Synthesis of ribonucleic acids in Escherichia coli irradiated with ultraviolet light

i88

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 8322.

S Y N T H E S I S OF R I B O N U C L E I C ACIDS IN E S C H E R I C H I A IRRADIATED

COLI

WITH ULTRAVIOLET LIGHT

A. SIBATANI

AND

NOBUKO MIZUNO"

Research Institute ]or Nuclear Medicine and Biology, Hiroshima University, Hiroshima (Japan) (Received April ioth, 1963)

SUMMARY

Sensitivity of the synthesis of various RNA components in Escherichia coli B (H) to ultraviolet irradiation was examined by chromatographic analysis of nucleic acid samples labelled with [~P]orthophosphate. After a dose of ultraviolet irradiation sufficient to block the DNA synthesis for 3o min, the synthesis of RNA components was not severely affected during the same period. With increasing doses of ultraviolet irradiation, RNA synthesis was also impaired progressively, and the order of its sensitivity was that of the increasing molecular weight for transfer RNA and ribosomal RNA. Thus, the synthesis of 23-S RNA was affected most readily, with corresponding inhibition of labelling of 5o-S ribosome subunit with [14C]uracil. Higher doses of ultraviolet irradiation completely blocked the synthesis of ribosomal RNA but still allowed some formation of transfer RNA and messenger RNA. No preferential inhibition of the synthesis was observed for any one of the chromatographically separated messenger RNA components.

INTRODUCTION

Irradiation of E. coli with small doses of ultraviolet irradiation causes a temporary cessation of DNA synthesis without markedly affecting overall synthesis of RNA and protein l& According to the current view, bacterial cells contain, along with tRNA and rRNA, an unstable minor RNA component designated generally as mRNA with a DNA-like base composition s& It is believed that mRNA is synthesized by RNA polymerase (EC 2.7.7.6) which requires, like DNA polymerase (EC 2.7.7.7), a DNA primerS, 6. Further recent findings suggest that the nucleotide sequence of both rRNA and tRNA is primarily determined by DNA of the bacterial chromosome 7-9. Since overall RNA synthesis of the cell is immediately blocked by actinomycin, a powerful inhibitor of DNA-dependent enzymic synthesis of RNA, it may be that all the cellular RNA components are synthesized by RNA polymerase on the DNA template 1°. However, the enzymic synthesis of tRNA and rRNA has not yet been Abbreviations: mRNA, messenger RNA; rRNA, ribosomal RNA; tRNA, transfer RNA. " On leave from Institute for Molecular Biology, Faculty of Science, Nagoya University, Nagoya. Present address: Cancer Research Institute, Kyushu University School of Medicine, Fukuoka (Japan).

Biochim. Biophys. Acta, 76 (1963) 188-2oo

RNA SYNTHESIS IN UV-IRRADIATED BACTERIA

189

demonstrated. It is now pertinent to ask whether syntheses of these RNA components show any difference in the sensitivity to ultraviolet irradiation, to throw some light on the mechanism of synthesis, or its regulation, of these RNA components in bacteria. In a recent report, WAINFAN et al. n indicated that an irradiation dose which partially inhibited rRNA synthesis did not affect tRNA synthesis. The present communication confirms this point and further extends the observation to higher doses of ultraviolet irradiation. MATERIALS AND METHODS

