- Email: [email protected]
Inhibition of the synthesis of 5-S ribosomal RNA in Escherichia coli by levallorphan
481
BIOCHIMICA ET BIOPHYSICA ACTA BBA 96195
I N H I B I T I O N OF T H E SYNTHESIS OF 5-S RIBOSOMAL RNA IN E S C H E R I C H I A COLI BY LEVALLORPHAN R. R O S C H E N T H A L E R * , M. A. D E V Y N C K * * , P. F R O M A G E O T A N D E. J. S I M O N " * Service de Biochimie, Ddpartement de Biologie, Centre d'Etudes Nucldaires de Saclay, 9I-Gi[-sur Yvette (France) (Received D e c e m b e r I9th, 1968)
SUMMARY
Levallorphan (1.54. lO-3 M) inhibits RNA biosynthesis by 7 ° %, whereas the synthesis of protein is inhibited by 14 %. The residual synthesis of tRNA under such conditions is greater by at least a factor of 2 than that of high molecular weight RNA. mRNA synthesis, on the contrary, seems to be only slightly inhibited or not at all. Levallorphan therefore exhibits effects similar to levorphanol. As the origin of 5-S RNA has been the subject of recent discussion we analyzed the extent of its synthesis in the presence of levallorphan. It has been found that 5-S RNA synthesis is inhibited bv the drug at least as profoundly as high molecular weight RNA, a result which puts the 5-S RNA in the same class as the ribosomal RNA's and suggests that the same type of control mechanism regulates the synthesis of 5-S, I6-S and 23-S RNA's.
INTRODUCTION
The structure of the 5-S RNA discovered in Escherichia coli by ROSSET AND MONIER1 and ROSSET et al3 has been elucidated 3, but its function is still unknown. It has been considered to be a ribosomal RNA. This conclusion could be specifically tested by examination of 5-S RNA synthesis in the presence of levorphanol, a drug which exhibits a selective inhibition of ribosomal RNA (16 S and 23 S) as reported by SIMON AND V A N PRAAG 4,5. Because of the difficulties experienced in obtaining narcotic drugs for purposes of investigation, levallorphan, a nonnarcotic N-allyl analogue of levorphanol, was used.
HO~N_CH2_CH=CH 2 L LLORPHAN EVA
* P r e s e n t address: c/o Prof. Liebermeister, K l i n i k u m r e c h t s der Isax, Munich, G e r m a n y . ** P r e - d o c t o r a l fellow of t h e C o m m i s s a r i a t tt l ' E n e r g i e A t o m i q u e . *** P r e s e n t a d d r e s s : D e p a r t m e n t of Medicine, N e w Y o r k U n i v e r s i t y Medical Center, 550 F i r s t A v e n u e , N e w York, N Y lOO16, U.S.A. "*" Career Scientist of t h e H e a l t h R e s e a r c h Council of t h e City of N e w York.
Biochim. Biophys. Acta, 182 (1969) 481-49 °
482
R. ROSCHENTHALERet al.
With this tool at hand, it was shown that the synthesis of 5-S RNA was inhibited as profoundly as that of the high molecular weight rRNA's, a finding consistent with the view that 5-S RNA is regulated in the same way as the other ribosomal RNA's. MATERIALSAND METHODS
Bacterial strains and media E. coli K12S was obtained from Dr. R. Devoret, C.N.R.S., Gif-sur-Yvette, and was used for the labeling experiments. Bacteria of a K~2 Hfr-strain obtained from Dr. G. Cohen were used as nonlabeled carrier cells. The cells were grown in a low phosphate medium buffered with triethanolamine at pH 8.1, as described by SIMON AND VAN PRAAG4. Sodium succinate (0. 5 %) served as carbon source, and the medium was supplemented with 0.2 % casamino acids (Difco). Materials Levallorphan was generously provided by Hoffmann-La Roche, Basel. E2-~4C]Uracil (47-4 mC/mmole) and [6-~H~uracil (24 C/mmole) were obtained from the D~partement des Radio616ments C.E.A., Saclay. Double-labeling experiments The double-labeling procedure minimizes apparent differences in the labeling of nucleic acid fractions due to differential losses during extraction and fractionation. Two cultures of I 1 each were incubated and allowed to grow to an absorbance at 547 m# of 0.300. At this absorbance, levallorphan in the appropriate concentration was added to one of the two log-phase cultures, followed after 5 rain by [3H]uracil (2 mC). In both cases, unlabeled uracil was added to give a final uracil concn, of 20/~g/ml. [14ClUracil (200 #C) was added to the control culture at the same time. Both cultures were then incubated for 15 min. 5 ml of a 1 % solution of unlabeled uracil was then added to each culture, and the radioactive uracil was "chased" for another lO rain. The bacteria were chilled by pouring frozen buffer (o.oi M Tris, o.oi M MgCI2, pH 7-5) into the culture and harvested in a Servall centrifuge at 2 °. The labeled cells were resuspended in the same buffer and mixed in such a way that the total ~H radioactivity versus the total 14C radioactivity incorporated were in a ratio of 3:1. In one experiment both cultures were centrifuged after the i5-min incubation resuspended in the original medium supplemented with unlabeled uracil (5° #g/ml) and nfixed immediately. This chase, made in the absence of levallorphan, was done to make certain that our results were not influenced by unequal rates of release of labeled uracil from mRNA in the inhibited and control cultures. Preparation o/nucleic acids Unlabeled carrier cells (5 g) were added to the mixture of labeled cells, and the whole suspension was washed twice with buffer. After grinding the bacteria with 15 g of alumina and removal of alumina and unbroken cells by centrifugation the Biochim. Biophys. Acta, 182 (1969) 481-49o
INHIBITION OF
RNA
SYNTHESIS BY LEVALLORPHAN
483
supernatant was made 0.2 % with respect to deoxycholate and the cell debris and precipitate were sedimented. The supernatant was either directly extracted with phenol, or subjected to high-speed centrifugation to collect the ribosomes from which the 5-S RNA was extracted as described b y COMB AND ZEHAVI-WILLNERs. The 4-S and 5-S RNA were separated from the bulk of rRNA b y precipitation of the high molecular weight RNA with 2 M NaC1.
Fractionation o~ nucleic acids The material prepared was analyzed by methylated serum albumin coated kieselguhr column chromatography as described by MANDELL AND HERSHEY~. Bovine serum albumin Fraction V (Calbiochem) was used to prepare the methylated serum albumin. The gradient used (total vol. 600 ml) was between 0.2 M and 1.2 M NaC1. Fractions of IO ml were collected. In some cases, Sephadex column chromatography was used. Sephadex G-75 (Pharmacia) suspended in potassium acetate (5 mM, p H 4.2) was made into a 2.5 cm × 14o cm column, and maintained at 4 °, and after addition of the sample, eluted with the same buffer at a rate of 6 ml/h, the volume of each fraction being 5 ml. The absorbance of the fractions is read at 260 m# with a Zeiss spectrophotometer PMQ II. The amounts of 3H and 14C, respectively, are measured with the usual precautions for double-labeling experiments s after precipitation of the sample with 5 % trichloroacetic acid and filtration on Millipore filters. The highest variation at the lowest 3H/14C ratios is 15 %.
