. Such characterizations have been interpreted in terms of the functional lifetime of m R N A and have been found to be on the order of a few minutes in bacteria 2-4 and a few hoursS, 6 or days 7-9 in animal cells. To date, however, very little evidence has accumulated concerning the characterization of m R N A in plant tissue 1°-12 and even less information is available concerning the m R N A stability 13-~5. A useful system for studying m R N A in plant tissue is based on the 4-fold increase in respiratory rate that develops in potato tuber slices incubated aerobically for 24 h; a process that depends on both RNA and RNAmediated protein synthesis 16. Furthermore, the stability can be compared at different rates of metabolism; namely an inactive (0-7 h) and an active ( > 18 h) state as measured by salt uptakO 7, protein synthesis le or carbohydrate metabolism as,19. The present communication is concerned with the determination of the functional stability of m R N A in these slices. * P r e s e n t address: D e p a r t m e n t of Zoology, U n i v e r s i t y of Wisconsin, Madison, Wisc., U.S.A. *~ Deceased: J a n u a r y 2I, 1965. Abbreviation: m R N A , m e s s e n g e r R N A .
Biochim. Biophys. Acta, 142 (1967) 403-409
404
R . E . CLICK, D. P. HACKETT
MATERIALS AND METHODS
Monthly shipments of potato tubers (Solanum tuberosurn, var. White Rose) were obtained monthly through the courtesy of Dr. H. TIMM, Department of Vegetable Crops, University of California at Davis and stored at 21 °. Cylinders of tissue were removed with a cork borer (I cm diameter) and I-ram-thick slices were cut therefrom using a hand microtome. The slices were washed for 25 rain in sterile, deionized water and then incubated in 0.02 M potassium phosphate (pH 6.7), I" Io -4 M CaSO~, I. Io -3 M MgC12, 50//g/ml dihydrostreptomycin and IOO units/ml penicillin at 21 ° in small petri dishes a6. Protein synthesis was determined by measuring the amount of !I-a4C]leucine incorporated into the slices. After appropriate intervals of incubation, the slices were removed from the radioactive solution and washed in 25 ml of non-radioactive leucine (0.2 mg/ml) for 30 rain to remove any readily exchangeable [aaClleucine. They were then extracted with 7° ~o ethanol for 8 h in a Soxhlet apparatus, after which they were rinsed briefly with absolute ethanol. The alcohol-insoluble residue was dried at 8o ° and counted directly in an automatic gas flow apparatus (NuclearChicago) with an efficiency of approx. 5 %. The alcohol-soluble material was concentrated and counted in a scintillation spectrometer with an efficiency of 55 %
RESULTS
The measurement of the metabolic stability of RNA can most easily be made in the absence of its synthesis. Therefore, actinomycin C1 (Merck, Sharp and Dohme), the classical inhibitor of RNA synthesis 2°, was employed in the experiments to be described. It will be assumed that actinomycin neither increased nor decreased the decay rate of m R N A 21-23 although there are reports challenging these assumptions 24,2~.
I. Actinomyein effects on respiration and RNA synthesis In light of the lethal action that actinomyein has on rats 9 and on mammalian tissue culture cells ~6, the toxicity of the antibiotic was examined by determining the TABLE I EFFECTS OF DINITROPHENOL ON FRESH SLICES AND SLICES AGED IN 25 p g / m l ACTINOMYCIN The r e s p i r a t o r y r a t e (#1 O2/h p e r g) w a s m e a s u r e d b y s t a n d a r d W a x b u r g m a n o m e t r i c t e c h n i q u e s a t p H 6.0 a f t e r a t o t a l i n c u b a t i o n t i m e of 24 h ( e x c e p t for t h e fre s h slices w h i c h w e r e a n a l y z e d imm e d i a t e l y ) . T h e c o n t r o l r a t e w a s d e t e r m i n e d p r i o r to t h e m e a s u r e m e n t in d i n i t r o p h e n o l (5" i o 5 M, p H 6.0).
Time o~ actionomycin addition
kd 02/h per g Control
DNP
A
F r e s h slices Initial 5 h I1 h Never
24 22 93 II 7 117
5° 46 122 143 145
26 24 29 26 28
Biochim. Biophys. Acta, 142 (1967) 403-409
MESSENGER RNA
4o7
described in other systems ~3-s7 or the activation of pre-existing non-translatable mRNA3S,39. It is rather unlikely that the first possibility is correct, since the percent inhibition of RNA synthesis increased directly with the length of incubation in the antibiotic. The pool size also is an unlikely factor since the control and treated tissue had equivalent isotopic material at the 48-h period, whereas the amount of isotope incorporated at this time is 2-fold higher in the control. Further experimental data will be required to understand this observation.