Cells, media and conditions [or irradiation and labelling E. coli strain B(H) received from Dr. M. NOMURA of Osaka University was used throughout the present study. Tris-salts medium contained 4.676 g NaC1, 1.491 g KC1, 1.o7o g NH4CI, 0.203 g MgCI,, o.o147 g CaC12, 0.0227 g Na2SO4, o.o871 g KH2PO4 and 0.0541 mg FeC13 in I 1 of o.I M Tris buffer (pH 7.8). Tris-glucose-Casamino acid medium was Trissalts medium containing o.2 % glucose and o.I % Difco Casamino acid. For lowphosphate growth medium, KH2P Q was omitted from Tris-glucose-Casamino acid medium. It still contained about IO/~g P per ml in the form of Pt originating from Casamino acid. Bacteria subcultured overnight in Tris-glucose-Casamino acid medium were inoculated at a density of about IOs cells/ml in the low-phosphate growth medium, and the culture was shaken at 37 ° . Exponentially growing bacteria (generation time 4 ° min) at a density of lO 9 cells/ml were harvested and resuspended in a cold Trissalts medium (without KH2PO4) at the same density. Cells were transferred, in portions of 25 ml, into I7-cm petri dishes and irradiated for desired periods under constant stirring at a distance of I m from a I5-W Toshiba germicidal lamp. Glucose and Casamino acid were added to control and irradiated cell suspensions to restore the composition of the low-phosphate growth medium, and incubation was continued for desired periods at 37 °. For labelling RNA, 1-2o/zC of carrier-free [32P]PI per ml or o.I #C of [2-14C~uracil (330 mC/mole, Daiichi Pure Chemicals Co.) per ml was added. At the end of the labelling period, the cultures were rapidly poured on to crushed ice and centrifuged. The cells were washed in the cold with i mM magnesium acetate in o.oi M Tris buffer (pH 7.4). Preparation o/ nucleic acids and cell extracts Nucleic acids were prepared according to ISHIHAMA et al. 12 by sodium dodecyl sulphate-phenol extraction after grinding the bacterial cells with quartz sand. For preparation of cell extracts, bacteria were washed twice with cold o.oi M Tris buffer (pH 7.4), containing an appropriate amount of magnesium acetate, and ground with quartz sand for 3 min. Disrupted cells were extracted with 3 volumes of the buffer solution. The extract was then clarified by centrifuging at 20 ooo×g for 15 rain, and. incubated for 15 rain at 37 ° to degrade mRNA. Chromatographic analysis o/ nucleic acids Nucleic acids were chromatographed on a methylated bovine serum albumin column is prepared by a simplified procedure. Hyflosupercel (IO g) in 50 ml of Biochim. Biophys. Acta, 76 (1963) 188-2oo

19o

a . SIBATANI, N. MIZUNO

o.I M NaCI was mixed with 2.5 ml of I °/o methylated bovine serum albumin solution to make a column of 1.8 × 15 cm, washed with 200 ml of 0.2 M NaCI, loaded with a sample of nucleic acids dissolved in 25 ml of 0.2 M NaC1 and washed with 50 ml of 0.2 M NaC1 to remove contaminating [3~P]PI. Nucleic acids were eluted with 50 ml of 0.4 M NaC1 followed by a linear concentration gradient (o.4-1.o M) of NaC1 in 0.05 M phosphate buffer (pH 6.7). Radioactivity of collected fractions was measured with I-ml liquid samples in metal planchettes, and expressed in

total

total counts/rain per fraction counts/min of 32p added to incubation medium

io 3

Nucleotide composition of m R N A fraction was determined by the distribution of ~2p according to ISHIHAMA et al. ~.

Sedimentation analysis A I-ml sample of cell extracts labelled with [~4C]uracil was layered on top of 29 ml of a linear sucrose density gradient (5-20 ~o in o.oi M Tris buffer (pH 7.4)) containing an appropriate amount of magnesium acetate, and centrifuged at 25 ooo rev./min using a swinging bucket rotor, Spinco type SW25, in a model L ultracentrifuge. Fractions were collected, after puncturing the bottom of the tube, and absorbancy at 260 m# was measured. Precipitate formed in each fraction by 0.5 N HC104 was washed with this acid on a membrane filter, dried and counted in a windowless gas-flow counter (Aloka, Tokyo). RESULTS

Pulse-labelling o/RNA~in ultraviolet-irradiated bacteria Under the conditions of ultraviolet irradiation employed, exposure of bacteria to ultraviolet light for 1.5 rain caused, on incubation in low-phosphate growth medium at 37 °, a temporary cessation of DNA synthesis lasting about 30 rain. During this period, the rate of RNA and protein increase was reduced only slightly in ultraviolet-irradiated bacteria. In order to test whether mRNA synthesis was inhibited by this dose of ultraviolet irradiation, control and irradiated bacteria were exposed to L32P]PI for 45 sec or 2 rain starting at the Ioth, 2oth, and 3oth rain of incubation which followed the irradiation, and nucleic acids were chromatographed on methylated bovine serum albumin columns. In accordance with the observation of 1SHIHAMA et al. 12, rapidly labelled RNA of the control culture was eluted at four regions, and considered to represent mRNA. It includes m R N A I eluting at tim DNA peak, and m R N A I I - I V eluting before, between and behind the I6-S and 23-S rRNA peaks, respectively. In the present work, labelling of m R N A I I - I V was followed, that of m R N A I being quantitatively insignificant. In repeated experiments, it was found that the labelling of mRNA peaks was extremely low in irradiated cultures. Results with control cultures were variable, however, labelling of m R N A sometimes being extensive but on other occasions only little higher than that of the irradiated cultures. The experimental conditions had possibly caused some delay in the uptake of external 32p by bacterial cells and/or by Biochim. Biophys. Acta, 76 (i903) i 8 8 - 2 o o

RNA SYNTHESIS IN U V - I R R A D I A T E D BACTERIA

191

RNA precursors, especially with irradiated bacteria. We therefore followed the uptake of [2-1aC]uracil by the cold trichloroacetic acid-insoluble fraction of control and irradiated bacteria immediately after irradiation. As shown in Fig. I, the incorporation of [laCluracil into what was presumably R N A was somewhat suppressed in the irradiated cells, but they showed a significant uptake of [14Cluracil even during the initial 2 rain. It was concluded that the technique of short pulse-labelling with 32p might not be adequate for determination of the rate of m R N A synthesis.

