The nature of high molecular weight fragments of ribosomal RNA

.J. .3lol. Biol.

11967) 29, 307-313

The Nature of High Molecular Weight Fragments of Ribosomal RNA HANNAH

Medical

J. GOULD

Research Council, Biophysics Research Unit, h’ing’s 26-29, Drury Lane, London, W.C.2, England? (Received 17 March

College

1967’)

A method for obtaining high molecular weight fragments of rabbit reticulocyte ribosomal RNA is reported. High molecular weight specific fragments accounting for approximately half of the 30 s component have been isolated from a T, ribonuclease digest and their properties studied. The molecular weights of the fragments fall in the range of 10,000 to 400,000. The hypochromicity measured at 280 and. 260 rnp as a function of temperature suggests that much of the original double-helical structure of the RNA within the fragments remains intact. The melting temperature (T,) is higher, and the profile somewhat sharper, than in the case of intact 30 s RNA, and the helical regions contain a significantly higher proportion of G-C base pairs. The fragments isolated thus appear to originate from the more thermostable G--C rich regions of the intact RKA, which give rise to the distinctive upper phase of the melting curve previously noted. They contain a small number of “hidden breaks,” i.e. discontinuities in the phosphodiester chain caused by enzymic attack within helical regions. These products have been analysed after heating the RNA to unfold the helical regions and dissociate t,he resulting fragments. The “hidden breaks,” resemble those in single-stranded regions, in that t,hey occur at specific and characteristic points of the molecule. The number of moles of each fragment per mole of 30 s ribosomal RNA has been calculated from the weight percentage of the fragment in the digest and its molecular weight. The values are in many cases around unity, suggesting that all the ribosomal RNA molecules within reticulocytes contain at least some common sequences. This supports the view that the cistrons controlling ribosomal Ii?\TA synthesis may likewise be identical.

1. Introduction Recent studies (McPhie, Hounsell & Gratzer, 1966; Gould, 1966a,b; Gould, Bonanou & Kanagalingham, 1966) have suggested the possibility of isolating specific fragments of ribosomal RNA in which large portions of the original primary and secondary structure remain intact. Several advances in technique have made this possible. First, the use of polyacrylamide gel electrophoresis has provided a method of uniquely high resolution for separating polynucleotide species according to molecular weight (Richards, Co11& Gratzer, 1965; McPhie et al., 1966). Secondly,through the application of this met,hod, it has been shown (McPhie et al., 1966; Gould, 1966a,b; Gould et (II., 1966) that nucleases attack ribosomal RNA with a high degree of specificity and that the products of a limited digestion include a number of fragments apparently monodisperse with molecular weights in the range 10,000 to 400,000. The present report describes the extraction from polyacrylamide gels of nine t Prcsellt Address: Medical Research Council, SIetabolic mere t, Imperial College, Imperial Institute Road, London, 307

Reactions Unit, Biochemistry S.W.7, England.

Depot-

308

H.

fragments limitctl pink

arising

from

tligcstion dyt

precise profiles

with

pyroninc rscision

individual of enzymic

the 30 s components T, rihonuclcasc.

y binds of specific

and t,he effect fragments attack

.J. C;OUJ>l)

to RNA

without,

zones after

of brief have

of rabbit)

Atlva.utagr apparently

separation

heating

rcticuloc>te

has l~ctr

i tlli('ll

;Llt,willg

has taken

ribosoma~l

ttS.\

of

1 IN* fwl

t Iral

//, . antI

I’xkGlit

t iw

l)lact*.

I\bsorba~icc~

to ‘30°C on the elcctrol,lloret’ic

been studied

in order

and on the propertics

mobility

on t lit‘

;I~
ItJclt i tjg of’ I II(,

to shed some lights on the spccii~icit?

of the intact

and dcgradcd

RN.1.

2. Materials and Methods (a) Preparation

of reticulocyte

ribosomes

Rabbits weighing 2 to 3 kg were made anaemic by five consecutive daily injections of 0.8 ml. of neutralized 2.5% phenylhydrazine, and were bled on the seventh day by heart, puncture. The reticulocytes were washed and lysed, and the ribosomes isolated from tht: lysate in the manner previously described (Amstein, Cox 6r Hunt, 1964; Arnstein, (‘OS. Gould & Potter, 1965). (b) isolation

of RNA

RNA was isolated by using guanidinium chloride to dissociate the ribonucleoprotein, and ethanol to precipitate the RNA according to the method of Cox (1966a). (c) Digestion

