The nucleotide base composition of ribonucleic acid from several microbial species

BIOCHIMICA ET BIOPHYSICA ACTA

513

BBA 8157

THE

NUCLEOTIDE FROM

BASE COMPOSITION SEVERAL

OF RIBONUCLEIC

MICROBIAL

ACID

SPECIES

J. E. M. MIDGLEY"

Carnegie Institution o/ Washington, Department o/ Terrestrial 3/Iagnetism, Washington, D. C. (U.S.A.) (Received March 23rd, 1962)

SUMMARY

The nucleotide base compositions ot alkali digests of various microbial RNA components have been measured by an isotope dilution technique and column chromatography. All bacterial samples for analysis were taken during the exponential growth of cultures in a simple chemically defined medium. The total triehloroacetic acidprecipitable RNA, the 3o-S, 5o-S and 7o-S ribonucleoprotein particles, and the soluble RNA have been measured in each of five bacterial species. The corresponding fractions in yeast have also been analyzed. In addition, the base composition of the rapidly labeled I4-S RNA fraction has been measured after short periods of 3~p incorporation into bacteria. Only in this fraction has any consistent correlation between RNA composition and the DNA composition of the bacterial species been found.

INTRODUCTION

The RNA of bacteria is remarkably invariable in nucleotide base composition, whilst the DNA nucleotide composition may vary widely from species to species. BELOZERSKY A N D SPIRIN1, on the basis of determinations of the total unfractionated cell RNA in many species, indicated that there might be a slight correlation between the composition of the unfractionated RNA and the DNA. MIURA2 has reported the finding of a similar slight correlation in the s-RNA within a group of six bacteria. Other workers have reported no~, 3 or a very marginaP correlation between the base composition of the ribosomal RNA in bacteria and the DNA. No compositional differences have been observed3, 5 in the compositions of the I6-S and the 23-S RNA particles resulting from the phenol treatment ot E. coli 7o-S ribosomes. There may exist a small fraction of the bacterial RNA which possesses a base composition like that of the DNA, uracil substituting for thymine 6. Reinforcing this possibility, the composition of newly formed RNA in bacteria and in yeast, as measured by the short exposure of cultures to [z~P]orthophosphate, bears some resemblance to the DNA of the organismsL8. However, in no case as yet has the composition of such fractions been reported to be identical with that of the DNA in steadily growing * Carnegie Institution Fellow, 196o-1962. Present address: Department of Biochemistry, University of Leeds, England.

Biochim. Biophys. Acta, 61 (1962) 513-525

514

J.E.M.

MIDGLEY

cells. HAYASHI AND SPIEGELMAN9 have reported the formation of an RNA with the base composition of the cell DNA in bacteria immediately after transfer of cells from broth to glucose media. The presence of an RNA fraction corresponding to infecting bacteriophage DNA has been reported during the infection of E. coli by T-2 bacteriophage 1°,11. A similar RNA fraction corresponding to T- 4 bacteriophage has been purified from infected cells of E. coli (see ref. 12). The bulk RNA of bacteria has at most a very small correlation with the DNA of the species. In E. coIi, SPAHR AND TlSSI~RES5 have reported small differences in the nucleotide base composition of the 3o-S and the 5o-S ribonucleoprotein particles. BOLTONls has also found compositional differences in these fractions and has shown differences in the oligonucleotide pattern resulting from digestion of the 3o-S and 5o-S particles with pancreatic ribonuclease. Differences in elution by NaC1 from DEAE-cellulose columns have been observed for phenol-extracted RNA from E. coli 3o-S and 5o-S particles 14. In the present work, five bacterial species and one of yeast have been examined to see if any consistent correlation between the RIqA and the DNA base composition exists in one or more of the RNA fractions which could be isolated. The technique of isotope dilution was used to determine the composition of these RI~A fractions with the greatest possible accuracy, so that even fairly small differences in base composition could be detected amongst the fractions of a given species. METHODS