1600

1200

E P

g

1/

iiI

O

80(3

O

/ii//// 400

n,"

0

0

2

4 6 Time (min)

8

10

Fig. I. E f f e c t s oI u m a v i o l e t i r r a d i a t i o n on i n c o r p o r a t i o n of ~x*C~uracil into t h e acid-insoluble fraction of E. coli cells. Cells irradiated for 1. 5 m i n ( © - © ) (survival ratio o.2 %), a l o n g with control cells ( O - O ) , were d i l u t e d w i t h 9 vol. of p r e w a r m e d f l e s h g r o w t h m e d i u m c o n t a i n i n g [14C]uracil. A t i n t e r v a l s p o r t i o n s of t h e s u s p e n s i o n were r e m o v e d for p r e c i p i t a t i o n in IO % trichloroacetic acid a n d filtration on m e m b r a n e filters.

We therefore followed the outcome of labelling with 32p during longer periods, when significant labelling of t R N A and rRNA would tend to obscure the m R N A peaks. In order to correct for the possible difference in the rate of 32p incorporation into the cells and/or RNA precursors, we tried to match the absorbancy and radioactivity curves for tRNA. As shown below, synthesis of tRNA was little affected by 1.5 min exposure to ultraviolet irradiation, so that the specific activity of tRNA would serve as a direct measure of the availability of a2p for m R N A synthesis in control and irradiated cells. To facilitate curve matching, tRNA was eluted with o.4 M NaC1 forming a sharp peak. In this way we ascertained that radioactivity of the m R N A - r R N A region of the elution diagram in irradiated culture was about 7 ° % of that in the control culture with pulse-labelling for 2 min starting from Ioth min of post-irradiation incubation. When the labelling time was extended to 3 or 4 min, RNA of the irradiated as well as the control culture was extensively labelled, and the presence of the m R N A - I V peak indicated the synthesis of m R N A in irradiated culture (Fig. 2). However, it was not possible to estimate the level of inhibition of m R N A synthesis from the present data. Nor could we obtain any indication that the synthesis of m R N A was severely inhibited by ultraviolet irradiation causing a temporary but complete cessation of DNA synthesis. Biochim. Biophys. Acta, 76 (I963) I 8 8 - 2 o o

192

A. SIBATANI, N. MIZUNO

I4]tRNA 1.2

DNA r'RNA(o--o) 16S

(a)

23S

i

6

iI 41 IL iJ

'0.8

5

4 ~,

6

g 0.6 e,

i iq

>~

,J!

0.4

0.2

21 1

20 40 60 Fr'Qction number"

80

(b) 1.4

I

15

mRNA (o-- -o)

1.2 !4 1.0 h

3~

f~

g

t~

5 q2 .~

0.4 0.2

0

0

--

20 40 60 Fraction numbeP

~

150

0

Fig. 2. F r a c t i o n a t i o n of 3~P-labelled nucleic acids f r o m c o n t r o l a n d i r r a d i a t e d E. coli cells b y m e t h y l a t e d b o v i n e s e r u m a l b u m i n c o l u m n c h r o m a t o g r a p h y , i o rain a f t e r t h e o u t s e t of p o s t i r r a d i a t i o n c u l t u r e , cells were l a b e l l e d w i t h 3~P(I mC/35 ml) for 4 rain. N uc l e i c a c i ds w e re e x t r a c t e d f r om IO m l of l a b e l l e d c u l t u r e m i x e d w i t h 4 ° m l of u n l a b e l l e d carrier-cell s u s p e n s i o n of t h e s a m e d e n s i t y . The scale of r a d i o a c t i v i t y was so chosen t h a t t h e c u r v e s for a b s o r b a n c y a n d r a d i o a c t i v i t y for t l Z N A were well m a t c h e d . (a), c o n t r o l ; (b), i r r a d i a t e d for 1. 5 nlin; s u r v i v a l r a t i o = o.I % .