of RNA

with

TI ribonuclense

The conditions for digesting ribosomal RNA with T, ribonucleaso were similar to those reported previously (Gould, 19666). The incubation mixture contained 3 ml. of RNA at 3.0 mg/ml. in distilled water, 0.15 ml. 2 nr-Tris buffer (l)H 7.6) and 0.3 ml. of T, ribonuclease (Sankyo Corp. Ltd.) at 1000 units/ml. At the end of the incubation, 0.4 ml. of 50% sucrose was added so that the sample could be layered beneath the reservoir buffer (see below) and 0.15 ml. of 1% pyronine Y was added to sbain t,he RNA migrating in the gel during electrophoresis. (d) Electrophoresis

of RNA

digest

Electrophoresis was carried out exactly as previously described (Gould, 1966a) except for the method of sample application. In this case, 0.65 ml. of sample was placed on top of each of 7 gels with the tubes positioned in the electrophoresis apparatus and the lowei reservoir buffer compartment filled. It was previously found that such large sample rolumes do not cause any deterioration (band broadening) of the gel pattern. The samples were carefully layered with reservoir buffer to the top of the tubes with a drawn-out Pasteur pipette, and then the upper reservoir buffer compartmcnt~ \s.as filled carefully to avoid disturbing the sample. Elect,rophoresis was carried out at room temperature for 1 hr at 125 v and a current of 5 ms/tube. Gels were expelled from the t’ubos after rimming with a needle by fitting a rubber bulb filled with water on one end and applying gcntlc pressure. One gel was stained for 24 hr in a solution of 17; acridine orange and 2’;;) lanthanum acetate in 1576 acetic acid and destained electrophoretically to provide a permanent record of the original digestion pattern. (e) Extraction

of RNA

from

pobyacrylamide

gels

In t’hese experiments the extraction of RNA was carried out immediately after clectrophoresis. The prestained gels may, however, be stored frozen and thawed out to be photographed or extracted later. Nine distinct zones could be seen in each of the gels. The zones were individually cut out with a razor blade and the corresponding slices were pooled. The pooled gels were homogenized with 2 ml. of distilled water in a gla,ss homogenizer and the suspension was centrifuged. The supernatant liquid was removed and the RNA was precipitated by the addition of 0.1 vol. 2 M-sodium acetate and 3 vol. ethanol. The precipitate was allowed to form overnight at -20°C and was then centrifuged for 20 min at 0°C in an MSE Mistral

RIBOSOMAL

RNA

31i!l

FFLAGfiII
centrifuge at 3000 rev./min. The RNA was redissolved in 1 ml. of distilled water and IVprecipitated wit11 sodium acetate and ethanol. The precipitate was redissolved in 0.4 ~111. rlistill<~tl \vntcr containing 0.1 m g of bentonitc/ml. A sc~ontl extraction of t,hc polyacr~~-

1am1d~ gull suspension with water in the majlncr alnount,

of RNA

Tile over-all digest., judging

account the small increment Sl jectra

of the extracted

violl:t-absorbing

tlescribed yic~ldetl less t,han IO”,, of t III-

obtained in the first extract. yield of cxtrackd RNA \~a:: approximately 30”;, of that in the original from the recovery of optical density units at 260 mp and not taking-into

material

(< 15%) in A,,,, on digestion. fragments

showed

no evidence

of contamination

which might have arisen from the polyacrylamide

with

ultrkn -

gel during

the isolation procedure: peak absorbances were around 260 rnp, minima occurred in tile spectra betnecn 230 and 240 mp, and the ratio A210/‘14260 was about 1.6, the sama as fol bob11 30 s and 19 s components of ribosomal RNA. Some of the spectral characteristic”< of t,lle fragments are summarized in Table 1. (f) Molecular

weights

of frrrgmelzts

The relation, Sg,,, = 13.75 - 11.26 R,, and mol. wt = (6.88 - 5.63 R,)2 x 104 (McPhio et crl., 1!)66) were used to calculate the sedimentation coefficient (S$o,W) and the

molecular

xveights, respectively,

of the RNA

species from the observed quantity

R, in

5(‘s polyacrylnmide gels under the conditions stated. Sedimentation coefficients of the ril)osomal fraglnents in a solution of 0.1 M-X&~, 0.02 M-Tris buffer (pH 7.6) and 0.1 mg brntonit.e/ml., u-ere measured using a Spinoo model E ultracentrifuge with ultraviolet, optics (Table 1). The experimental results verify the relation between RF and Sg, reported by Mcl’hic rt (II. (196G). (g) Test for

“hidden

breaks”