The bacterial species Pseudomonas aeruginosa A.T.C.C. 9027, Aerobacter aerogenes A.T.C.C. 211, Escherichia coli ML 30, Bacillus subtilis A.T.C.C. 6051, and Proteus vulgaris A.T.C.C. 4669 were used. The yeast used was Saccharomyces cerevisiae A.T.C.C. 2338. The DNA (guanylic acid+cytidylic acid)/(adenylic acid+thymidylic acid) ratios of these organisms lie in a range from 1.75 (Pseudomonas) to 0.6 (Proteus and Saccharomyces) 1. All bacterial cultures were grown in aerated media at 37 ° containing o.oi M Tris adjusted to pH 7.2 with HC1, o.oi M Na2S04, o.oi M MgCI~, NH4C1, 5 g/l, and sodium and potassium phosphates to give a concentration of 0.0002 M with respect to PO4s-. Yeast was grown in a medium containing 1 % (w/v) bactopeptone, o.I % (w/v) yeast extract, o.oi M MgC12, o.oi M Na2SO4, 5 % (w/v) glucose, and I g/1 each of NaC1 and KC1. The cultures were aerated and grown at 3 o°. For the production of RNA for base-composition analyses, the bacteria and yeast were grown in the presence of EsZPlorthophosphate for several hours in the logarithmic phase. They were then harvested at cell densities of about I g/1 and were washed three times in o.oi M Tris buffer (pH 7.3) containing o.oi M MgC12. The pellet was resuspended in the Tris-MgC12 buffer and the cells were broken in the French pressure cell at 15 ooo lb/in *. The cell extract was then centrifuged at lO5 ooo × g for 2 rain to remove cell walls and unbroken cells. The supernatant was further centrifuged at lO5 ooo × g for 45 min to pellet the 7o-S ribosomes. The pellet was then washed with o.oi M Tris-o.oI M MgC1, buffer (pH 7.3), resuspended and repelleted by a further centrifugation for 45 min. In this way a purified sample of 7o-S ribosomes was prepared. E. coli ML 30 unlabeled 7o-S ribosomes were also prepared from one batch of cells by the same method. 3o-S and 5o-S ribosomes derived from the 7o-S particles were purified by the use of the sucrose density-gradient sedimentation method 15. A small quantity of Biochim. Biophys. Acta, 61 (1962) 513-525

RNA

COMPOSITION

515

labeled 7o-S ribosomes (less than o.I rag) was suspended in o.oooi M MgC12-Tris buffer, and was centrifuged at 37 ooo rev./min in the swinging bucket rotor for 16o rain, through a 5-2o % (w/v) sucrose density gradient containing o.oooi M MgeleTris buffer. At this RNA concentration, the resolution of the 3o-S and 5o-S ribosomes was sufficient to allow samples to be taken without cross-contamination (Fig. I). Pro/eus

/ [

Vu/garL~ 32p labeled extrocl 50S

1

50S

g 8 0._ ,~ 5 X I0 5

2

4

6

8

fO

12

14

~6

18

Fraction number Fig. I. Sucrose d e n s i t y - g r a d i e n t s e d i m e n t a t i o n p a t t e r n of a2P-labeled 3o-S a n d 5o-S r i b o s o m e s of P. vulgaris. Sucrose c o n c e n t r a t i o n 5 - 2 o % in o.oooi M MgC12-Tris buffer. C e n t r i f u g a t i o n a t 37 ooo r e v . / m i n for 16o m i n a t 4 °.

s-RNA was purified by further centrifugation of the bacterial extract from which the 7o-S particles had been removed (24° min at lO5 ooo ×g). The supernatant was carefully pipetted off and was then treated with phenol and 2 % sodium dodecyl sulphate 16 after the manner of KIRBY1L After precipitation by 3 vol. of cold 95 % ethanol, the s-RNA was dissolved in o.oi M Tris-o.oI M MgC12 buffer (pH 7.3) and was adsorbed on DEAE-cellulose. It was then eluted in a linear NaC1 gradient (o.2-i.o M) TM. s-RNA eluted at o.5 M NaC1, and any degraded ribosomal RNA not pelleted by centrifugation eluted at o.8-1.o M NaC1 (Fig. 2). Unfractionated cell RNA was obtained by precipitating labeled cells in cold 5 % (w/v) trichloroacetic acid solution and filtering off the material on Millipore filters TM.

~-

S - RNA ~'

105

~" - I0

~J~

I

I,

8XlO4

0.8 "~

l

~.~ 6X104 m C 4XlO4

8L)

2XlO4

'

',

//i

,"/

./// 5

o6 g

IO

RNA

15

o.4 8

", \ . . j ,-.~. .... . 02 9 = 20

25

30

35

40

45

Fraction number

Fig. 2. E l u t i o n b y NaC1 f r o m a D E A E - c e l l u l o s e c o l u m n of p h e n o l - t r e a t e d s u p e r n a t a n t of s2p. labeled B. subtilis cell e x t r a c t , o b t a i n e d a f t e r c e n t r i f u g a t i o n a t lO 5 ooo × g for 24o rain. L i n e a r g r a d i e n t of NaC1 (o.2-1.o M ) in o.oi M T r i s - o . o I M MgCI, b u f f e r (pH 7.3). 0 - 0 , 3~p c o u n t s / m i n ; 0 - O , NaC1 c o n c e n t r a t i o n , M .

Biochim. Biophys. Acta, 61 (1962) 5 1 3 - 5 2 5

516

J.E.M.