I I I . Actinomycin e/]ects on the increase in respiratory activity The final criterion used to measure the functional half-life is perhaps the most meaningful, namely the loss of the capacity to increase enzymatic activity. The enzyme(s) analyzed in this tissue are those involved in the development of the respiratory capacity. As discussed earlier, actinomycin has no effect on the respirat o r y rate of the tissue at the time of its addition (Table I); furthermore, puromycin, a protein synthesis inhibitor, also has no effect on the rate TM. These findings suggest that the enzyme(s) responsible for the increased respiratory rate were metabolically stable proteins. Thus analysis of the respiratory rate can be determined at a single time (all measurements were made after a total incubation of 24 h). The effect on the respiratory rate of adding actinomycin at different times of incubation is given in Fig. 3- If added at zero time, the increase in the respiratory i
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I 4
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I 8
i
] 12
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Fig. 3. The effect of delayed additions of a c t i n o m y c i n (to 25 ~g/ml) and p u r o m y c i n (to 5" lO-4 M) on the d e v e l o p m e n t of the r e s p i r a t o r y capacity. The inhibitors were added after o, 2.5 and 5 h of incubation. The r e s p i r a t o r y rate was m e a s u r e d at the end of 24 h. Q - O , control; [ ~ - - - [ ~ , a c t i n o m y c i n ; A- - - A, puromycin.
rate was completely suppressed. However, if added after 2.5 or 5 h of incubation, 31 9/0 and 61 °/o of the maximal increase occurred respectively. However, the minim u m time required for actinomycin to completely prevent the respiratory increase, when added at either 2.5 or 5 h, was constant, requiring 4-4-5 h (calculated from Fig. 3). These results suggest that the half-life of the RNA required for the synthesis of the respiratory enzyme(s) was approx. 1. 7 h, a value comparable to the functional life-time of m R N A utilized for bulk protein synthesis during this phase of metabolism. Biochim. Biophys. Acta, 142 (1967) 4o3-4o9
40~
R . E . CLICK, D. P. HACKETT
Recent evidence suggests that enzymes in both bacterial 21 and mammalian cells e° may have a time differential between their synthesis and the appearance of activity. Since any discernable lag between these two processes would lead to inaccurate estimates of the RNA half-life, they were analyzed in the absence of (net) protein synthesis. Addition of puromycin within 2.5 h after the initiation of the incubation period resulted in complete suppression of the development of respiration (Fig. 3) while actinomycin required 4 h for complete suppression. If enzyme activation itself does not require protein synthesis these results suggest that, at this time, enzyme activation (after polypeptide formation) cannot influence stability measurements. Thus the inhibition of the respiratory enhancement by actinomycin is a reliable measure of the mRNA required for this phenomenon. While addition of puromycin after 2.5 h did allow significant augmentation of the respiratory capacity, these results are most readily explained by the lack of total inhibition of protein synthesis (Fig. 2) and not by a time lag between the appearance of activity and synthesis de novo.
CONCLUDING REMARKS
The present estimates of a short-lived (1.6 h half-life) functional mRNA that is required for protein synthesis and for the development of respiratory capacity during the metabolically inactive period of potato tuber slices are in accord with estimates made for pulse-labeled RNA in soybean hypocotyl tissuel4, ~6. However, a much shorter half-life has been reported for pea roots a5 (IO min) and a much longer one has been reported for germinating cotton embryos 13 ( > 16 h). The latter functional time, however, was estimated in the presence of RNA synthesis (37 °/1, of the control). In view of the differential inhibition of RNA synthesis by actinomycia (mRNA being the least affected), the value obtained by WATERS AND DURE13 may be over-estimated. Nevertheless, at the moment, insufficient evidence is available to make firm conclusions concerning the uniformity or variation in functional half-life of mRNA either between different plant species, different tissues within a single plant, or different cells within a single tissue. Ill addition, the possibilities of variation of mRNA stability in response to environmental shifts are yet to be explored with plant material.
ACKNOWLEDGEMENTS
We wish to thank Dr. D. SONNEBORN for critically reading the manuscript. This investigation was supported by National Science Foundation Grant GB-I75I. One of us (R.E.C.) was a Predoctoral Fellow of the Public Health Service. REFERENCES i 2 3 4
F. C. D. D.
JACOB AND J. ~IONOD, J. Mol. Biol., 3 (I96I) 318LEVINTHAL, A. KEYNAN AND A. HIGA, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1631. NAKADA AND B. MAGASANIK, J. Mob. Biol., 8 (1964) lO5. P. FAN, J. Mol. Biol., 16 (1966) 164 .