0.1 -ml. portit)ns containing 50 to 100 pg of RNA extracted from the acrylamide gels and dissolved in an aqueous suspension of 0.1 :A bentonite were heated for 30 set in a water bath at WCC and then plunged into ice. Sucrose was added to the samples before layering on t,hc acrylnmitle gels, and electrophorcsis carried out as described. It is known from the mehing cur\.e of the intact RNA as well as the frugmcnts (Fig. 1) that this temperature sholdtl be sufficient to melt out essentially all secondary structure and so reveal ang “hidden breaks” in hclica,l regions. It was also ascertained that 30-set heating was sufficient tirncx for the sanlple to reach 9O”C, and that intact ribosomul RPiA was not thermall,r, degratLed under t,hese conditions. (h) Melting

projile

of RXL4 frugments

T~:ruperaturc-absorbance profiles were measured with a Beckman DU manual instru nlent, w&h a thermostatically controlled cell-housing supplied from a Haake-Fe circulating therlllost,at. Tile temperature was measured with a mercury thermomet,er in a separate cell. Sa,~r~ples lvere dissolved in 0.01 M-phosphate buffer (pH 7.6) at a concentration -;uffiraicmt, to gi\ e m absorbance reading of 0.3 to 0.4 at 260 II+ in a cell of 1 cm path lengt,h. C&z~a.stopperetl c:ells were fillccl leaving only a small air space at the top, and se&d with K’T\. hilicone rubber to prcvent loss of liquid.

3. Results Limited ‘I’, ribouuclease digestion of reticulocyte ribosomal RNA results in a number of stable digestion intermediates of high molecular weight which can be gels (Gould, 1966u,b; Gould et al., 1966). Plate 1 separated on ;iO$ p 01y acrylamide ~1~0~s the distribution of these fragments formed over a wide range of conditions. Plate I I shows the results of extractming RNA fragments from polyacrylamide gels. .%t.the ext’reme left is the pattern obtained when one of seven identical gels was stained ;lfttlr elcctrophoresis of an RNA digest prepared as described in Plate I(b). Nine prestail~cd zones were cut out individually from the other six gels and the corresponding zones were pooled. After extracting the RNA, the fractions were re-run on gels under

H. ,J. GOULII

310

the same conditions as the original digest. The cxt’ract’ed samples cuntaiucg I a iiigll proportion of material with the sam1 mobility as the, corrcspontling 1:,.X.1 it1 t II{, original gel. Some cross-contamin&on with other fragrntuts ant1 wit IL IO\V itloicc,ll!;n weight RNA (R, ::= 1), presumably arisitlg from degratla&)n of ttrca fi~agmclrl li, \\‘a~ also apparent’. The hypochromicity of the fragments is substantial (20 to 30’); at both 260 and 280 mp). The fragments contain a higher proportion of relatively- thcrmostablc secondarystructure (T,> GO%), melt for the most part over a smaller temperature rangt?, ant1 (Table 1) compared to tllc tot’al exhibit higher values ,for the ratio d~,,,/dd,,, RNA. TABLE

1

Properties of isolated ribosomal RNA fragments

1 2 3 4 5 6 7 8 9 30sRNA

104 74 70 67 104 136 134 243 1750 -

9.7 4.0 2.0 30.0

0.587 0.500 0.513 0.575 0,540 0.570 0.575 0.523 0.482 0.515

0.605 0.787 0.7i5 0.710 0.720 0.642 O.ilO 0.675 0.605 0.553

0.282 0.252 0.218 0.173 0.180 0.164 0.168 0.209 0.145 0.290

0.314 0.277 0.261 0.215 0.227 0.188 0.187 0.207 0.112 0.249

0.898 0.804 0.835 0.805 0.893 0.870 0.900 1.01 1.29 1.16

The electrophoretic patterns obt,ained after heating the fragments reveal the presence of a number of “hidden breaks” (Plate III). Fragment 1 is extensively degraded to material of less than 10,000 molecular weight. In the case of the other fragments, heating is seen to generate a number of fragments smaller t,han the original fragment, but still of quite high molecular weight. A high degree of specilicit,y in t)he position of enzymic attack in helical regions is evident from the observed patterns. 4. Discussion Many of the bulk properties of ribosomal RNA, in particular, molecular weight and base composition (Petermann, 1964), nearest-neighbour frequency (Widnell KS Tata, 1966), helix content (Spirin, 1963) and the average composition and length of the helical regions (Cox, 1966a), have been fully explored. In terms of fine structure, only the end groups (Hunt, 1965; Midgeley & McIlreavy, 1966) and the terminal sequences (Takanami, 1967) have been studied. In the present work, an attempt is made to bring physical and chemical studies of ribosomal RNA to a higher level of resolution by specific fragmentation and isolat’ion and characterization of the fragments. The fragments of reticulocyte ribosomal RNA which can be resolved by electrophoresis on 5% polyacrylamide gels account for half the total mass of the 30 s component (Table 2). The molecular weights are in the range of 10,000 to 400,000, which is still larger