MIDGLEY

The rapidly labeled I4-S RNA component was isolated by several methods to be described later. All samples to be analyzed were precipitated by cold 5 % trichloroacetic acid and filtered before alkaline hydrolysis was carried out. Repeated washings of the filter with 5 % trichloroacetic acid effectively removed contaminating 5'-nucleotides arising from the pool of RNA precursors in the cells. Hydrolysis of the RNA samples was carried out by treatment of the filters with 0.38 M KOH for 15 h at 37 °. Excess unlabeled E. coli ML 30 7o-S RNA, prepared by phenol treatment of 7o-S ribosomes, was routinely added. The soluble brown material produced by the dissolution of the filter in the alkali did not interfere with the analyses. Excess alkali was neutralized by I.O M perchloric acid and the resulting precipitate was centrifuged. The 2'- and 3'-nucleotides were adsorbed on a 0.5 × 15 cm Dowex-I-formate column (200-400 mesh) s° and elution was effected by a nonlinear gradient .1 of formic acid (0-4 M) ss so that the 2'- and 3'-isolners of adenylic and guanylic acids were partially resolved. In this way, a check was kept on the hydrolytic procedure and on 5'-nucleotide contaminations, by a comparison of the specific activities of the 2'- and 3'-isomers. No compositions have been quoted in this paper in which any differences in 2'- and 3'-nucleotide specific activities occur. The technique of isotope dilution allowed the minimization of some of the more likely errors in base-composition determination when simple summation of nucleotide absorbancies or asp counts are used for base-analysis measurements. The sample of unlabeled RNA was used to supply effectively all the ultraviolet absorbancy of the eluted 2'- and 3'-nucleotides, and many determinations of the ratio of radioactivity of the sample to ultraviolet absorption were made for each nucleotide in each analysis, contaminating material being readily detected. Further, inaccuracies in the determinations due to incomplete digestion of the RNA, to selection of some of the nucleotides during the mechanics of transfer and to possible nucleotide interconversions or dephosphorylations during hydrolysis or preparation of the RlffA fractions are greatly minimized. However, for accurate absolute determinations of the nucleotide base compositions of the labeled samples, the base composition of the E. coli 7o-S RNA used as unlabeled carrier must be accurately determined. The accuracy of this determination does not affect the relative compositions of any two or more labeled samples. RESULTS

Analysis o] the standard E. coli R N A

The composition of the single batch of E. coli ML 30 7o-S RNA used as unlabeled carrier in all subsequent determinations was measured by alkaline hydrolysis of a sample, colunm chromatography and summation of the ultraviolet absorbancies obtained from the elution of each nucleotide being used. In the digests, approx. 98 % of the material hydrolyzed was recovered from the column. These measurements were checked against the result obtained by the summation of the 3sp counts/min contained in each nucleotide after hydrolysis and column chromatography of a labeled sample of E. coli 7o-S RNA, prepared in the same way. Finally, to check the validity of the absorbancy coefficients used in the calculations of base composition throughout, labeled E. coli RNA was hydrolyzed by alkali in the presence of an excess of unlabeled material. All fractions collected were acidified to pH 2.0 with o.I M HCI Biochim. Biophys. Acta, 61 (1962) 513-525

RNA COMPOSITION

517

before determining the ultraviolet absorbancy. Readings of absorbancy in the range 256-280 m# were made on a Zeiss spectrophotometer, and specific activities were measured only in those fractions having absorbancies of between 0.8 and 3.0 at the wavelength of m a x i m u m absorbancy for each nucleotide at p H 2.0. Using the millimolar extinction coefficients at p H 2.0: cytidylic acid, 13.o at 280 m/~; adenylic acid, 15.1 at 257 m#; guanylic acid, 12.2 at 256 m#; uridylic acid, IO.O at 262 m/~, the specific activities of the nucleotides were found to be constant to within 1 % . The possibility of the fractionation of the standard RNA by the phenol procedure was also checked by comparison of the composition determined from phenol-extracted 7o-S RNA and from trichloroacetic acid-precipitated 32P-labeled 7o-S ribosomes of E. coli. No significant differences could be detected.

The composition o[ bulk R N A components in the cell Table I indicates the nucleotide base composition of the E. coli ML 30 7o-S RNA as determined by two methods. The results are the mean of several determinations b y each method. TABLE I DETERMINATIONS

OF THE

BASE COMPOSITION

O F E s c h e r i c h i a coli

7o-S

RNA

Several d e t e r m i n a t i o n s b y each of t h e t w o m e t h o d s were carried out. T h e m e a n nucleotide base c o m p o s i t i o n u s e d in e x p e r i m e n t s was: cytidylic acid, 21.9 mole %, adenylic acid, 25.1 mole o/ /o, g u a n y l i c acid, 32.6 mole %, uridylic acid, 20. 4 mole %. All nucleotide b a s e - c o m p o s i t i o n a n a l y s e s are a c c u r a t e to ~ 1.5 %. • Nucleotide

Cytidylie Adenylic Guanylic Uridylic

acid acid acid acid

Summation o! a2p counts in nucleotides

Summation o] ultraviolet absorbancies o/ nucleotides

(mole %)

at p H 2 (mole °~o)