RESULTS
Before investigating the effects of levallorphan on the synthesis of 5-S RNA, we have established that this drug inhibits the growth of E. coli in a similar way to that of levorphanol ~. Until a concn, of 1.6 mM levallorphan is reached, viable cell counts show that there is little or no killing of bacteria. Furthermore the incorporation of [14C]uracil into nucleic acids decreases sharply with an increase in levallorphan concentration, whereas the incorporation of [l*C]phenylalanine is, under the same conditions, only slightly affected, as demonstrated in the case of levorphanol.
TABLE I EFFECTS OF LEVALLORPHAN ON [14C]PHENYLALANINE AND [aH]URACIL INCORPORATION IN E. coli I0/zC (200/~g) ESH]uracil and 0.I/*C [x4C]phenylalanine were added to I0 ml of bacterial culture. Aliquots were t a k e n at various intervals and the labels incorporated were measured. The d a t a give the percentage of incorporation versus the control.
Conch. o/ levallorphan
Incorporation o/ [liC]phenylalanine (%)
Incorporation of [SH]uracil (%)
ioo
IOO
(raM) o
1.39 1.54
96 86
42 28
Biochim. Biophys. Acta, 182 (1969) 481-49 °
R. ROSCHENTHALERgt al.
484
Comparison o/the inhibition by levallorphan o/the synthesis oI high molecular weight rRNA, mRNA and soluble RNA The profile of the 3H/~4C ratio in the eluate from the methylated albuminkieselguhr column constitutes a measure of the relative rates of synthesis of the nucleic acid fractions from drug-treated and control cells.
0,60
16
112 0.40
:1o 2-
-s u
g
-<. 0.20
:4 ._o
o
......° o ° \ b ........
' ........
4'0 .................
Fractions
6'0 ........
' .....
8b
.......
Fig. I. Influence of levallorphan on the synthesis of the various classes of E. coli nucleic acids. E. coli was g r o w n in a m e d i u m containing 1.3o mM levallorphan and pH]uracil. I t w a s mixed w i t h bacteria g r o w n in the absence of the drug, b u t otherwise u n d e r identical conditions, and la.beled w i t h [14C]uracil. A s u p e r n a t a n t was p r e p a r e d after 2. 5 it centrifugation at lO 5 ooo × g in order to reduce tile a m o u n t of r i b o s o m e s present, and analyzed on a m e t h y l a t e d albumin-kieselg u h r column. O - O , absorbaxtce at 260 m/,; [2]- - -E], 3H/14C ratio. The sedimented material has also been analyzed b y the same technique, and the ~H114C ratio observed for the r R N A is identical w i t h those s h o w n in tile figure.
A typical elution profile of a cell extract is shown in Fig. i. It can be seen that the highest isotope ratio occurs at or near the DNA peak. In view of this very reproducible observation and the previous finding that DNA synthesis is not significantly inhibited b y levorphanol 4 under the conditions used, the isotope ratio in DNA was taken as IOO % for the purpose of calculating percent inhibition in other fractions. Purification of the material in this DNA peak by the procedure of SCHMIDT AND THANNHAUSER 10 did not alter the isotope ratio. The lowest 3H/14C ratio was observed in the region of the I6-S and 23-S rRNA. The average isotope ratio in the 4-S RNA region was always 2-3 times higher. Thus, in the experiment shown in Fig. I, the synthesis of I6-S and 23-S RNA was inhibited 80-90 ~o, while that of 4-S RNA was inhibited 50-60 ~o. This difference increased with further purification of the fractions. A number of experiments supporting this conclusion are shown later in this paper (see Table II). The question of the inhibition of m R N A synthesis was not extensively investigated in this work. The two peaks of radioactivity occurring just before and after the DNA peak are sensitive to ribonuclease I. These RNA peaks are located in one of the regions where labeled RNA is found in extracts from cultures submitted to a brief pulse of radioactive RNA precursor 11. This evidence together with the insensitivity of protein synthesis make it likely that levallorphan does not modify greatly the synthesis of mRNA. Biochim. Biophys. Acta, 182 (1969) 481-49o
INHIBITION OF R N A SYNTHESIS BY LEVALLORPHAN
485
The evidence presented indicates that the inhibitory effect of levallorphan is predominantly on the synthesis of rRNA (16 S and 23 S), as has previously been reported for levorphanol.
El/ect o/ levallorphan in the synthesis ol 5-S r R N A Attempts to separate 5-S RNA from tRNA (4 S) were first made by the technique of ROSSET et al. 2. The small peak following closely the 4-S RNA peak in the elution profile from a methylated albumin-kieselguhr column is usually considered to be mainly 5-S RNA. However, this second peak was not obtained reproducibly and disappeared if the fibrous material present during the alcohol precipitation step was removed by means of a rotating glass rod (Fig. 2). It appeared also that the size of this second peak was variable according to the time that elapsed during the preparation of the nucleic acids. Furthermore, in cases when this second peak was well identified, a decrease in the 3H/14C ratio was always observed just prior to the appearance of the presumed 5-S RNA peak. When a soluble RNA preparation was used which lacked the second peak, the second half of the 4-S RNA peak exhibited a much lower 3H/14C ratio (Fig. 2). These observations led us to the preliminary assumption that the second peak, when present, consisted mainly of aggregates of tRNA. Elution of 5-S RNA presumably occurs between the two peaks. After the second peak, the 3H/14C ratio decreases again. This might indicate the presence of another form of 5-S RNA. COMBAND ZEHAVIWILLNER6 have observed a second peak containing 5-S RNA on elution from a methylated albumin-kieselguhr column. This could be seen if the end groups of the 5-S RNA were modified by treatment with periodate. AUBERT et al. 1~ also showed two 5-S RNA peaks after urea denaturation.
0'40t
]
1
1000 m 50
,< u .9
~:
0"30
N
40 50 6'0 3'0 4b Frect[ons
50
Fig. 2. E l u t i o n profiles and 3H/14C ratios of m a t e r i a l f r o m the 4 S-5 S region. On the left, a p r e p a r a t i o n of 4-S-5-S R N A w h i c h had been k e p t in a frozen s t a t e for several days. On the right, p r e p a r a t i o n f r o m which the fibrous material had been removed. The d o t t e d lines designate the fractions which were pooled. The corresponding c o l u m n s below give the 3H11'C ratio of this material. The concn, of levallorphan in the inhibited culture was 1.41 mM.
Biochim. Biophys. Acta, 182 (1969) 481-49 °
486
g. ROSCHENTHALERet al.
To obtain further evidence on the nature of this second peak, the nucleic acids present in a lO5 o o o × g supernatant were purified by passage through a DEAEcellulose column, and the tRNA fraction recovered. This material was filtered through a Sephadex G-75 column (magnesium acetate 5.1o -3 M, pH 4.2) and a part of the tRNA was eluted with the void volume, indicating aggregation. This aggregated material was kept frozen for several months, and chromatographed on a methylated albumin-kieselguhr column. The elution pattern shown in fig. 3 showed the normal tRNA peak and a large peak at the position usually assigned to the 5-S RNA. Obviously this latter peak is due to some form of tRNA and not to 5-S RNA. It will be noticed also that an important part of the RNA was eluted up to regions normally occupied by I6-S and 23-S RNA. These results show that either aggregation or conformational changes of low molecular weight RNA can cause difficulties in obtaining a good resolution of 5-S RNA from 4-S RNA. We therefore utilized the more effective method described by COMB AND Z E H A v I - W I L L N E R ~.