Biochim. Biophys. Acta, 142 (1967) 403-409
MESSENGER RNA
4o5
response of the potato slices to dinitrophenol, an agent that uncouples oxidative phosphorylation. Incubation of fresh slices in the antibiotic for 24 h resulted in tissue that retained the respiratory properties of freshly sliced tissue (Table I), namely the normal and uncoupled rates. Addition of the antibiotic after 5 or I I h of incubation, which normally permits some increase in the respiratory capacity 16, has no effect on the respiratory response to dinitrophenol. These results demonstrate that actinomyGin has no detrimental effect on the processes directly involved in oxygen consumption and, therefore, is presumed to be non-toxic to this tissue. RNA synthesis in potato tuber slices is effectively inhibited b y low levels of actinomycin 16. The action of the drug apparently is on the DNA-dependent RNA polymerase 27,2s since there is little effect of the inhibitor on the synthesis of nucleotide precursors in this tissue TM. The kinetics of inhibition (Fig. I) indicate that uracil 4-
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Fig. I. T h e effect of a d d i n g a c t i n o m y c i n (to 25 # g / m l ) , a f t e r 5.5 a n d 24 h of i n c u b a t i o n , on t h e i n c o r p o r a t i o n of [14C!uraciL I n c u b a t i o n m e d i u m c o n t a i n e d 1.25" lO s d i s i n t . / m i n p e r 8 m].
incorporation was quickly inhibited b y 25 #g/ml of actinomycin (the residual incorporation after 24 h m a y represent DNA labeling29). When the drug is added after 5 h of incubation, a loss of radioactive polymeric material occurred, followed, however, b y a subsequent increase. In this case, the loss of label is considered an unreliable measurement of m R N A half-life in view of both the low level of incorporation observed and the possible involvement of labeling other classes of RNA. Others, also have questioned the reliability of this technique 3°-32.
II. A ctinomycin el/ects on bulk protein synthesis Estimates of the functional life-time of the messengers can presumably be made b y studying the kinetics of protein synthesis after the addition of actinomycin*. Fig. 2 shows the effect of the antibiotic on the incorporation of El*C]leucine into protein during the metabolically inactive phase. As shown, 4 h were required to completely inhibit the incorporation, suggesting that the RNA required for protein synthesis at this stage of incubation had a half-life of about 1.6 h (assuming an exponential decay). Biochim. Biophys. Acta, 142 (1967) 4 o 3 - 4 o 9
400
R . E . CLICK, D. P. HACKETT 34 ~
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Fig. 2. The effect of a d d i n g a c t i n o m y c i n (to 25/*g/ml) a n d p u r o m y c i n (to 5" IO 4 M), a f t e r 5 h of i n c u b a t i o n , on t h e i n c o r p o r a t i o n of ~14Clleucine. I n c u b a t i o n m e d i u m c o n t a i n e d o.41. lO s d i s i n t . / m i n per 8 ml.
The stability of mRNA was also determined during the metabolically active period following the completion of the respiratory rise (24 h). Evaluated from pulselabeling experiments (compared with the previous continuous-labeling experiments) protein synthesis decreased to 50 °o of the control 4 h after addition of actinomycin (Table I1L a result similar to that found for the inactive period. In the subsequent TABLE II CAPACITY FOR PROTEIN SYNTHESIS AFTER ACTINOMYCIN ADDITION T w e l v e slices ( a b o u t 0.83 g) were a g e d p e r e x p e r i m e n t for 18 h. A t t h i s t i m e , a c t i n o m y c i n D (to 25 u g / m l ) w a s a d d e d to o n e - h a l f of t h e s a m p l e s (6 dishes). A t t h e d e s i g n a t e d t i m e s one c o n t r o l s a m p l e a n d one a c t i n o m y c i n - t r e a t e d s a m p l e were t r a n s f e r r e d t o s t e ri l e s o l u t i o n s c o n t a i n i n g [14C]leucine (5.6- i o 5 d i s i n t . / m i n per 8 ml) a n d p u l s e d for 2 h.
Incubation time prior to pulse labeling
Counts~rain per g in protein
Disint./min per g in soluble pool*
(h)
Control
A ctinomycin
Control
A ctinomycin
18 22 26 32 4° 48
2966 3612 4557 5776 9344 12 600
3900 2o75 19o7 3790 5025 6989
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48 44 ° 24 75 ° 22 27 ° 12 61o 797 ° 8630
980 59o ioo 640 040 74 °
* C o r r e c t e d to i o o °/o c o u n t i n g efficiency.