21. 7 25.2 32.8 20. 3

22.o 25.1 32.4 20. 5

The base compositions of the unfractionated cell RNA precipitable by cold 5 % trichloroacetic acid, the 7o-S, 5o-S and 3o-S ribosomes, and the s-RNA in the five bacterial species are given in Tables I I - V I . In comparison, the base composition of the 8o-S, 6o-S and 4o-S ribosomes of yeast, and the s-RNA is given in Table VII. The slight differences observed in the 3o-S and the 5o-S ribosomes nucleotide base composition in a given species are reproducible to better than I °/o. As, in several of the determinations, the compositions of the RNA in the 5o-S and 3o-S particles differ in individual nucleotides b y as much as lO-15 ~o in a single species, these differences are probably real. Neither the unfractionated cell RNA, the 7o-S RNA, nor the s-RNA were found to possess a definite correlation with the DNA for any species. In fact, the compositions of these fractions in the five bacterial species are all invariable within the limits of the experimental error of determination. Yeast has a ribosomal RNA and total-cell RNA base composition basically unlike that of bacteria. The results for yeast ribosomal and s-RNA can be compaced with those of MONIER, STEPHENSON AND ZAMECNIK~3. If there exists in these fractions an RNA with a composition like that of the DNA, the accuracy of measurement b y the isotope dilution technique cannot permit it to be more than IO % of the RNA. B i o c h i m . B i o p h y s . A c t a , 61 (1962) 5 1 3 - 3 2 5

518

J.E.M.

MIDGLEY

TABLE II COMPOSITIONS OF R N A FRACTIONS OF P s e u d o m o n a s

a e r u g i n o s a ATCC 9027

D N A composition: adenylic acid = t h y m i d y l i c acid, 18 mole %; guanylic acid = cytidylic acid, 32 mole %. The underlined values in the 3o-S and 5o-S b a s e - c o m p o s i t i o n analyses are t h o s e which are different in the t w o s u b u n i t s f r o m the bacterial species. All nucleotide base-composition a n a l y s e s are a c c u r a t e to 4- 1.5 %. Nadeotide

Cytidylic acid Adenylic acid Guanylic acid Uridylic acid Purine Pyrimi dine Guanylic acid + cytidylic acid Adenylic acid + uridylic acid

Total RNA

7o-S

5o-S

3o-S

22.2

21. 7 25.7 31.6 21.o

21.2 26.3 31.2 21. 3

21.6 25.1 32.8 20. 5

25.7 31.3 20.8

s-RNA

28.3 2o.8 33.8 17.1

1.33

1.35

1.35

1.36

1.2o

1.15

1.14

I.IO

1.19

1.64

TABLE III COMPOSITION OF R N A FRACTIONS OF A e r o b a c t e r aerogenes ATCC 211 D N A composition: adenylic acid = thyrnidylic acid, 22 mole %; guanylic acid = cytidylic acid, 28 mole %. The underlined values in the 3o-S and 5o-S b a s e - c o m p o s i t i o n analyses are those which are different in the t w o s u b u n i t s f r o m the bacterial species. All nucleotide base-composition analyses are accurate to 4- 1. 5 %. Nueleotide

Total RNA

Cytidylic acid Adenylic acid Guanylic acid Uridylic acid Purine "Pyrimidine

22.6 25.o 31.7 20. 7

Guanylic acid + cytidylic acid Adenylic acid + uridylic acid

7o-S

5o-S

3o-S

s-RNA

21.9 25. 5 31.5 21.1

22.o 25.6 31.2 21.2

22. 4 25.3 3o.8 21. 5

29.2 19.7 32.3 18.8

1.32

1.33

1.32

1.27

I.IO

1.19

1.15

1.14

1.15

1.6o

TABLE IV COMPOSITIONS OF R N A FRACTIONS OF E s c h e r i c h i a coli NiL 3 ° D N A composition: adenylic acid = thymidylic acid, 24 mole %; guanylic acid = cytidylic acid, 26 mole %. The underlined values in the 3o-S and 5o-S b a s e - c o m p o s i t i o n analyses are t h o s e which are different in the t w o s u b u n i t s f r o m the bacterial species. All nucleotide base-composition analyses are accurate to 4- 1.5 %. Nucleotide

Cytidylic acid Adenylic acid Guanylic acid Uridylic acid Purine Pyrimidine Guanylic acid + cytidylic acid Adenylic acid + uridylic acid

Total RNA

22.1 25.2 32.5 20.2

7o-S

5o-S

3o-S

s-RNA

21.9 25.1 32.6 20. 4

21.5 25.4 33.5 19.6

22.7 24.8 31.° 21.5

29.5 19-7 33 .8 17.o

1.37

1.36

1.44

1.26

i.i 7

1.2o

1.2o

1.22

1.16

1.71

Biochim.

Biophys.