This method removes mRNA and most of the tRNA from isolated ribosomal particles. RNA is then extracted, and the largest part of the high molecular weight RNA is removed. The remaining material is chromatographed on a methylated albumin-kieselguhr column whose elution profile and 3H/14C ratio are given in Fig. 4. The 5-S RNA peak is easily identified and resolved from residual high molecular weight RNA. The ~H/14C value corresponding to the 5-S RNA is remarkably low even more so than the ratio corresponding to the I6-S peak. It is important to point out that the 3H/14C ratio of the residual high molecular weight fraction present is the same as in the original extract (Fig. I). By comparison with Fig. I, it appears that the inhibition observed for the 5-S RNA synthesis in the presence of levallorphan is close to 9 ° °/o when compared with the control.
D.3
~
30
~'", ,~o.O. ° o~
°
0.05
o
o
eg ...... 5b.... 4o
~
Fractions 6'0 .....
ab " 9'o
Fractions
Jo...... 3'o...... , i b
5b...... g6 ...... 7~...... 8b
~
Fig. 3. E l u t i o n pro file from a m e t h y l a t e d a l b u m i n - k i e s e l g u h r - c o l u m n c h r o m a t o g r a p h y of t R N A a g g r e g a t e s , t R N A were p u r i f i e d b y D E A E - c e l l u l o s e a n d S e p h a d e x G-75 c h r o m a t o g r a p h y . O n l y t h e m a t e r i a l e l u t i n g w i t h t h e v o i d v o l u m e f r o m t h e S e p h a d e x w a s a p p l i e d on t h e m e t h y l a t e d a l b u m i n - k i e s e l g u h r co lumn. Fig. 4. E l u t i o n profile from R N A e x t r a c t e d from p u r i f i e d r i b o s o m a l p a r t i c l e s . R i b o s o m e s coll e c t e d from c o n t r o l a n d 1. 3 miV[ l e v a l l o r p h a n - t r e a t e d c u l t u r e s , w e r e d i a l y z e d a g a i n s t a Tris buffer, o . o i M (pH 7.2), o . i mM Mg 2+, a n d w a s h e d w i t h t h e s a m e buffer. The r i b o s o m a l p a r t i c l e s w e re extracted with phenol and the high molecular weight R N A deliberately incompletely precipit a t e d w i t h 2 M NaC1. The r e m a i n i n g R N A was a p p l i e d t o a m e t h y l a t e d a l b u m i n - k i e s e l g u h r colu m n . T h e first A2e o m/~ p e a k c o n s i s t s of 5-S R N A , a n d t h e s e c ond is m o s t l y I6-S R N A , s i nc e 23-S R N A p r e c i p i t a t e s m o r e r e a d i l y .
Biochim. Biophys. Acta, 182 (I969) 481-49o
INHIBITION OF
RNA
SYNTHESIS BY LEVALLORPHAN
487
Evidence/or the absence o/ a ~tee 5-S RNA pool The low 8H/14C ratio in the 5-S RNA isolated from ribosomes may merely reflect the absence of new ribosomes to which newly formed 5-S RNA can attach in the levallorphan-treated cells. It therefore became essential to investigate the possibility of the existence of a free 5-S RNA pool in levallorphan-treated bacteria. To investigate this question it was necessary to use a method which gave better and more consistent separation of 4-S and 5-S RNA in total extracts and in lO5 ooo x g supernatants than the methylated albumin-kieselguhr columns. We chose the method of separation on Sephadex G-75 used in this laboratory for a number of years (M. A. DEVYNCK AND Y. COURTOIS,
unpublished results).
Supernatants from the centrifugation of labeled, levallorphan-treated cell extracts at lO5 ooo x g with added, unlabeled 5-S RNA and total cell extracts with no added 5-S RNA carrier were chromatographed on Sephadex G-75. Fig. 5 B shows that total extracts exhibited 8H and 14C peaks which coincided with the 260 m# absorbance peak of 5-S RNA, indicating that both control and inhibited cells produced 5-S RNA. The minimum of the 3H/14C ratio also occt, rred at the maximum of the absorbance peak, indicating inhibition of 5-S RNA synthesis by levallorphan.
1800.
$5
1600 ~- 140C
o
1200 0
4'0
Fractions
5'0
3°°°1~5°°° .-ii.I
"~£ 2 000110000 ir
"
!.m.ml A %'~
4'0
3'0 6
Fractions
SO
000_ _30 000.? E
£ta, ooo ~oooo .~
il
•
/ '..
B
_5
.. "
4
o
2 ooo ,o ooo 0.0
2'7
,
3'7 Fractions
t ,
47
P
Fig. 5. A b s e n c e of a free pool of 5-S R N A in n o r m a l a n d l e v a l l o r p h a n - t r e a t e d E. coli cells. Control a n d l e v a l l o r p h a n - t r e a t e d E. coli c u l t u r e s w e r e m i x e d a n d d i v i d e d i n t o t w o e q u a l parts. A lO5 ooo × g s u p e r n a t a n t w a s p r e p a r e d f r o m t h e first part. U n l a b e l e d 5-S R N A w a s a d d e d , a n d t h e m i x t u r e w a s c h r o m a t o g r a p h e d o n S e p h a d e x a s d e s c r i b e d in MATERIALS AND METHODS. T h e u p p e r c u r v e s h o w s t h e s e p a r a t i o n of 4-S a n d 5-S R N A . T h e c u r v e s in A g i v e t h e a m o u n t s of [SH]- a n d [l~Cluracil i n c o r p o r a t e d a n d t h e i s o t o p e ratio. T h e s e c o n d ham of t h e cells w a s u s e d for t h e p r e p a r a t i o n of a w h o l e n u c l e i c acid e x t r a c t ; t h e c u r v e s i n B give a/so t h e a m o u n t s of [SH]- a n d /]4C/uracil i n c o r p o r a t e d as w e l l as t h e i r i s o t o p e ratio. Q - C ) , 3~ r a d i o a c t i v i t y (counts/ min); 0-0, 14C r a d i o a c t i v i t y ( c o u n t s / m i n ) ; I . . . m , 8H/14C ratio. Biochim. Biophys. Acta, 182 (1969) 481-49 o
R. ROSCHENTHALERet al.
488
The Sephadex eluates of the lO5 ooo × g supernatant, however, showed no radioactivity peaks in the 5-S RNA region for either control or treated cells (Curves in Fig. 5A). The isotope ratio merely decreased to a value of about 4-5, characteristic of the 4-S RNA region, and remained constant throughout that region. From this we conclude that neither control nor levallorphan-treated cells contain detectable free 5-S RNA in the supernatant fraction. These findings are in agreement with data from other laboratories showing that 5-S RNA does not accumulate in the free state. This appears to be true even for cells treated with levallorphan in which the number of new ribosomes formed is greatly reduced. Identical results were obtained in an experiment in which the isotopes for the control and inhibited cultures were interchanged. The possibility was considered that 5-S RNA might be present in treated cells in a form bound weakly to ribosomes. After dialysis of ribosomes against buffer containing o.I mM Mg ~+, careful examination of the dialysate revealed no free 5-S RNA.