Inhibition o/ RNA synthesis with higher doses o/irradiation We then studied the effects of different doses of ultraviolet irradiation on the syntheses of major :RNA components of bacterial cells. Bacteria irradiated for different lengths of time were grown for 30 rain in the presence of [3~P]PI. R N A cornBiochim. Biophys. Acta, 76 (1963) 188-2oo

RN&

193

SYNTHESIS IN U V - I R R A D I A T E D BACTERIA

ponents were separated by column chromatography on methylated bovine serum albumin. Results in Fig. 3 show that RNA synthesis was severely impaired by higher doses of irradiation, and the extent of the impairment differed for individual RNA components. (0)

1.0

~

(

12~

0.6

6

~

0.4

4

0.2

2

mRNA 5Z 819

~

,.6[

4 •

0 O.e

(d)

mRN~ IV

o.8[ (el

o.,FIt Fraction number

]

o.~ (f)

40 50 Fraction number mRNA

2

60

0

7

0,4

O.2

} - ~10 50 60 Fraction number mRNA

h

,/t 4J°'to2t tt

I.\/ ,{

L,o F r a c.ot i o n n.o'oo umber

,o°''

-,o

AtI1

).1

.o.°o

Fraction number

Fig. 3. F r a c t i o n a t i o n of nucleic acids f r o m E. coli cultures, i r r a d i a t e d for v a r i o u s periods, b y m e t h y l a t e d b o v i n e s e r u m a l b u m i n c o l u m n c h r o m a t o g r a p h y . Cells were i r r a d i a t e d for (a) o m i n (control); (b) 1. 5 rain; (c) 3 rain; (d) 6 rain; (e) 12 m i n ; a n d (f) 3 ° min, a n d i n c u b a t e d a f t e r 2-fold dilution for 3 ° rain w i t h I/zC of 32p p e r ml.

The synthesis of 23-S RNA was most affected. In fact, irradiation for 6 min almost completely blocked the labelling of this component, while it permitted a significant labelling of I6-S RN'A and tRNA (Fig. 3, d). When bacteria were irradiated for 12 or 30 min, labelling of I6-S RNA was also blocked, although there was still a slight but significant uptake of 82p by tRNA (Fig. 3, e and f). Synthesis of mRNA, indicated by the characteristic position of mRNA II-IV-peaks in the elution diagram, could hardly be detected in the control cells and cells irradiated for 1. 5 min (Fig. 3, a, b), since they were entirely masked by an extensive labelling of rRNA. However, when bacteria were irradiated for 3 min or longer (Fig. 3, c-f), synthesis of rRNA was progressively suppressed, and labelled mRNA peaks became apparent. Thus, in bacteria irradiated for 12 and 30 min, a small amount of radioactivity was present in tRNA and three major peaks of mRNA appeared. In all these cases, there was a well defined peak of mRiXrA IV, which tended to disappear under certain conditions such as treatment with chloramphenicol 1~. The impression was gained that irradiation caused no differential blocking of the formation of various mRNA components. This was confirmed by a separate experiment with bacteria irradiated for 12 min and grown for 30 rain in the presence of a higher 3~p content. Biochim. Biophys. Aaa. 76 (9163) 188-2oo

194

A. SIBATANI, N. MIZUNO TABLE I ~UCLEOTIDn COMPOSITION OF m R N A IN ULTI~AVIOLE'r-IRRADIATED E . coli (mole %)

Cells were i r r a d i a t e d for 12 m i n a n d g r o w n for 3 ° rain in t h e p r e s e n c e of i o / * C [a'PiP1 pe r ml. V a l u e s o t h e r t h a n line I were t a k e n f r o m ISHIHAMA et al. 12. Source

Fraction

I r r a d i a t e d cells N o r m a l cells N o r m a l cells N o r m a l cells

mlR.NA I V mRNA IV rRNA DNA

A

G

24.1 25.0 24.1 24-25

C

25. 5 25.1 31.9 25-26

U(T)

26. 7 24. 4 21.5 25-26

Pyrimidine: purine

23. 7 25.5 22.8 24-25

I.OI I.OO 0.79 I.OO

(A + U ( T ) ) : (G+C)

0.92 1.o2 0.88 o.92-1.oo

Again there was no radioactive peak corresponding to rRNA. The central part of the mRNA-IV peak was pooled and its nucleotide composition was estimated by 32p distribution (Table I). It was thus found that this peak had a typical DNA-like nucleotide composition, indicating a synthesis of mRNA and a complete blockage of the synthesis of 23-S rRNA in these cells during 30 rain following irradiation. The base analysis of other mRNA peaks was not undertaken because any residual synthesis of I6-S rRNA might have obscured the picture.