A c t a , 61 (1962) 513-525

R N A COMPOSITION

519

TABLE V COMPOSITION OF R N A FRACTIONS OF B a c i l l u s s u b t i l i s ATCC 6o51 D N A composition: adenylic acid = thymidylic acid, 29 mole %; guanylic acid = cytidylic acid, 21 mole %. The underlined values in the 3o-S and 5o-S b a s e - c o m p o s i t i o n analyses are those which are different in the two s u b u n i t s f r o m the bacterial species. All nucleotide base-composition analyses are accurate to ± 1.5 °o. Nudeotide

Cytidylic Adenylic Guanylic Uridylic

acid acid acid acid

Purine Pyrimidine Guanylic acid + cytidylic acid Adenylic acid + uridylic acid

Total RNA

7o-S

5o-S

3o-S

s-RNA

22.1 25. 5 31.4 21.o

22.3 25.9 31.o 20.8

22.5 26.5 32.0 19.3

22.3 26.5 29.6 21.6

28.3 2o.2 33-9 17.6

1.32

1.32

1.39

1.28

1.17

1.17

1.15

1.2o

1.o8

1.65

T A B L E VI COMPOSITION OF I I N A FRACTIONS OF P r o t e u s v u l g a r i s ATCC 4669 D N A composition: adenylic acid = t h y m i d y l i c acid, 3 I m o l e %; guanylic acid = cytidylic acid, 19 mole %. The underlined values in the 3o-S and 5o-S base-composition analyses are those which are different in the two s u b u n i t s f r o m the bacterial species. All nucleotide base-composition analyses are accurate to 4- 1.5 %. Nucleotide

Total RNA

7o-S

5o-S

3o-S

s- RNA

Cytidylic acid Adenylic acid Guanylic acid Uridylic acid I'urine l ~yrimidine

22.6 24.6 32.0 20.8

21. 7 26.2 31.4 20. 7

2t. 3 26. 5 31-4 20.8

23.o 24. 7 31,9 20. 4

29.3 19. I 33-3 18. 3

G~mnylic acid + cytidylic acid Adenylic acid + uridylic acid

i. 3 °

1.35

I. 37

i, 3 o

I. Li

1.21

1.13

I.II

1.22

1.67

TABLE VII COMPOSITIONS OF R N A FRACTIONS OF ~ a c c h a r o m y c g s cerevisiae DNA composition: adenylic acid = thymidylic acid, 3 2 mole %; guanylic acid ~ cytidylic acid, 18 mole %. The underlined values in the 4o-S and 6o-S base-composition analyses are those which are different in the t w o s u b u n i t s from the bacterial species. All nucleotide base-composition analyses are accurate to i 1.5 %. Nucleotide

Total RNA

8o-S

6o-S

4o-S

s-RNA

Cytidylic acid Adenylic acid Guanylic acid Uridylic acid Purine Pyrimidine

19.4 26.8 28. 3 25.5

19.2 27.2 28.2 25.4

I9.o 27.9 28. 4 24-7

19.1 25.2 28. 4 27.3

26.3 19.2 34.3 20.2

Guanylic acid + cytidylic acid Adenylic acid + uridylic acid

1.23

i .24

1.29

1.15

1.15

O.91

0.90

0.9 °

O.91

1.55

Biochim.

Biophys.

A c t a , 61 (1962) 513-525

520

J.

E. M. MIDGLEY

The composition o/ she z4-S RNA ]raction It has been established that the first detectable labeled polynucleotide material formed during the incorporation of [s,p]_ or [laC]uracil into bacterial RNA has different sedimentational and chromatographic properties from the RNA detectable by ultraviolet absorption 24. It has also been found that most of the [aaC]uracil which is incorporated into this fraction is eventually incorporated into the RNA of the ribosomes25, ~6. McCARTHY, BRITTEN AND ROBERTS~5,~6 have termed this fraction the "eosome". As this material accounts for effectively all the 3~P-labeled RNA present in short periods of isotope incorporation, its base composition should be similar to that of unfractionated cells at these times. The five species of bacteria used in the bulk RNA studies above were exposed to short periods of E32P]orthophosphate incorporation during exponential growth. The cells were then squirted into lO % cold trichloroacetic acid and filtered on Millipore filters. Many washes of trichloroacetic acid were given to remove most of the 5'nucleotides on the filter. From an aliquot of cells which had been poured onto crushed ice rather than into trichloroacetic acid, I4-S RNA was then isolated. The analyses of the pulse-labeled RNA in the five species are given in Table V l U . The extracts from the cells poured onto crushed ice were adsorbed on DEAEcellulose and eluted by a linear NaC1 gradient of o.2-1.o M NaC1 in o.oi M Triso.oi M MgCI~ buffer (pH 7.3)- Fig. 3 shows a typical elution pattern. It can be seen TABLE

VIU

COMPOSITIONS OF LABELED R N A FORMED DURING SHORT EXPOSURE OF BACTERIA TO [aSPJORTHOPHOSPHATE All n u c l e o t i d e b a s e - c o m p o s i t i o n a n a l y s e s are accurate t o ±

Species

Labeled RNA composition (trichloroacetic acid-precipitable) (mole %)

Time o/ labeling with isotope (rain)

Ps. aeruginosa ,4. aerogenes E. coli 13. subtilis P. vulgaris

Cytidylic acid

4 4 2 2 4

Adenylic acid

25.4 23. 4 22.9 23.3 22.2

Guanylic acid

21.1 24.8 25.0 25.6 26. 7

31.9 30.3 29.5 27.7 27.0

Ribosomes

104 - T2.O ~-

E

o'..