4 h, no further change was observed. However, after a further 8 h incubation in the antibiotic, protein synthesis increased to a level comparable to that occurring after 18 h incubation. These changes are not readily explainable at present, but may be due to the "apparently incomplete" inhibition of RNA synthesis by actinomycin (Fig. I), the differences in the transfer of radioactive amino acid from the medium into the soluble pool (Table II), selectivity of RNA inhibition by actinomycin Biochim. Biophys. Acta, 142 (1967) 403-409
MESSENGER R N A
409
5 E. REICH, R. M. FRANKLIN, A. J. SHATKIN AND E. L. TATUM, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1238 . 6 S. PENMAN, K. SCHERRER, Y. BECKER AND J. E. DARNELL, Proc. Natl. Acad. Sci. U.S., 49 (1963) 654. 7 p. R. GRoss AND G. H. COUSINEAU, Biochem. Biophys. Res. Commun., io (1963) 321. 8 T. HUMPHREYS, S. PENMAN AND E. BELL, Biochem. Biophys. Res. Commun., 17 (1964) 618. 9 M. REVEL AND ~-I. H. HIATT, Proc. Natl. Acad. Sci. U.S., 51 (1964) 81o. IO U. E. LOENING, Nature, 195 (1962) 467 . i i R. ]3. VAN HUYSTEE AND J. H. CHERRY, Biochem. Biophys. Res. Commun., 23 (1966) 835. 12 C. Y. LIN, J. L. KEY AND C. E. ]3RACKER, Plant Physiol., 41 (1966) 976. 13 L. C. WATERS AND L. S. OURE, J. Mol. Biol., 19 (1966) i. 14 J. INGLE, J. L. KEY AND R. E. HOLM, J. Mcl. Biol., I I (1965) 73 ° . 15 U. E. LOENING, Proc. Roy. Soc., London Ser. B, 162 (1965) 121. 16 R. E. CLICK AND D. P. HACKETT, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 243. 17 F. C. STEWARD AND J. F. SUTCLIFFE, in F. C. STEWARD, Plant Physiology, Academic Press, N e w York, 1959, p. 25318 T. Ap F.EES AND H. BEEVERS, Plant Physiol., 35 (196o) 839. 19 J. A. ROMBERGER AND G. NORTON, Plant Physiol., 36 (1961) 20. 20 J. M. KIRK, Biochim. Biophys. Acta, 42 (196o) 167. 21 L. I-I. HARTWELL AND B. MAGASANIK, J. Mol. Biol., 7 (1963) 4 °122 A. C. TRAKATELLIS, ]~. HEINLE, M. MONTJAR, i . E. AXELROD AND W . N. JENSEN, Arch. Biochem. Biophys., 112 (1965) 89. 23 H. CHANTRENNE, Biochim. Biophys. Acta, 95 (1965) 351. 24 G. Acs, E. REICH AND S. VALANJU, Biochim. Biophys. Acta, 76 (1963) 68. 25 i . C. TRAKATELLIS, A. E. AXELROD AND M. MONTJAR, Nature, 203 (1964) 1134. 26 R. WII~2SNER, G. ACS, E. REICH AND A. SHAFIQ, J. Cell. Biol., 27 (1965) 47. 27 I. H. GOLDBERG AND M. RABINOWITZ, Science, 136 (1962) 315 • 28 J. HURWlTZ, J. J. FURTH, M. MALAMY AND M. ALEXANDER, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1222. 29 R. E. CLICK AND D. P. HACKETT, J. Mol. Biol., 17 (1966) 279. 3 ° F. GROS, J. M. DUBERT, A. TISSlI~RES, S. BOURGEOIS, M. MICHELSON, R. SOFFER AND L. LEGAULT, Cold Spring Harbor Syrup. Quant. Biol., 28 (1963) 299. 31 D. KENNELL, J. Mol. Biol., 9 (1964) 789 . 32 N. S. GIRIJA AND A. SREENIVASAN, Biochem. Biophys. Res. Commun., 21 (1965) 404 . 33 G. P. GEORGIEV, Q). P. SAMARINA, M. I. LERMAN, M. N. SMIRNOV AND A. N. SEVERTZOV, Nature, 200 (1963) 1291. 34 M. R. POLLOCK, Biochim. Biophys. Acta, 76 (1963) 80. 35 l{. P. PERRY, P. R. SRINIVASAN AND D. E. KELLEY, Science, 145 (1964) 504 . 36 J. L. KEY AND J. INGLE, Proc. Natl. Acad. Sci. U.S., 52 (1964) 1382. 37 D. P. TSCHUDY, H. S. MARVER AND A. COLLINS, Biochem. Biophys. Res. Commun., 2i (1065) 480. 38 J. BRACHET, H. DENIS AND F. DE VITY, Develop. Biol., 9 (1964) 398. 39 P. R. GROSS, L. I. MALKIN AND VV'. A. MOYER, Prec. Natl. Acad. Sci. U.S., 51 (1964) 407 • 4 ° G. GUIDICE, F. T. KENNEY AND G. n . NOVELLI, Biochim. Biophys. Acta, 87 (1964) 171.
Biochim. Biophys. Acta, 142 (1967) 403 409