Dose-e[]ect curves/or the synthesis o/ RNA components The extent of inhibition of synthesis of individual RNA components caused by various doses of irradiation was next studied. As noted earlier, there may have been a delay in a,p incorporation into irradiated cells, but this may be neglected for a labelling period of 30 rain, especially since it is unlikely to affect the comparative synthesis of different RNA components. Since the yield of RNA extracted from ground cells may have varied, and since there was a net synthesis of RNA during the labelling period, neither total nor specific radioactivity of separated RNA components can be used for estimating the amount synthesized during the postirradiation period. W~ therefore calculated the fractional increment of RNA components as defined by Am/too, where m 0 and Am are the amounts respectively of a certain RNA component present before irradiation and of that formed during the post-irradiation incubation, per unit volume of bacterial culture. We ascertained in separate experiments that irradiation for 30 rain neither reduced the RNA content of bacteria nor induced subsequent breakdown of RNA (uniformly labelled with [14Cjuracil) which was present prior to irradiation. Also, [14C]uracil incorporated into total RNA during 30 rain following 30 rain irradiation did not show any significant difference in extractability by sodium dodecylsulphate-phenol from unlabelled RNA that existed in the cell prior to irradiation. Then, in each elution diagram of Fig. 3,

Am

z

mo

bA

(I)

-- z

where z is the radioactivity in counts/min per ml in the effluent fraction at the absorbancy peak of an RNA component, A is the absorbancy of this fraction at 260 m/~, and b is defined by b --

Pi* e Pt Biochim.

(2) Biophys.

Acta,

76 (1963) 188-2oo

RNA

SYNTHESIS IN U V - I R R A D I A T E D BACTERIA

195

in which e is the absorbancy coefficient (Ig RN'A-P/cm 3) of this RNA component in the same fraction (in 0.05 M phosphate buffer (pH 6.7), containing a certain concentration of NaC1), and Pi* and Pi are the total radioactivity (counts/rain) and amount of P (g) in the form of Pi, respectively, in a unit volume of culture medium. We determined e for tRNA, I6-S RNA and 23-S RNA at the respective peaks of a representative chromatogram of E. coli RNAs, and obtained values of 2.33" lO5 for tRNA and 1.99- lO 5 for both I6-S and 23-S RNA. Using these and other values obtained in the experiments illustrated in Fig. 3, we calculated, according to Eqn. I, fractional increments of tRNA and I6-S and 23-S rRNAs in control and in irradiated cultures. In Fig. 4 they are plotted against the dose of ultraviolet irradiation. Here, the increments for rRNAs are obviously

< z n,"

!

-

\

"-o.

-re

0

E

b

.c_

~-2 i

.9

"'"'10... o

-2

P J

o

,

I

,

I

10 20 Ul~aviolet dose (rain)

t

I

30

Fig. 4. Dose-effect c u r v e for fractional i n c r e m e n t of t R N A , I6-S a n d 23-S r R N A in ultraviolet. i r r a d i a t e d E. coll. O - O , t R N A ; 0 - 0 , I6-S lZNA; I ) - ~ , 23-S R N A ; O - ' - O , viability.

too high at higher doses, because small amounts of radioactivity present in the respective fractions were largely due to mRNA, for which no objective correction was available. For lower doses of irradiation, radioactivity of mRNA could be neglected because of the extensive net synthesis of rRNA. Therefore, the dose-effect curves for rRNAs should be steeper at higher doses than those illustrated in Fig. 4. Inspection of Fig. 4 shows several things. First, different RNA components were reproduced in the control culture to the same extent and had nearly doubled their amounts. Second, each curve had a shoulder, indicating that their hit numbers were higher than unity. Third, the sensitivity to irradiation of the synthesis of these RNA components increased in the order of increasing molecular weight. Apparently, tRNA had some components whose synthesis was more resistant to irradiation than that of Other tRNA components, but whether this was due to the heterogeneity at the cellular or molecular level is not clear. Such consideration was not feasible for rR1VA for the reason mentioned above. The bacterial survival curve, drawn at the position convenient for comparison, declined more steeply than that of 23-S rRNA, but the latter should have been steeper, so that again the comparison was not feasible. It would be of interest to obtain similar curves for mRNA components. Comparison of the chromatograms in_Fig. 3, d-f, suggests that mRNA synthesis was also Biochim. Biophys. Acta, 76 (1963) 188-2oo

196

A. SIBATANI, N. MIZUNO

progressively impaired by increasing irradiation doses, but it was not possible to assess the extent of inhibition of their synthesis. This is because, as noted earlier, the short-pulse experiment failed to give an adequate estimate of the magnitude of mRNA synthesis in irradiated bacteria, and also because of the variation in the rate of renewal and the extent of recycling of material for these unstable RNA components. However, irradiation with higher doses gave labelling only in tRNA and mRNA, so that the synthesis of mRNA must have been more resistant to irradiation than that of rRNA. But whether it was more resistant than tRNA synthesis could not be determined.