Inhibition o/di]/erent classes o / R N A by increasing concentralions o/levallorphan Table I I summarizes the results of a number of experiments carried out with concentrations of levallorphan ranging between I and 1. 5 mM. The values for I6-S and 23-S RNA are listed in a single column because the inhibition of synthesis of these two species of rRNA is virtually identical. The synthesis of 5-S RNA is inhibited to the same extent as (or slightly more than) the high molecular weight rRNA. t R N A (4 S) is always considerably less affected than the three types of rRNA.
TABLE THE
II
RELATIVE
SYNTHESIS
OF
RNA
CLASSES
AT VARIOUS
CONCENTRATIONS
OF LEVALLORPHAN
Concn. o/ levallorphan (mM)
4-SRNA
I6-S and 23-SRNA synthesis* (%)
5-SRNA
i.o i.i 1.2 1.5
87 65 4° 26
55 3° I7 12
34 28 IO io
* C a l c u l a t e d f r o m 8H#4C r a t i o s . T h e i s o t o p e r a t i o i n D N A w a s t a k e n a s i o o % .
DISCUSSION
The studies of SIMON9 establish that levorphanol, a narcotic drug for higher animals, inhibits E. coli growth at a mean concn, of 3-4 mM at neutral pH. At p H 8.5, some inhibition of growth is observed with lower concentration (o. I raM). A closer examination of the properties of this drug by SIMON AND VAN PRAAG4,5 showed that at p H 8.2, 1-2 mM levorphanol inhibits 80-90 % the synthesis of RNA in E. coli W, whereas DNA synthesis continues unaffected, at least for one generation, and protein synthesis is only inhibited b y about 50 %. Biochim. Biophys. Aeta,
182 (1969) 4 8 1 - 4 9 °
INHIBITION OF R N A
SYNTHESIS BY LEVALLORPttAN
489
The effect of levorphanol essentially corresponds to an inhibition of high molecular weight rRNA synthesis, tRNA synthesis being less affected and that of mRNA even less so. It was of considerable interest to determine whether levallorphan, a structural analog of levorphanol devoid of narcotic properties, would manifest similar effects. Levallorphan has been reported by SIMON9 to inhibit the growth of E. coli, to a greater extent than the parent compound. G R E E N E AND MAGASANIK la extensively studied the effects of levallorphan at the rather high concentration of 5 mM at pH 7. They observed a total inhibition of macromolecular synthesis, and interestingly enough, a rapid leakage and destruction of cellular ATP. The latter result is comparable to the observation of SIMON et al. 14 on the leakage of polyamine from levorphanol-treated E. coli cells. These indications promoted a closer analysis of the susceptibility of different classes of nucleic acids to the inhibition by levallorphar. To attain higher sensitivity, the analysis has been performed with a double-labeling technique, using [3HI- and EJ*C]uracil, respectively. It appears first that the drug depresses most of the RNA synthesis, whereas during the assay DNA synthesis seems to be unaffected. For the purpose of standardization, the isotope ratio 3H/14C found in DNA has been arbitrarily taken as the reference. The total amount of DNA present in control and levallorphan-treated cultures of identical absorbance, supports this contention. The synthesis of 4-S RNA and I6-23-S RNA can thus be compared. It is clear that the high molecular weight rRNA is more inhibited than the 4-S RNA. As the levallorphan concentration increases from I to 1.5 mM, the inhibition becomes more pronounced, and it is to be expected that for still higher amounts of the drug, total inhibition will be found, as described by GREENE AND MAGASANIK13 for bacteria and by NOTEBOOM AND MUELLERls for HeLa cells. The behavior of mRNA has not been thoroughly investigated. However, 3H/14C ratio of the rapidly labeled RNA peak located after the 23-S RNA peak is not altered if the pulse of labeled precursor is made in the presence of levallorphan under the conditions described here. Since no direct interaction between DNA and these drugs has ever been detected, as neither levorphanol nor levallorphan is active in an RNA-synthesising system in vitro, it is likely that their influence is indirect and due to the pertubation of control mechanisms. Since the control mechanisms for 4-S RNA, rRNA and mRNA are affected to different extents by levallorphan, the possibility arises of using this drug to define the mechanism controlling the synthesis of a given RNA molecule. We applied this approach to establish that the 5-S RNA discovered by ROSSET AND MONIER1 and ROSSET et al. 2 is an RNA whose biosynthesis is controlled by a mechanism specific for rRNA. This conclusion is based on the comparable inhibition of I6-23-S RNA and 5-S RNA, and on the fact that no free pool of 5-S RNA can be detected in normal or levallorphan-treated cells. The similarity between 5-S RNA and high molecular weight RNA regulation of synthesis is in agreement with the observations of HECHT et al. 1~, which showed the similarity between the kinetics of formation of 5-S RNA and mature I6-S RNA, as well as with the findings of MORELL et al. ~7 which located the 5-S RNA cistron close to the I6-S and 23-S cistrons. The way levallorphan affects the regulation of rRNA's is still unknown. However, the effects of levallorphan on the synthesis of various classes of RNA are comparable to those obtained in a "shifted-down" culture. How far such a comparison can hold is under investigation. Biochim. t~iophys. Acta, 182 (1969) 481-49 o
R. ROSCHENTHALER et al.
49 °
REFERENCES I 2 3 4 5 6
7 8 9 io II 12 13 14 I5 16 17
ROSSET AND R. MONIER, Biochim. Biophys. dcta, 68 (1963) 653ROSSET, R. MONIER AND J. JULIEN, Bull. Soc. Chim. Biol., 46 (1964) 87. G. BROWNLEE, F. SANGER AND B. G. BARRELI, Nature, 215 (I967) 735. J. SIMON AND D. VAN PRAAG, Proc. Natl. Acad. Sci. U.S., 51 (1964) 877. J. SIMON AND D. VAN PRAAG, Proc. Natl. dead. Sci. U.S., 51 (1964) 1151. D . G. COMB AND T. ZEHAvI-WILLNER, J. Mol. Biol., 23 (1967) 441. J. D. MANDELL AND a . D. HERSHEY, Anal. Biochem., i (196o) 66. K. A. O. ELLEM, Biochim. Biophys. Acla, 149 (1967) 74. E. J. SIMON, Science, 144 (1964) 543. G. SCHMIDT AND S. J. THANNHAUSER, J. Biol. Chem., 161 (1945) 83. A. ISHIHAMA, N. MIZUNO, M. TAKARI, E. OTAKA AND S. OSAWA, J. Mol. Biol., 5 (1962) 251. M. AUBERT, J. F. SCOTT, M. REYNIER AND M. MONIER, Proc. Natl. Aead. Sci. U.S., 61 (I968) 292. R. GREENE AND B. ~][AGASANIK, Mol. Pharmacol., 3 (1967) 453. E. J. SIMON, S. S. COHEN AND A. RAINA, Bioehem. Biophys. Res. Commun., 24 (1966) 482. W. D. NOTEBOOM AND G. C. MUELLER, Mol. Pharmacol., 2 (1966) 534. N. B. HECHT, M. BLEYMAN AND C. R. WOESE, Proe. Natl. Acad. Sci. U.S., 59 (1968) I278P. MORELL, I. SMITH, D. DUBNAU AND J. MARMUR, Biochemistry, 6 (1967) 258.