S

B~

~

~.~

Guanylic acid + cylidylie acid Adenylic acid+uridylic acid

21.6 21.5 22.6 23.4 24.1

1.34 1.16 i.io 1.o4 o.97

Newly formed RNA

'

'~ /

t

',

t° 'L

/

0.4 o° t

v

Uridylic acid

1. 5 % .

0

~

/

20

r ,

"~

40

0.2 Z

60

80

Fraction number

F i g . 3. E l u t i o n b y N&CI f r o m a D E A E - c e l l u l o s e c o l u m n of a cell e x t r a c t f r o m an E. coli culture labeled for 3 mill b y asp. L i n e a r NaC1 g r a d i e n t ( o . 2 - 1 . o M ) i n o . o i M Tris--o.oI M MgCli buffer (pH 7.3)----, NaCI c o n c e n t r a t i o n , M O - O , trichloroacetic acid-precipitable a~p counts/rain; O - O , u l t r a v i o l e t a b s o r p t i o n a t 260 m # .

Biochim. Biophys, Acta, 61 (1962) 5 1 3 - 5 2 5

RNA COMPOSITION

521

that only one labeled component, not tracking with a n y of the ultraviolet-absorbing material, elutes at 0.6 M NaC1. This material was pooled, trichloroacetic acid precipitated and collected by filtration. Analysis of the filters gave the compositions listed in Table IX. In each of the species examined, the base composition of the I4-S or "eosome" obtained in this way is identical within experimental error to that of the total-cell labeled RNA at this time. TABLE IX COMPOSITION OF THE I 4 - S (EOSOME) R N A COMPONENT OF BACTERIA P U R I F I E D BY D E A E - c E L L U L O S E CHROMATOGRAPHY

All n u c l e o t i d e b a s e - c o m p o s i t i o n a n a l y s e s are a c c u r a t e t o ~: 1. 5 % .

Species

Ps. aeruginosa E. coli B. sztblilis P. vztlgaris

Time o/labeling with isotope (rain)

4 2 2 4

z4-S RNA composition Cytidylic acid

Adenylie acid

25.6 22.7 22.5 21.9

20.8 25.I 25.3 27.0

Guanylic acid

31. 7 29.1 28.0 27.6

Uridylic acid

Guanylic acid+cytidylic acid Adenylic acid+uridylic acid

21.9 23.1 24.2 23.5

1.31 1.o 7 1.o2 0.98

A culture of B. subtilis was given a 3-rain labeling period with ~2p during exponential growth. The base composition of the total-cell labeled RlgA was measured, and a sample of the cell juice was treated with phenol, and after alcohol precipitation and dissolving the RNA in o.oi M Tris-o.oI M MgC12 buffer (pH 7.3), it was then adsorbed on a methylated serum albumin coated kieselguhr column ~7. The RNA was eluted by a linear gradient of NaC1 from o.4-1.1 M in 0.04 M phosphate buffer (pH 6.7). The elution pattern is shown in Fig. 4. The labeled RNA does not track exactly with the I6-S and 23-S RNA produced from the bulk of the RNA components of the cell. There are three radioactive peaks, but analysis of each showed that there was no difference in base compositions of any

I

600

S. RNAI

analysed Fractionspooledand

600 ~" 400 "~

o

Jo

20

30

40

50

Fraction number Fig. 4- E l u t i o n b y NaC1 from a m e t h y l a t e d s e r u m a l b u m i n c o a t e d k i e s e l g u h r c o l u m n of a cell e x t r a c t from an B. subtilis c u l t u r e l a b e l e d for 3 rain b y 3~p. The cell e x t r a c t w a s t r e a t e d w i t h p h e n o l to r e m o v e p r o t e i n f r o m t h e r i b o s o m e s before a d s o r p t i o n on t h e c o l u m n . L i n e a r g r a d i e n t of NaC1 (o.4-1. I M) in 0.04 M p o t a s s i u m p h o s p h a t e buffer, p H 6. 7. O - - - 0 , t r i c h l o r o a c e t i c a c i d - p r e c i p i t a b l e a2p c o u n t s / m i n ; O - O , u l t r a v i o l e t a b s o r p t i o n a t 260 m/~.

Biochim. Biophys. Acta, 61 (1962) 513 525

522

J. E, M. MIDGLEY

one peak from the composition of the material eluted at o.6 M NaC1 from DEAEcellulose or from the total-cell labeled RNA at this time. It is evident that under these conditions no further fractionation of the newly formed RNA labeled with sap has been achieved. The I4-S component of E. coli labeled for 3 rain by s2p was isolated by sucrose density-gradient centrifugation in the swinging bucket. After centrifugation at 37 ooo rev./min for 16o rain a peak sedimenting at about 14 S was clearly resolved by its radioactivity (Fig. 5). This peak was collected and trichloroacetic acid precipitated.