Sedimentation analyses o/ cell extracts Since 23-S RNA is derived from 5o-S ribosome subunit alone 14, it is expected that the irradiation dose that preferentially blocks the synthesis of 23-S RNA will preferentially suppress the formation of 5o-S subunit while permitting the synthesis of 3o-S ribosome subunit. To confirm this, we conducted sedimentation analyses of ribosomes and their subunits labelled with E14Cluracil for 30 min following irradiation for 6 rain. In these analyses we employed crude bacterial extracts obtained with two levels of Mg 2+ concentration. Under these experimental conditions, mRNA would be degraded into small fragments of 8-14 S (ref. 15) but still might be attached to the ribosome at higher Mg ~+ concentrations. We therefore incubated the crude extract at 37 ° for 15 rain prior to sedimentation analysis to eliminate, from ribosomes, mRNA (see ref. 16), which would have incorporated radioactivity and complicated the picture. Fig. 5 shows that, in irradiated cells, 3o-S but not 5o-S subunits incorporated a significant amount of the label. In addition, there was an indication that a certain radioactive component was present between 3o-S particle and tRNA of irradiated cells. This might be either degraded mRNA or an anomalous precursor of 5o-S subunit containing a RNA component chromatographically indistinguishable from

1.• (o)

50S

1.2

I 1°I b' 6000 "~

~L 3 0 0 0

1.21 2000 E

0.~

tooo°o,

o

o

lo00 0

°6

lb

Fraction number

Fraction number

~o

Fig. 5. Sedimentation analyses of cell e x t r a c t s at o.25 mM Mg *+ from control and irradiated cultures. Cell suspensions irradiated for o and 6 min were incubated at 37 ° for 3 ° rnin with [14C]uracil, harvested and washed w i t h buffer containing o.I mM Mg a+. Period of centrifuging was 4 h.

Biochim. Biophys. Acta, 76 (9163) 188-2oo

RNA

197

SYNTHESIS IN U V - I R R A D I A T E D BACTERIA

I6-S rRNA. Fig. 6 shows that the preincubated ribosomes were partially dissociated into subunits, even at 5 mM Mg~+. Here, 7o-S ribosome of the irradiated cells contained some of the label, and 3o-S subunit was the main labelled subunit. This indicates that some of the newly synthesized 3o-S subunits had been exchanged with pre-existing ones in 7o-S ribosomes, rather than that they had been associated with newly produced 5o-S subunits, to form the labelled 7o-S ribosomes, since few 5o-S subunits had been formed under these conditions. 1.0 (a)

1.C 70S 50S 30S

-(b) 2000

4000

0.~

~

0.8 15oo I "2

"~ 0.6 o

20OO>

0.4

I000~

0.4

0

~d

looo 0.2 ._8

0,2

~~"

500 ~ o n-

10

2'0

0

Fraction number

10 Fraction number

20

O

Fig. 6. S e d i m e n t a t i o n a n a l y s e s of cell e x t r a c t s a t 5 InM Mg z+ f r o m c o n t r o l a n d irradiated cultures. Period of c e n t r i f u g i n g was 3.5 h. O t h e r c o n d i t i o n s were t h e s a m e as for Fig. 5-

DISCUSSION

The semi-conservative model of DNA replicationlL 18 that is most widely accepted, requires a complete strand separation of parent duplex molecule. It is known that ultraviolet irradiation causes inter- or intramolecular cross-linking of DNA19, 2°, probably due to the formation of thymine dimers 21, and also cross-linking between cellular DNA and proteins 2z. Such cross-links occurring in DNA would prevent a regular strand separation and might lead to cessation of DNA synthesis. Different RNA components of the cell appear to be synthesized on chromosomal DNA 5-9. Since individual RNA molecules would require only short segments of DNA strand as templates, a few cross-links occurring in DNA would not seriously interfere with RNA synthesis, whether or not partial strand separation of DNA is necessary for RNA synthesis (see refs. 23 and 24). If such a concept is correct, the higher sensitivity to ultraviolet irradiation of DNA synthesis than RNA synthesis can be explained. The base sequence 25, and to a lesser extent the base composition ~6also, appear to be significantly different between RNAs of 3o-S and 5o-S ribosome subunits of E. coli. This would indicate that 23-S r]ZNA as the component of 5o-S particles, and I6-S rRNA derived from 3o-S particles ~4, are formed by separate cistrons. The different irradiation sensitivity of the labelling of I6-S and 23-S rRNA or 3o-S and 5o-S ribosome subunits, as observed in the present paper, is consistent with such an idea. However, our results alone can certainly not exclude the alternative possibility Biochim. Biophys. Acta, 76 (1963) 188-2oo