R. R. G. E. E.
Biochim. Biophys. Acta, 182 (1969) 481-49 °
BIOCHIMICA ET BIOPHYSICA ACTA BBA 96195
I N H I B I T I O N OF T H E SYNTHESIS OF 5-S RIBOSOMAL RNA IN E S C H E R I C H I A COLI BY LEVALLORPHAN R. R O S C H E N T H A L E R * , M. A. D E V Y N C K * * , P. F R O M A G E O T A N D E. J. S I M O N " * Service de Biochimie, Ddpartement de Biologie, Centre d'Etudes Nucldaires de Saclay, 9I-Gi[-sur Yvette (France) (Received D e c e m b e r I9th, 1968)
SUMMARY
Levallorphan (1.54. lO-3 M) inhibits RNA biosynthesis by 7 ° %, whereas the synthesis of protein is inhibited by 14 %. The residual synthesis of tRNA under such conditions is greater by at least a factor of 2 than that of high molecular weight RNA. mRNA synthesis, on the contrary, seems to be only slightly inhibited or not at all. Levallorphan therefore exhibits effects similar to levorphanol. As the origin of 5-S RNA has been the subject of recent discussion we analyzed the extent of its synthesis in the presence of levallorphan. It has been found that 5-S RNA synthesis is inhibited bv the drug at least as profoundly as high molecular weight RNA, a result which puts the 5-S RNA in the same class as the ribosomal RNA's and suggests that the same type of control mechanism regulates the synthesis of 5-S, I6-S and 23-S RNA's.
INTRODUCTION
The structure of the 5-S RNA discovered in Escherichia coli by ROSSET AND MONIER1 and ROSSET et al3 has been elucidated 3, but its function is still unknown. It has been considered to be a ribosomal RNA. This conclusion could be specifically tested by examination of 5-S RNA synthesis in the presence of levorphanol, a drug which exhibits a selective inhibition of ribosomal RNA (16 S and 23 S) as reported by SIMON AND V A N PRAAG 4,5. Because of the difficulties experienced in obtaining narcotic drugs for purposes of investigation, levallorphan, a nonnarcotic N-allyl analogue of levorphanol, was used.
HO~N_CH2_CH=CH 2 L LLORPHAN EVA
* P r e s e n t address: c/o Prof. Liebermeister, K l i n i k u m r e c h t s der Isax, Munich, G e r m a n y . ** P r e - d o c t o r a l fellow of t h e C o m m i s s a r i a t tt l ' E n e r g i e A t o m i q u e . *** P r e s e n t a d d r e s s : D e p a r t m e n t of Medicine, N e w Y o r k U n i v e r s i t y Medical Center, 550 F i r s t A v e n u e , N e w York, N Y lOO16, U.S.A. "*" Career Scientist of t h e H e a l t h R e s e a r c h Council of t h e City of N e w York.
Biochim. Biophys. Acta, 182 (1969) 481-49 °
482
R. ROSCHENTHALERet al.
With this tool at hand, it was shown that the synthesis of 5-S RNA was inhibited as profoundly as that of the high molecular weight rRNA's, a finding consistent with the view that 5-S RNA is regulated in the same way as the other ribosomal RNA's. MATERIALSAND METHODS
Bacterial strains and media E. coli K12S was obtained from Dr. R. Devoret, C.N.R.S., Gif-sur-Yvette, and was used for the labeling experiments. Bacteria of a K~2 Hfr-strain obtained from Dr. G. Cohen were used as nonlabeled carrier cells. The cells were grown in a low phosphate medium buffered with triethanolamine at pH 8.1, as described by SIMON AND VAN PRAAG4. Sodium succinate (0. 5 %) served as carbon source, and the medium was supplemented with 0.2 % casamino acids (Difco). Materials Levallorphan was generously provided by Hoffmann-La Roche, Basel. E2-~4C]Uracil (47-4 mC/mmole) and [6-~H~uracil (24 C/mmole) were obtained from the D~partement des Radio616ments C.E.A., Saclay. Double-labeling experiments The double-labeling procedure minimizes apparent differences in the labeling of nucleic acid fractions due to differential losses during extraction and fractionation. Two cultures of I 1 each were incubated and allowed to grow to an absorbance at 547 m# of 0.300. At this absorbance, levallorphan in the appropriate concentration was added to one of the two log-phase cultures, followed after 5 rain by [3H]uracil (2 mC). In both cases, unlabeled uracil was added to give a final uracil concn, of 20/~g/ml. [14ClUracil (200 #C) was added to the control culture at the same time. Both cultures were then incubated for 15 min. 5 ml of a 1 % solution of unlabeled uracil was then added to each culture, and the radioactive uracil was "chased" for another lO rain. The bacteria were chilled by pouring frozen buffer (o.oi M Tris, o.oi M MgCI2, pH 7-5) into the culture and harvested in a Servall centrifuge at 2 °. The labeled cells were resuspended in the same buffer and mixed in such a way that the total ~H radioactivity versus the total 14C radioactivity incorporated were in a ratio of 3:1. In one experiment both cultures were centrifuged after the i5-min incubation resuspended in the original medium supplemented with unlabeled uracil (5° #g/ml) and nfixed immediately. This chase, made in the absence of levallorphan, was done to make certain that our results were not influenced by unequal rates of release of labeled uracil from mRNA in the inhibited and control cultures. Preparation o/nucleic acids Unlabeled carrier cells (5 g) were added to the mixture of labeled cells, and the whole suspension was washed twice with buffer. After grinding the bacteria with 15 g of alumina and removal of alumina and unbroken cells by centrifugation the Biochim. Biophys. Acta, 182 (1969) 481-49o
INHIBITION OF
RNA
SYNTHESIS BY LEVALLORPHAN
483
supernatant was made 0.2 % with respect to deoxycholate and the cell debris and precipitate were sedimented. The supernatant was either directly extracted with phenol, or subjected to high-speed centrifugation to collect the ribosomes from which the 5-S RNA was extracted as described b y COMB AND ZEHAVI-WILLNERs. The 4-S and 5-S RNA were separated from the bulk of rRNA b y precipitation of the high molecular weight RNA with 2 M NaC1.
Fractionation o~ nucleic acids The material prepared was analyzed by methylated serum albumin coated kieselguhr column chromatography as described by MANDELL AND HERSHEY~. Bovine serum albumin Fraction V (Calbiochem) was used to prepare the methylated serum albumin. The gradient used (total vol. 600 ml) was between 0.2 M and 1.2 M NaC1. Fractions of IO ml were collected. In some cases, Sephadex column chromatography was used. Sephadex G-75 (Pharmacia) suspended in potassium acetate (5 mM, p H 4.2) was made into a 2.5 cm × 14o cm column, and maintained at 4 °, and after addition of the sample, eluted with the same buffer at a rate of 6 ml/h, the volume of each fraction being 5 ml. The absorbance of the fractions is read at 260 m# with a Zeiss spectrophotometer PMQ II. The amounts of 3H and 14C, respectively, are measured with the usual precautions for double-labeling experiments s after precipitation of the sample with 5 % trichloroacetic acid and filtration on Millipore filters. The highest variation at the lowest 3H/14C ratios is 15 %.