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Fig. 5- Sucrose d e n s i t y - g r a d i e n t s e d i m e n t a t i o n of a cell e x t r a c t f r o m E. coli labeled for 3 rain b y 32p. Sucrose c o n c e n t r a t i o n 5 - 2 0 % in o.oooi M MgCl=-Tris buffer. C e n t r i f u g a t i o n a t 37 ooo r e v . / m i n for 16o rain a t 4 °, O - O , u l t r a v i o l e t a b s o r p t i o n a t 26o m p ; 0 - 0 , trichloroacetic acid-precipitable 32p c o u n t s ] r a i n .

Its analysis showed that it was identical to the total-cell labeled RNA at this time, and to the material eluted from DEAE-cellulose at o.6 M NaC1. This would indicate that the eosome or I4-S RNA can be isolated as a discrete object without measurable change in base composition and that column chromatography either by the MANDELL AND HERSHEY column ~7 of phenol-treated RNA, or by DEAE-cellulose of untreated cell extracts, does not result in the isolation of newly formed RNA with a base composition any different from that obtained by trichloroaeetic acid precipitation of unfractionated labeled cells. DISCUSSION

The analyses of the bulk RNA components in the five species of bacteria used indicate no obvious relationship in the nucleotide base composition of the various purified RNA fractions to the DNA. The composition of the RNA which comprises most of this material in the cells, the 7o-S ribosomes, is extremely invariable in composition from species to species. The s-RNA also appears to be very constant in composition (Fig. 6). The composition obtained for purified E. coli s-RNA agrees with the results obtained by DUNN, SMITH AND SPAHR2s and by ZILLIG et al. ~9. Subfractionation of the 7o-S component of bacteria into 5o-S and 3o-S or of yeast 8o-S into 6o-S and 4o-S has brought to light some differences in base composition of the two fractions. In general, purine contents are higher, pyrimidines lower in the larger (5o-S or 6o-S) than in the smaller (3o-S or 4o-S) ribosomal subunits. There is, however, no uniformly consistent relationship in composition between Biochim. Biophys. Acta, 61 (1962) 5 1 3 - 5 2 5

RNA COMPOSITION

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Fig. 6. Comparison of (guanylic a c i d + c y t i d y l i c acid)/(adenylic acid+uridylic acid) values for s-RNA and 7o-S RNA, from bacteria with DNA (guanylic a c i d + c y t i d y l i c acid)/(adenylic acid + thymidylic acid) values ranging from o.6 to 1.75. O-C), s-RNA values; O - O , ?o-S ribosomal 1RNA values.

the DNA, and either 3o-S or 5o-S in the bacterial species. The weak relationship between the DNA and s-RNA in bacteria reported by MIURA8 is possibly due to contamination of the soluble fraction by the I4-S RNA component stripped from the 7o-S ribosomes during washing in o.14 M NaC1. The I4-S RNA component of bacterial cells has been detected by short periods of [14C]uracil incorporation into growing cells of E. c0li25,~,3°, 31. It has been variously ascribed the role of "messenger" RN'A3°,31 and of "ribosomal RNA precursor"Zs,ze. By present theories, these two roles would predict base compositions of the I4-S component of two types. The "messenger" theory, assuming that the RNA carries genetic information, postulates that the base composition of this RNA fraction is like that of the DNA in the cell, whilst the "ribosomal precursor" theory25,~6 would predict the composition to be like that of the ribosomes. The I4-S fraction has been found to be very different in base composition from the normal total trichloroacetic acid-precipitable RNA in the cell or from the ribosomes. Figs. 7 and 8 indicate a possible relationship between the DNA composition of the bacteria, the I4-S RNA and 7o-S RNA base compositions in each of the five i.¢ .-V

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l~'ig• 7. Comparison of (guanylic acid + cytidylic acid)/(adenylic acid +uridylic acid (thymidylic acid)) values for 7o-S ribosomal I~NA, I4-S 1RNA and DNA from bacteria with DNA (guanylic acid + cytidylic acid)/(adenylic acid + thymidylic acid) values ranging from o.6 to 1.75. + - + , DNA values; C)-O, newly formed (I4-S) RNA values; Q - Q , 7o-S ribosomal RNA values. Biochim. Biophys. Acta, 61 (1962) 513-525

524

J. E. M. MIDGLEY

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Fig. 8. Graphical representation of nucleotide base composition of D N A , I4-S R N A and 7o-S ribosomal RNA in each of the five bacterial species used. Ordinate, nucleotide base composition (mole ~o). On the left ordinate of each graph, D N A base composition, on the right ordinate, 7o-S R N A base composition. I4-S R N A composition as best fit between these two compositions.