198

A. SIBATANI, N. MIZUNO

that ultraviolet irradiation interferes with the dimerization of I6-S rRNA subunits into 23-S rRNA and their incorporation into 5o-S ribosome subunits, although they are entities distinct from I6-S rRNA to be incorporated into 3o-S ribomes. They might correspond to the additional radioactive component noticed between 3o-S particles and tRNA in the irradiated cells (Fig. 5, right). The formation of 5o-S ribosome subunit or 23-S rRNA is also preferentially suppressed by fluoro-uracil or chloramphenico127-2% 33. It appears that the synthesis of 23-S rRNA is generally more vulnerable than that of I6-S rRNA or tRNA. We have further demonstrated that the sensitivity to ultraviolet irradiation of the synthesis of tRNA and rRNAs decreased in the order of decrease in their molecular weight. This may be readily explained by the different target sizes of their DNA templates, which should be proportional to the molecular weight of the RNA that they code. If the same hit number be necessary to block the synthesis of different RNAs, then the slope of the dose-effect curve for RNA synthesis would be a simple function of the molecular weight of RNA. As can be seen from Fig. 7, such a model o tRNA

-1

._m

Q; >

~-~ m

S rRNA

.-1

23-S~

rRNA/

\

Radiation dose in arbitrary

unit

Fig. 7. H y p o t h e t i c a l dose-effect c u r v e s for t h e s y n t h e s i s of t R N A a n d I6-S a n d 23-S r R N A on t h e a s s u m p t i o n t h a t t h e ability to s y n t h e s i z e each R N A c o m p o n e n t is i n a c t i v a t e d b y a single h i t on its c o r r e s p o n d i n g cistron, t h e size of w h i c h is p r o p o r t i o n a l to t h e m o l e c u l a r w e i g h t of R N A (tI~NA 25ooo , I6-S r R N A 560 ooo, 23-S r R N A 11oo ooo).

would yield very different slopes for tRNA and I6-S RNA, while the difference between I6-S and 23-S RN'As would be much smaller. In actual observation (Fig. 4), the increment of rRNA was overestimated at higher doses. Further, had ultraviolet irradiation interfered with dimerization of I6-S RNA subunits into 23-S RNA, the slope for I6-S RNA would have been still steeper. However, the rather small difference in the slope between tRNA and 23-S rRNA at'lower doses may be evidence against the simple model as represented by Fig. 7. The behaviour of mRNA in irradiated cells poses another problem. Although Biochim,--Biophys. Acta, 76 (1963) 188-2oo

Rl~A SYNTHESIS IN UV-IRRADIATED BACTERIA

199

we could not exactly assess the sensitivity of its synthesis to ultraviolet irradiation, evidence was obtained that it was less sensitive than rRNA synthesis. However, within chromatographically separated components of mRNA, we observed no preferential inhibition of the synthesis of any particular component. This, together with the fact that the sedimentation coefficients of these RNA components ranged between 12 and 30 S (ref. I2), would indicate that the mRNA synthesis falls outside the regularity noticed between the molecular size and sensitivity of the synthesis to irradiation. If this be so, mRNA and other cellular RNA would be synthesized by somewhat different mechanisms. However, the possibility cannot be excluded that the mRN_A_ peaks in the methylated bovine serum albumin chromatogram are association products among mRNAs or with rRNA 29, although separate peaks, especially mRNA IV, are not a simple chromatographic artifact'5, 30 but represent compounds pre-existent in the cell in varying amounts under different biological conditions12, ~1. If cistrons for average mRNA are actually smaller than that for I6-S rRNA, our observations on the irradiation sensitivity of mRNA synthesis may be reconciled with the regularity noted above for tRN'A and rRNA. In any event, the present observation that mRNA synthesis is relatively resistant to irradiation is in line with the recent findings z2 that the potency of fl-galactosidase synthesis is retained by irradiated E. coll. After completion of this manuscript, we have learned that R{}RSCH and coworkers33, ~ made similar observations on the differential sensitivity to irradiation of the formation of ribosome subunits and RNA components in E. coll. Their conclusion, as well as that of WAINFAN et al. n, is in accord with ours reported h e r e ACKNOWLEDGEMENTS

We express our gratitude to Dr. M. Fox, Massachusetts Institute of Technology, Dr. G. S. STENT, University of California, and Dr. M. TAKANAMI and Mr. T. 0KA-MOTO of this Institute for helpful discussion during the early part of this investi-. gation. Thanks are further clue to Dr. A. RbRSCH, Medisch Biologisch Laboratorium R V 0 - T N 0 , Rijswijk (Netherlands), for informing us of his work before publication. This work was supported in part by a Grant from the Scientific Research Funds of the Ministry of Education. REFERENCES 1 z 3 4 5 7 s * as 11 lz l~ 14 15