RESULTS
Before investigating the effects of levallorphan on the synthesis of 5-S RNA, we have established that this drug inhibits the growth of E. coli in a similar way to that of levorphanol ~. Until a concn, of 1.6 mM levallorphan is reached, viable cell counts show that there is little or no killing of bacteria. Furthermore the incorporation of [14C]uracil into nucleic acids decreases sharply with an increase in levallorphan concentration, whereas the incorporation of [l*C]phenylalanine is, under the same conditions, only slightly affected, as demonstrated in the case of levorphanol.
TABLE I EFFECTS OF LEVALLORPHAN ON [14C]PHENYLALANINE AND [aH]URACIL INCORPORATION IN E. coli I0/zC (200/~g) ESH]uracil and 0.I/*C [x4C]phenylalanine were added to I0 ml of bacterial culture. Aliquots were t a k e n at various intervals and the labels incorporated were measured. The d a t a give the percentage of incorporation versus the control.
Conch. o/ levallorphan
Incorporation o/ [liC]phenylalanine (%)
Incorporation of [SH]uracil (%)
ioo
IOO
(raM) o
1.39 1.54
96 86
42 28
Biochim. Biophys. Acta, 182 (1969) 481-49 °
R. ROSCHENTHALERgt al.
484
Comparison o/the inhibition by levallorphan o/the synthesis oI high molecular weight rRNA, mRNA and soluble RNA The profile of the 3H/~4C ratio in the eluate from the methylated albuminkieselguhr column constitutes a measure of the relative rates of synthesis of the nucleic acid fractions from drug-treated and control cells.
0,60
16
112 0.40
:1o 2-
-s u
g
-<. 0.20
:4 ._o
o
......° o ° \ b ........
' ........
4'0 .................
Fractions
6'0 ........
' .....
8b
.......
Fig. I. Influence of levallorphan on the synthesis of the various classes of E. coli nucleic acids. E. coli was g r o w n in a m e d i u m containing 1.3o mM levallorphan and pH]uracil. I t w a s mixed w i t h bacteria g r o w n in the absence of the drug, b u t otherwise u n d e r identical conditions, and la.beled w i t h [14C]uracil. A s u p e r n a t a n t was p r e p a r e d after 2. 5 it centrifugation at lO 5 ooo × g in order to reduce tile a m o u n t of r i b o s o m e s present, and analyzed on a m e t h y l a t e d albumin-kieselg u h r column. O - O , absorbaxtce at 260 m/,; [2]- - -E], 3H/14C ratio. The sedimented material has also been analyzed b y the same technique, and the ~H114C ratio observed for the r R N A is identical w i t h those s h o w n in tile figure.
A typical elution profile of a cell extract is shown in Fig. i. It can be seen that the highest isotope ratio occurs at or near the DNA peak. In view of this very reproducible observation and the previous finding that DNA synthesis is not significantly inhibited b y levorphanol 4 under the conditions used, the isotope ratio in DNA was taken as IOO % for the purpose of calculating percent inhibition in other fractions. Purification of the material in this DNA peak by the procedure of SCHMIDT AND THANNHAUSER 10 did not alter the isotope ratio. The lowest 3H/14C ratio was observed in the region of the I6-S and 23-S rRNA. The average isotope ratio in the 4-S RNA region was always 2-3 times higher. Thus, in the experiment shown in Fig. I, the synthesis of I6-S and 23-S RNA was inhibited 80-90 ~o, while that of 4-S RNA was inhibited 50-60 ~o. This difference increased with further purification of the fractions. A number of experiments supporting this conclusion are shown later in this paper (see Table II). The question of the inhibition of m R N A synthesis was not extensively investigated in this work. The two peaks of radioactivity occurring just before and after the DNA peak are sensitive to ribonuclease I. These RNA peaks are located in one of the regions where labeled RNA is found in extracts from cultures submitted to a brief pulse of radioactive RNA precursor 11. This evidence together with the insensitivity of protein synthesis make it likely that levallorphan does not modify greatly the synthesis of mRNA. Biochim. Biophys. Acta, 182 (1969) 481-49o
INHIBITION OF R N A SYNTHESIS BY LEVALLORPHAN
485
The evidence presented indicates that the inhibitory effect of levallorphan is predominantly on the synthesis of rRNA (16 S and 23 S), as has previously been reported for levorphanol.
El/ect o/ levallorphan in the synthesis ol 5-S r R N A Attempts to separate 5-S RNA from tRNA (4 S) were first made by the technique of ROSSET et al. 2. The small peak following closely the 4-S RNA peak in the elution profile from a methylated albumin-kieselguhr column is usually considered to be mainly 5-S RNA. However, this second peak was not obtained reproducibly and disappeared if the fibrous material present during the alcohol precipitation step was removed by means of a rotating glass rod (Fig. 2). It appeared also that the size of this second peak was variable according to the time that elapsed during the preparation of the nucleic acids. Furthermore, in cases when this second peak was well identified, a decrease in the 3H/14C ratio was always observed just prior to the appearance of the presumed 5-S RNA peak. When a soluble RNA preparation was used which lacked the second peak, the second half of the 4-S RNA peak exhibited a much lower 3H/14C ratio (Fig. 2). These observations led us to the preliminary assumption that the second peak, when present, consisted mainly of aggregates of tRNA. Elution of 5-S RNA presumably occurs between the two peaks. After the second peak, the 3H/14C ratio decreases again. This might indicate the presence of another form of 5-S RNA. COMBAND ZEHAVIWILLNER6 have observed a second peak containing 5-S RNA on elution from a methylated albumin-kieselguhr column. This could be seen if the end groups of the 5-S RNA were modified by treatment with periodate. AUBERT et al. 1~ also showed two 5-S RNA peaks after urea denaturation.
0'40t
]
1
1000 m 50
,< u .9
~:
0"30
N
40 50 6'0 3'0 4b Frect[ons
50
Fig. 2. E l u t i o n profiles and 3H/14C ratios of m a t e r i a l f r o m the 4 S-5 S region. On the left, a p r e p a r a t i o n of 4-S-5-S R N A w h i c h had been k e p t in a frozen s t a t e for several days. On the right, p r e p a r a t i o n f r o m which the fibrous material had been removed. The d o t t e d lines designate the fractions which were pooled. The corresponding c o l u m n s below give the 3H11'C ratio of this material. The concn, of levallorphan in the inhibited culture was 1.41 mM.
Biochim. Biophys. Acta, 182 (1969) 481-49 °
486
g. ROSCHENTHALERet al.
To obtain further evidence on the nature of this second peak, the nucleic acids present in a lO5 o o o × g supernatant were purified by passage through a DEAEcellulose column, and the tRNA fraction recovered. This material was filtered through a Sephadex G-75 column (magnesium acetate 5.1o -3 M, pH 4.2) and a part of the tRNA was eluted with the void volume, indicating aggregation. This aggregated material was kept frozen for several months, and chromatographed on a methylated albumin-kieselguhr column. The elution pattern shown in fig. 3 showed the normal tRNA peak and a large peak at the position usually assigned to the 5-S RNA. Obviously this latter peak is due to some form of tRNA and not to 5-S RNA. It will be noticed also that an important part of the RNA was eluted up to regions normally occupied by I6-S and 23-S RNA. These results show that either aggregation or conformational changes of low molecular weight RNA can cause difficulties in obtaining a good resolution of 5-S RNA from 4-S RNA. We therefore utilized the more effective method described by COMB AND Z E H A v I - W I L L N E R ~.