HIBOSOMAL

RNA TABLE

Molar

yields

2

of rihosomal

RNA

0.294

254,000

0.380 043.5 @500 0~594 04m

206,WO

0.710 0.850 0.920 1 .oo -

21 I

FItAGMEXTS

178.000 148.000 112,000 92,000 i2.000 36,000 22.000

fractions

Il.l,59 (I.129 0.111 0~093 (14170 O~ll;iX 41.016 0422 II411

I.(111 0.33 ( I .; x O.&S 1 .o’( O.il’ 1.9s R.J(l ;.(I!\

10,000 1,600,OOO

I 400

t,halI that of transfer RNA, the largest RNA whose sequence has been determined. Howe-\-cr, it has been shown that these fragments contain “hidden breaks” at specific points and that smaller fragments can be obtained merely by heating t,he RNA. In this way also a partial ordering of ribosomal RNA fragments is achieved which might 1~ usc~ful in t’hf, study of the primary structure. A similar strategy might be applicable to oi her high molecular weight RNA, such as viral RNA, or indeed, t’o DXA. Tht~ absorbance-melting profiles reflect t,he secondary structure of the fragments. It sllould be pointed out that all the samples contain some low molecular weight contaminants. The effect of such material would be to decrease the value of T, and to broatlon t(he rnelt’ing curve. Nevertheless, the hypochromicity is comparable to t,hat of intact RXA, the values of T, are higher, and in some cases the melting profiles are considerably sharper, although not obviously co-operative to the extent, of DSA. ‘Claus it stems likely that the fragments contain a large proportion of double-helix-al KS, \ C’os (196Ab) has shown that the base composition of double-helical regions in R?JA ~nay IV> descrihcd by the relation, fGc = l/(0*81 (d=l,,,/diz,,,) - 1*4ti), assuming ilrc:rl~ is no contribution to the absorbance changes on heating due to the formation (jr unfolding of single-stranded stacked structures. This assumpt’ion appears to be subst,antially justified for intact ribosomal RNA (Gratzer, 1966; Cox 8; Gould, ~mpublished results). Since one is considering here quite large fragments of ribosomal RNLI, each consistring of many short helical regions for the most part unaffected by I 11~t%nz.vme, treatment of the present data, in these terms is surmised to be similar1.v ,)11:;li tifd ‘IYIn ratio d.l,,,/dL42s0 on heating the fragments is in each case lower than t’hc> (,oi,rc,s~)r)Ilding values for intact RNA (Table 1). This indicates that the helical regions (of’ tl P fra,gmenta are enriched in (:-C base pairs, and the calculated caompositions are ~,i\-cs~rin ‘l’nbl(~ I . ‘l’hc existence of G-C-rich hclices in intact 30 s RNA may b(: ri~~.c),:t\i+~c(Ifrom11the melting curve (Fig. 1). ‘l’h(~ (i-(! content of the RNA rc+cteti b\~ i II{‘ Ill(*ltiltg (:UI’VC above GO”C is W”/,, compared to 5CV& for the lower-melting phascb. ‘i’hc cctmpositjion, thermal stability and yield of the fragments suggest that they ma>’ (Iriginate fir2rn t,he regions of intact RNA which melt above 60°C.

Temperature

eC)

FIG. 1. Melting profiles of the fragments shown in I’latc II. The fragmcnt.s were dissolved in 041 M-phosphate buffer (pH 7’0) and t,11e absorba.ncY? Clltl.ll~~x on heating were measured as described in tho text. The melting profile of the intart 30 s ~~~~~nlxnont of rabbit reticulocyte ribosomes is shown ill the upper left-most, lxu~el for comparisoll.