F. M. HAROLD AND Z. Z. ZIPORIN, Biochim. Biophys. Acta, 29 (i958) 439. p. HANAWALT AND R. SETLO~,V, Biochim. Biophys. Acta, 41 (196o) 283. S. BRENNER, F. JACOB AND M. MESELSON, Nature, 19o (1961) 576. F. GROS, H. HIATT, W. GILBERT, C. KURLAND, H. RISEBROUGH AND J. D. WATSON, Nature, 19o / I 9 6 I ) 581. j . j . FURTH, J. HURWITZ AND M. GOLDMAN, Biochem. Biophys. Res. Commun., 4 (1961) 362. S. ]3. W E i s s AND Y. NAKAMOTO, Proc. Natl. Acad. Sci. U.S., 47 (1961) 14oo. S. A. YANKOFSKY AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 48 (1962) lO67. H. M. GOODMAN AND A. RICH, Proc. Natl. Acad. Sci. U.S., 48 (i962) 21Ol. D. GIACOMONI AND S. SPIEGELMAN, Science, 138 (1962) 1328. j . HURWlTZ, J. j . FURTH, M. MALAMY AND M. ALEXANDER, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1222. E. WAINFAN, L. R. MANDEL AND E. ]3OREK, Biochem. Biophys. Res. Commun., IO (1963) 315 . A. ISHIHAMA, N. MIZUNO, M. TAKAI, E. OTAKA AND S. OSAWA, J. Mol. Biol., 5 (1962) 251. I . D. MANDELL AND A. D. HERSHEY, Anal. Biochem., I (196o) 66. C. G. KURLAND, J. Mol. Biol., 2 (196o) 83. R. MONIER, S. NAONO, D. HAYES~F. HAYES AND F. GROS, J. Mol. Biol., 5 (1962) 311.

Biochim. Biophys. Acta, 76 (1963) 188-2oo

20o

A. S I B A T A N I ,

N. MIZUNO

Proc. Natl. Acad. Sci. U.S., 47 (1961) 1588. j . D. WATSON AND F. H. C. CRICK, Cold Spring Harbor Syrup. Quant. Biol., 18 (1953) I23. M. MESELSON AND F. W. STAHL, Proc. Natl. Acad. Sci. U.S., 44 (1958) 671. A. WACKER, H. DELLWEG AND D. WEINBLUM, Naturwiss., 41 (196o) 477. j . MARMUR ANDL. GROSSMAN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 778. l~. BEUKERS AND XV. BERENDS, Biochim. Biophys. Acta, 41 (196o) 550. K. C. SMITH, Biochem. Biophys. Res. Commun., 8 (1962) 157. W. ]~. WOOD AND 1:) BERG, Proc. Natl. Acad. Sci. U.S., 48 (1962) 94. j. HURWIT2, J. J. FURTH, M. ANDERS AND A. EVANS, J. Biol. Chem., 237 (1962) 3752. 2~ A. I. ARONSON, J. Mol. Biol., 5 (1962) 453. 26 j. E. M. MIDGLEY, Biochim. Biophys. Acta, 61 (1962) 513 . 27 F. GROS, VV'. GILBERT, n . H. HIATT, G. ATTARDI, P. F. SPAHR AND J. D. \VATSON, Cold Spring Harbor Syrnp. Q**ant. Biol., 26 (1961) i i i . 2s F. GROS, S. NAONO, O. HAYES, F. HAYES AND J. D. WATSON, Colloq. Intern. Centre Nat. Rech. Sci. Paris, lO6 (1961) 437. 29 j. E. M. MIDGLEY AND B. J. McCARTHY, Biochim. Biophys. Acta, 61 (1962) 696. 30 t3. P. SAGICK, M. H. GREEN, M. HAYASHI AND S. SP1EGELMAN, Biophys. J., 2 (1962) 402. 31 T. KANO-SUEOKA AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1942. 32 S. \V. BOWNE AND P. ROGERS, J. Mol. Biol., 5 (1962) 90. 33 A. IZbRSC~, A. EDELMAN AND J. A. COHEN, Biochim. Biophys. Acta, 68 (1963) 271. 3~ A. RORSCH, naanuscript in preparation.

le M. W . ~]'IRENBERG AND J. H. MATTHAEI,

17 18 19 2o 21 22 23 24

Biochim. Biophys. Acta, 76 (1963) I88-2oo