species. It can be seen (Fig. 7) that if the I4-S fraction were in fact composed of two entities of RI~A with different base compositions corresponding to either the DNA or to the ribosomal RNA, then in each case the I4-S RNA would be made up of approx. 33 ~/o DNA-like and 67 % ribosomal RNA-like material. Alternatively, the I4-S component might be a homogeneous molecule with a composition intermediate between that of the DNA and the ribosomal RNA in each species. In a following paper 32 it has been observed that the base composition of the newly formed RNA after lO-15 sec a~p incorporation into growing bacterial ceils is still very like the base composition measured after as long as 4 min incorporation, in each of the five species. As this material is equivalent to the I4-S material in the cells, the proportions of DNAlike and ribosomal RNA-like structures in the component shown above probably also exist at these very brief incorporation periods. It must be emphasized that the base compositions of the I4-S components of bacterial cells given above represent only apparent compositions as probably the pool of material in the I4-S component has not been saturated with a2p at the times above. An absolute base composition of this RNA Call only be obtained by isolation of the fraction at incorporation times known to be adequate to saturate its pool. Further experiments to this end will be reported in a subsequent paper% REFERENCES 1 A. N. BELOZERSKY AND A. S. SPTRIN, in J. N. DAVlDSON AND E. CHARGAFF, The Nucleic Acids, Vol. 3, Academic Press, Inc., New York, 196o, p. 147. 2 I"{. I. MIURA, Biochim. Biophys. Aeta, 55 (1962) 62. 3 S. SPII~GELMAN, Cold Spring Harbor Symposium, 1961, p. 75. i C. 1~. WOESE, Nature, 189 (1961) 920. 5 p. F. SPAHR AND A. TlSSI~RES, J. Mol. Biol., i (1959) 237. e F. JAcoB AND J. MONOD, J. Mol. Biol., 3 (1961) 318. 7 M. YCAS AND W. S. VINCENT, Proe. Natl. Acad. Sci. U.S., 46 (196o) 804. 8 L. ASTRACHAN AND T. M. FISHER, Federation Proc., 20 (1961) 359. 0 3gL HAYASHI AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1564. 10 M. NOMURA, B. D. HALL AND S. SPIEGELMAN, J. Mol. Biol., 2 (196o) 306. I I E . VOLKIN AND L. ASTRACHAN, Virology, 2 (1956) 149. 12 E. K. V. BAUTZ AND •. D. HALL, Abstracts o/ Papers, 6th Annual Meeting, Biophysical Society (I962), 13 E. T. BOLTON, Carnegie Institution o/ Washington Year Book, 58 (1959) 275. 14 B. J. McCARTHY AND A. I. ARONSON, Carnegie Institution ol Washington Year Book, 59 (196°) 247.

Biochim. Biophys. Acta, 61 (1962) 513-525

R N A COMPOSITION

525

J. BRITTEN AND R. B. ROBERTS, Science, 131 (196o) 32. G. I{URLAND, J. Mol. Biol., 2 (196o) 83. S. KIRBY, Biochem. J., 64 (1956) 405 . T. BOLTON, R. J. BRITTEN, D. B. COWIE AND R. B. ROBERTS, Carnegie Institution o/Washington Year Book, 57 (1958 ) 14°. 16 R. J. BRITTEN, R. B. ROBERTS AND E. F. FRENCH, Proc. Natl..dead. Sci. U.S., 41 (1955) 863. 2o VV. E. COHN, in J. N. DAVlDSON AND E. CHARGAFF, The Nucleic Acids, Vol. i, Academic Press, Inc., New York, 1955, P. 21421 E. A. PETERSON AND I-[. A. SOBER, Anal. Chem., 31 (1959) 857. 22 E. T. BOLTON, u n p u b l i s h e d experiments. 23 R. MONIER, IV[. L. STEPHENSON AND P. C. ZAMECNIK, Biochim. Biophys. Acta, 43 (196°) i. 26 R. B. ROBERTS, R. J. BRITTEN AND E. T. BOLTON, Microsomal Particles and Protein Synthesis, P e r g a m o n Press, 1958, p. 84. 25 B. J. McCARTHY AND R. J. BRITTEN, Biophys. J . , 2 (1962) 3526 B. J. MCCARTHY, R. J. BRITTEN AND R. B. ROBERTS, Biophys. J . , 2 (1962) 57. 27 j . ]). MANDELL AND A. D. HERSHEY, Anal. Biochem., I (196o) 66. 2s D. B. DUNK, J. D. SMITH AND P. F. SPAHR, J. J~/Iol. Biol., 2 (196o) 113. ~9 \V. ZILLIG, D. SCHACTSCHABEL AND V¢. KRONE, Z. physiol. Chem. Hoppe-Seylers, 318 (196o) ioo. 30 S. BRENNER, F. JACOB AND M. MESELSON, Nature, 19o (1961) 576. 31 F. GROS, I7[. HIATT, W. GILBERT, C. G. KURLAND, t{. W. ~{ISEBROUGH AND j. V. WATSON, Nature, 19o (1961) 581. 32 j . E. M. MIDGLEY AND B. J. MCCARTHY, Biochim. Biophys. Acta, I 6 (1962) 696.

15 16 17 16

R. C. K. t~.

Biochim. Biophys. Acta, 6i (I962) 513-525