The results are consistent with the observations of Delihas & Bertmen (1966) that the over-all composition of T,-resist,ant fragments of ribosomal RN4 from rat liver is enriched in G and C. It can be inferred that sequences rich in these bases are present, in reticulocyte and liver ribosomal REA. These sequences appear t,o be of sucll structure that they are unusually resistant, to T, ribonuclease. Further work is being carried out on the fragments which should reveal t,he struct)ural features responsible for the cnzymic specificit,y. It is now known that multiple cistrons for ribosomal RNB exist in a wide variety of organisms (Spiegleman & Yanofsky, 1965). The number of cistrons appears to be approximately the same for both ribosomal components, but increases in proportion to the tot,al amount of DNA per cell. The number in higher organisms is therefore extremely large. It has been postulated that the cistrons may diKer and t,l~ corresponding ribosomal variants may serve different and complernent~arg functious. If this not’ion is correct, one might expect to find an overwhelming variety of fragmttnts upon nuclease digestion of ribosomal RNA, each in small molar J-i(xlds normalized to the moles of undegradetl RNA originally pr~~nt. An attempt has therefort: been matlc to c~saminc tllc mc,lar yield of’ fragments in t11c present stud?-. A microdensitometc*r trarirl~ of the: ~)olyac:l~?-l~~~niclcgel dt~llictc~tl ii, Plate I(b) is shown in Plate IV. Thr: molar ~-i&l of fragmc,nt,a has been calculatctc 1 from the relative weight of the fragments in the digest, together wit,11 the estjirnatctl

Mol. wt x IO-3

RF

0o-

o-2 -O-2

400

300

F 0.4

-+

0.6

-

200

-

100

c

0.8

--

I.0 i-

50 50

IO

f111 ,,r,,

,,

i

Mol. ffF

wt

x 10-3 O-

400

0,2-

-

300

04-

-

200

0.6.

0,8--

I.0 -

-

100

-

50

-

IO

Moi.wt x IO-3

4 O-

9.2 -

-

400

-

300

0 4-m

200

0.6-

0.8-

PO-

-

100

-

50

IO

3 5

I 2

o-2

04

II

4 ,

t

0.6

60 I

0.8

L-

RIBOSOMAL

RNA

3 13

FRAGMENTS

molecular weights. The values presented in Table 2 vary between 0.3 and 7.0. The yields of the smallest two fragments, 7 and 8, were greater than one mole/mole 30 s RNA. This may be explained by postulating repeating sequences in ribosomal RNA or else het’erogeneity in these fractions. From the lowest relative yield of O-33, assuming that the remainder of the fragments are, as they appear to be, monodisperse, an upper limit of three different 30 s ribosomal RNA species can be inferred. The indicated homogeneity of ribosomal RNA has far-reaching implications. On the assumption that there are indeed numerous genes coding for ribosomal RNA w&h equal efficiency in reticulocytes, the results suggest that these genes may be identical or closely similar. It, was previously found that rabbit liver and rabbit reticulocyte ribosoma RKA give the same pattern on digestion with T, ribonuclease, whereas ribosomal RNA’s isolated from a number of closely related species each gave a characteristic and distinctive pattern (Gould et al., 1966). The result’s taken together imply that certain mutations in the template for ribosomal RN-4 are viable. The results also suggest that the majority of cistrons for ribosomal RN14 enumerated by hybridization experiment,s may be copied from one or a few genes which alone replicate at cell division. I i hank inten~st~.

Sir *John

Randall

and

Professor

M. H.

Ii’. Wilkins

for

their

support

ant1

REFERENCES rImstein, H. R. V., Cox, R. A., Gould, H. J. 8: Potter, H. (1965). Biochem. J. 96, 500. Arnstein, H. R. V., Cox, R. A. & Hunt, J. A. (1964). Biochem. J. 92, 1966. (lox, R. A. (1966a). Biochemical Preparations, vol. 2, p. 104. New York: John Wiley. (lox, R. A. (19663). Biochem. J. 98, 841. Delihas, N. & Bertman, J. (1966). J. Mol. Bid. 21, 391. Gould, H. J. (1966a). Biochemistry, 5, 1103. (:ould, H. J. (1966b). Biochim. biophys. Acta, 123, ~'1. Gould, H. J., Bonanou, S. & Kanagalingham, K. (19t :). J. Mol. BioZ. 22, 397. Gratzer, W. B. (1966). Biochim. biophys. Actu, 123, 431. Hunt, J. A. (1965). Biochem. J. 95, 541. McPhie, P., Hounsell, J. & Gratzer, W. B. (1966). Biochemistry, 5, 988. Midgley, J. E. M. & McIlreavy, D. J. (1966). Biochem. J. 101, 32. Petermann, M. (1964). The Physical and Chemical Properties of Ribosomes. Amsterdam: Elsevier. Richards, E. G., Coll, J. A. & Gratzer, W. B. (1965). AnuZyt. Biochem. 12, 452. Hpiegleman, S. & Yanofsky, S. A. (1965). EvoZving Genes, ed. by V. Bryson & H. J. Vogel. New York: Academic Press. Spirm, A. S. (1963). Progr. NucZeic Acid Rea. 1, 301. Takanami, M. (1967). J. Mol. BioZ. 23, 135. Widnell, C. C. & Tata, J. R. (1966). Biochim. biophys. Actu, 123, 478.