This method removes mRNA and most of the tRNA from isolated ribosomal particles. RNA is then extracted, and the largest part of the high molecular weight RNA is removed. The remaining material is chromatographed on a methylated albumin-kieselguhr column whose elution profile and 3H/14C ratio are given in Fig. 4. The 5-S RNA peak is easily identified and resolved from residual high molecular weight RNA. The ~H/14C value corresponding to the 5-S RNA is remarkably low even more so than the ratio corresponding to the I6-S peak. It is important to point out that the 3H/14C ratio of the residual high molecular weight fraction present is the same as in the original extract (Fig. I). By comparison with Fig. I, it appears that the inhibition observed for the 5-S RNA synthesis in the presence of levallorphan is close to 9 ° °/o when compared with the control.
D.3
~
30
~'", ,~o.O. ° o~
°
0.05
o
o
eg ...... 5b.... 4o
~
Fractions 6'0 .....
ab " 9'o
Fractions
Jo...... 3'o...... , i b
5b...... g6 ...... 7~...... 8b
~
Fig. 3. E l u t i o n pro file from a m e t h y l a t e d a l b u m i n - k i e s e l g u h r - c o l u m n c h r o m a t o g r a p h y of t R N A a g g r e g a t e s , t R N A were p u r i f i e d b y D E A E - c e l l u l o s e a n d S e p h a d e x G-75 c h r o m a t o g r a p h y . O n l y t h e m a t e r i a l e l u t i n g w i t h t h e v o i d v o l u m e f r o m t h e S e p h a d e x w a s a p p l i e d on t h e m e t h y l a t e d a l b u m i n - k i e s e l g u h r co lumn. Fig. 4. E l u t i o n profile from R N A e x t r a c t e d from p u r i f i e d r i b o s o m a l p a r t i c l e s . R i b o s o m e s coll e c t e d from c o n t r o l a n d 1. 3 miV[ l e v a l l o r p h a n - t r e a t e d c u l t u r e s , w e r e d i a l y z e d a g a i n s t a Tris buffer, o . o i M (pH 7.2), o . i mM Mg 2+, a n d w a s h e d w i t h t h e s a m e buffer. The r i b o s o m a l p a r t i c l e s w e re extracted with phenol and the high molecular weight R N A deliberately incompletely precipit a t e d w i t h 2 M NaC1. The r e m a i n i n g R N A was a p p l i e d t o a m e t h y l a t e d a l b u m i n - k i e s e l g u h r colu m n . T h e first A2e o m/~ p e a k c o n s i s t s of 5-S R N A , a n d t h e s e c ond is m o s t l y I6-S R N A , s i nc e 23-S R N A p r e c i p i t a t e s m o r e r e a d i l y .
Biochim. Biophys. Acta, 182 (I969) 481-49o
INHIBITION OF
RNA
SYNTHESIS BY LEVALLORPHAN
487
Evidence/or the absence o/ a ~tee 5-S RNA pool The low 8H/14C ratio in the 5-S RNA isolated from ribosomes may merely reflect the absence of new ribosomes to which newly formed 5-S RNA can attach in the levallorphan-treated cells. It therefore became essential to investigate the possibility of the existence of a free 5-S RNA pool in levallorphan-treated bacteria. To investigate this question it was necessary to use a method which gave better and more consistent separation of 4-S and 5-S RNA in total extracts and in lO5 ooo x g supernatants than the methylated albumin-kieselguhr columns. We chose the method of separation on Sephadex G-75 used in this laboratory for a number of years (M. A. DEVYNCK AND Y. COURTOIS,
unpublished results).
Supernatants from the centrifugation of labeled, levallorphan-treated cell extracts at lO5 ooo x g with added, unlabeled 5-S RNA and total cell extracts with no added 5-S RNA carrier were chromatographed on Sephadex G-75. Fig. 5 B shows that total extracts exhibited 8H and 14C peaks which coincided with the 260 m# absorbance peak of 5-S RNA, indicating that both control and inhibited cells produced 5-S RNA. The minimum of the 3H/14C ratio also occt, rred at the maximum of the absorbance peak, indicating inhibition of 5-S RNA synthesis by levallorphan.
1800.
$5
1600 ~- 140C
o
1200 0
4'0
Fractions
5'0
3°°°1~5°°° .-ii.I
"~£ 2 000110000 ir
"
!.m.ml A %'~
4'0
3'0 6
Fractions
SO
000_ _30 000.? E
£ta, ooo ~oooo .~
il
•
/ '..
B
_5
.. "
4
o
2 ooo ,o ooo 0.0
2'7
,
3'7 Fractions
t ,
47
P
Fig. 5. A b s e n c e of a free pool of 5-S R N A in n o r m a l a n d l e v a l l o r p h a n - t r e a t e d E. coli cells. Control a n d l e v a l l o r p h a n - t r e a t e d E. coli c u l t u r e s w e r e m i x e d a n d d i v i d e d i n t o t w o e q u a l parts. A lO5 ooo × g s u p e r n a t a n t w a s p r e p a r e d f r o m t h e first part. U n l a b e l e d 5-S R N A w a s a d d e d , a n d t h e m i x t u r e w a s c h r o m a t o g r a p h e d o n S e p h a d e x a s d e s c r i b e d in MATERIALS AND METHODS. T h e u p p e r c u r v e s h o w s t h e s e p a r a t i o n of 4-S a n d 5-S R N A . T h e c u r v e s in A g i v e t h e a m o u n t s of [SH]- a n d [l~Cluracil i n c o r p o r a t e d a n d t h e i s o t o p e ratio. T h e s e c o n d ham of t h e cells w a s u s e d for t h e p r e p a r a t i o n of a w h o l e n u c l e i c acid e x t r a c t ; t h e c u r v e s i n B give a/so t h e a m o u n t s of [SH]- a n d /]4C/uracil i n c o r p o r a t e d as w e l l as t h e i r i s o t o p e ratio. Q - C ) , 3~ r a d i o a c t i v i t y (counts/ min); 0-0, 14C r a d i o a c t i v i t y ( c o u n t s / m i n ) ; I . . . m , 8H/14C ratio. Biochim. Biophys. Acta, 182 (1969) 481-49 o
R. ROSCHENTHALERet al.
488
The Sephadex eluates of the lO5 ooo × g supernatant, however, showed no radioactivity peaks in the 5-S RNA region for either control or treated cells (Curves in Fig. 5A). The isotope ratio merely decreased to a value of about 4-5, characteristic of the 4-S RNA region, and remained constant throughout that region. From this we conclude that neither control nor levallorphan-treated cells contain detectable free 5-S RNA in the supernatant fraction. These findings are in agreement with data from other laboratories showing that 5-S RNA does not accumulate in the free state. This appears to be true even for cells treated with levallorphan in which the number of new ribosomes formed is greatly reduced. Identical results were obtained in an experiment in which the isotopes for the control and inhibited cultures were interchanged. The possibility was considered that 5-S RNA might be present in treated cells in a form bound weakly to ribosomes. After dialysis of ribosomes against buffer containing o.I mM Mg ~+, careful examination of the dialysate revealed no free 5-S RNA.
Inhibition o/di]/erent classes o / R N A by increasing concentralions o/levallorphan Table I I summarizes the results of a number of experiments carried out with concentrations of levallorphan ranging between I and 1. 5 mM. The values for I6-S and 23-S RNA are listed in a single column because the inhibition of synthesis of these two species of rRNA is virtually identical. The synthesis of 5-S RNA is inhibited to the same extent as (or slightly more than) the high molecular weight rRNA. t R N A (4 S) is always considerably less affected than the three types of rRNA.
TABLE THE
II
RELATIVE
SYNTHESIS
OF
RNA
CLASSES
AT VARIOUS
CONCENTRATIONS
OF LEVALLORPHAN
Concn. o/ levallorphan (mM)
4-SRNA
I6-S and 23-SRNA synthesis* (%)
5-SRNA
i.o i.i 1.2 1.5
87 65 4° 26
55 3° I7 12
34 28 IO io
* C a l c u l a t e d f r o m 8H#4C r a t i o s . T h e i s o t o p e r a t i o i n D N A w a s t a k e n a s i o o % .
DISCUSSION
The studies of SIMON9 establish that levorphanol, a narcotic drug for higher animals, inhibits E. coli growth at a mean concn, of 3-4 mM at neutral pH. At p H 8.5, some inhibition of growth is observed with lower concentration (o. I raM). A closer examination of the properties of this drug by SIMON AND VAN PRAAG4,5 showed that at p H 8.2, 1-2 mM levorphanol inhibits 80-90 % the synthesis of RNA in E. coli W, whereas DNA synthesis continues unaffected, at least for one generation, and protein synthesis is only inhibited b y about 50 %. Biochim. Biophys. Aeta,
182 (1969) 4 8 1 - 4 9 °
INHIBITION OF R N A
SYNTHESIS BY LEVALLORPttAN
489
The effect of levorphanol essentially corresponds to an inhibition of high molecular weight rRNA synthesis, tRNA synthesis being less affected and that of mRNA even less so. It was of considerable interest to determine whether levallorphan, a structural analog of levorphanol devoid of narcotic properties, would manifest similar effects. Levallorphan has been reported by SIMON9 to inhibit the growth of E. coli, to a greater extent than the parent compound. G R E E N E AND MAGASANIK la extensively studied the effects of levallorphan at the rather high concentration of 5 mM at pH 7. They observed a total inhibition of macromolecular synthesis, and interestingly enough, a rapid leakage and destruction of cellular ATP. The latter result is comparable to the observation of SIMON et al. 14 on the leakage of polyamine from levorphanol-treated E. coli cells. These indications promoted a closer analysis of the susceptibility of different classes of nucleic acids to the inhibition by levallorphar. To attain higher sensitivity, the analysis has been performed with a double-labeling technique, using [3HI- and EJ*C]uracil, respectively. It appears first that the drug depresses most of the RNA synthesis, whereas during the assay DNA synthesis seems to be unaffected. For the purpose of standardization, the isotope ratio 3H/14C found in DNA has been arbitrarily taken as the reference. The total amount of DNA present in control and levallorphan-treated cultures of identical absorbance, supports this contention. The synthesis of 4-S RNA and I6-23-S RNA can thus be compared. It is clear that the high molecular weight rRNA is more inhibited than the 4-S RNA. As the levallorphan concentration increases from I to 1.5 mM, the inhibition becomes more pronounced, and it is to be expected that for still higher amounts of the drug, total inhibition will be found, as described by GREENE AND MAGASANIK13 for bacteria and by NOTEBOOM AND MUELLERls for HeLa cells. The behavior of mRNA has not been thoroughly investigated. However, 3H/14C ratio of the rapidly labeled RNA peak located after the 23-S RNA peak is not altered if the pulse of labeled precursor is made in the presence of levallorphan under the conditions described here. Since no direct interaction between DNA and these drugs has ever been detected, as neither levorphanol nor levallorphan is active in an RNA-synthesising system in vitro, it is likely that their influence is indirect and due to the pertubation of control mechanisms. Since the control mechanisms for 4-S RNA, rRNA and mRNA are affected to different extents by levallorphan, the possibility arises of using this drug to define the mechanism controlling the synthesis of a given RNA molecule. We applied this approach to establish that the 5-S RNA discovered by ROSSET AND MONIER1 and ROSSET et al. 2 is an RNA whose biosynthesis is controlled by a mechanism specific for rRNA. This conclusion is based on the comparable inhibition of I6-23-S RNA and 5-S RNA, and on the fact that no free pool of 5-S RNA can be detected in normal or levallorphan-treated cells. The similarity between 5-S RNA and high molecular weight RNA regulation of synthesis is in agreement with the observations of HECHT et al. 1~, which showed the similarity between the kinetics of formation of 5-S RNA and mature I6-S RNA, as well as with the findings of MORELL et al. ~7 which located the 5-S RNA cistron close to the I6-S and 23-S cistrons. The way levallorphan affects the regulation of rRNA's is still unknown. However, the effects of levallorphan on the synthesis of various classes of RNA are comparable to those obtained in a "shifted-down" culture. How far such a comparison can hold is under investigation. Biochim. t~iophys. Acta, 182 (1969) 481-49 o
R. ROSCHENTHALER et al.
49 °
REFERENCES I 2 3 4 5 6
7 8 9 io II 12 13 14 I5 16 17
ROSSET AND R. MONIER, Biochim. Biophys. dcta, 68 (1963) 653ROSSET, R. MONIER AND J. JULIEN, Bull. Soc. Chim. Biol., 46 (1964) 87. G. BROWNLEE, F. SANGER AND B. G. BARRELI, Nature, 215 (I967) 735. J. SIMON AND D. VAN PRAAG, Proc. Natl. Acad. Sci. U.S., 51 (1964) 877. J. SIMON AND D. VAN PRAAG, Proc. Natl. dead. Sci. U.S., 51 (1964) 1151. D . G. COMB AND T. ZEHAvI-WILLNER, J. Mol. Biol., 23 (1967) 441. J. D. MANDELL AND a . D. HERSHEY, Anal. Biochem., i (196o) 66. K. A. O. ELLEM, Biochim. Biophys. Acla, 149 (1967) 74. E. J. SIMON, Science, 144 (1964) 543. G. SCHMIDT AND S. J. THANNHAUSER, J. Biol. Chem., 161 (1945) 83. A. ISHIHAMA, N. MIZUNO, M. TAKARI, E. OTAKA AND S. OSAWA, J. Mol. Biol., 5 (1962) 251. M. AUBERT, J. F. SCOTT, M. REYNIER AND M. MONIER, Proc. Natl. Aead. Sci. U.S., 61 (I968) 292. R. GREENE AND B. ~][AGASANIK, Mol. Pharmacol., 3 (1967) 453. E. J. SIMON, S. S. COHEN AND A. RAINA, Bioehem. Biophys. Res. Commun., 24 (1966) 482. W. D. NOTEBOOM AND G. C. MUELLER, Mol. Pharmacol., 2 (1966) 534. N. B. HECHT, M. BLEYMAN AND C. R. WOESE, Proe. Natl. Acad. Sci. U.S., 59 (1968) I278P. MORELL, I. SMITH, D. DUBNAU AND J. MARMUR, Biochemistry, 6 (1967) 258.
R. R. G. E. E.
Biochim. Biophys. Acta, 182 (1969) 481-49 °