Properties of Ribosomes and RNA Synthesized by ... - Science Direct

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(nwthyL3H cts/min incorporat'ed per =I 260nm unit of ribosomes) in sucrose ...... Markey. F., Sims, P. F. G. & Wild. D. G. (1976). Biochem. J. 158, 451-456. .Moole.
J. Mol. B&d. (1979) 127, 375-395

Properties of Ribosomes and RNA Synthesized by Escherichia coli Grown in the Presence of Ethionine V.t Methylation

Dependence of the Assembly of E. coli 50 S Ribosomal Subunits JEAK-HERVI?

AIIX

ANI) DONBL

HAYES

lnstitut de Biologic l’hysico-chimique Lahoratoire de Chimie Cdlulaiw 13 Rue Pierre et Ma& Curie 7SOO5 Paris, Fraszw ASD KNUD

Nax

H. XIERIIAUS

Plan(:k LMitut fiir Nolekularr Abteilung Wittmawt Berlin,- Dahlem, Qeerwi tq

Genetik

.41l othionine-containing submethylated particle related t,o the 50 8 ribosomal subunit has been isolated from Escherich,ia coli gro\+~~ in t,he presence of ethionine. This particle (E-50s) lacks Llfi, contains reduced amounts of L6, L27, L28 and IJO and possesses a more labile and flexible st,rurtrlre t)han the normal 50 S subnnit. The E-50X particle has defective associatjion properties and is incapable of pcJptide bond formation. It can be converted to an active 50 S ribosomal subunit, when ethionine-treated bacteria are incubat,ed under conditions which permit methylation of submethylated cellular components (presence of methionine) in the absence of de novo protein and RNA synthesis (presence of rifampicin). Total reconstitution of 50 S ribosomal subunits in z&-o Ilsing normal 23 S and 5 S ribosomal RNA and proteins prepared front E-508 particles yields active subunits only if L16 is also added. Ttlc hypothcasis that E-50s particles accumulate in ethionine-treated bacteria because the absence of methylation of one or more of their components blocks a latr stage (I,16 integration) in the normal 50 S assemb1.y process is discussed.

1. Introduction Ethionine (a-amino-y-ethylmercaptobut8yric acid). an analogue of methionine (aamino-y-methylmercaptobutyric acid) can fully replace the latter in all the basic reactions of protein synthesis (Trupin et al.. 1966; Old & Jones, 1976a; Brown, 1973) and proteins containing ethionyl instead of methionyl residues have been found to function normally (Yoshida 85 Yamasaki, 1959; Old & #Jones, 1976b). In contrast, ethionine is not accepted by procaryotic methionine adenosyltransferase (ATP: Lmethionine-S-adenosyl-transferasr, EC 2.5.1.6) the product8 of which, S-adenosylI,-methionine, is used for most cellular methylation reactions. Undermethylated, cellular components can therefore be produced by culture of methionine-requiring &rains of Escherichia coli in minimal medium supplemented with ethionine. By using t Paper IV in this series is Lederer et rrl., 1977. 375 002~-~s:~0/79/04037~-~1

%02.00/0

Q 1979 Academic

Press Inc.

(London)

Ltd.

376

J.-H.

,1LIS.

I).

HAYEH

;1NrJ

I(.

H. NIERHAVS

t’his technique, we have shown (Alix $ Haye, q, 1974) t’hat t,hree rihosomal prot,eins of t,he 50 S subunit, namely Lll . L3 and L5, are modified by methglat~ion. M&hylation of other ribosomal or ribosome-associat,~?(l proteins, namely SI 1, I,lli. L33 and lF3. of t)echniqucs such as starvation has subsequently been detected, bJ7 using a variety of a Met- strain of E. coli by methionine deprivation (Chang & Chang, 1974), growth of E. coli in a medium containing [1-14C]methionine and [methyl-31-I]methionine (Chang et al., 1974; Chang $ Chang, 1975), and protein sequence studies (Terhorst, et al., 1972 ; Chen et al., 1977; Brauer & Wittmann-Liebold, 1977 ; Dognin & WittmannLiebold, 1977; Chen & Chen-Schmeisser, 1977). Recently, E. coli mutants deficient in ribosomal protein methylation have been discovered (Colson & Smith, 1977; Lhoest & Colson, 1977). The role of these met’hylations as well as that, of those previously known tjo occur in E’. co2i ribosomal RNA (Fellner, 1969) are not yet understood. 50 S ribosomal subunits from an Lll methylation deficient mutant show the same activity as fully methylated subunits (Colson & Smith, 1977). In contrast, the E-5OSt subunit is inactive. In this paper we analyse the properbies of this particle, describe its activation by methylation in vivo, and present the results of total reconstitution experiments performed to elucidate a possible role for methylation of protein Lll.

2. Materials and Methods (a) Reagents,

buffers

and bacterial

strains

chloramphenicol DL-Ethionine was supplied by Serlabo (Paris, France) ; rifampicin, by Boehringer (Mannheim, Germany) ; and puromycin by and S-adenosyl-L-methionine Serva (Heidelberg, Germany). The following buffers were used : Buffer 1: 10 mM-Tris.HCl (pH 7.4,0’%), 60 mM.ammonium chloride, 10 mM-magnesium acetate, 6-mM-fl-mercaptoethanol. Buffer 2: 10 mr\l-Tris.HCl (pH 7.4, OV). ti0 mM-ammonium chloride, 10 mM-magnesium acetate, 400 mM-sodium chloride, 6 mM-j%mercaptoethanol. Buffer 3: 10 mi\l-Tris.HCl (pH 7.4, O”C), 60 mM-ammonium chloride, 0.1 mM-magnesium acetate, 6 mM-fl-mercaptoethanol. E. coZiD10 (RNase I- ; Met- ; rel A- ; Gesteland, 1966) was used for the formation of ethionine particles; mutant prm-1 deficient in Lll methylation (Colson & Smith, 1977) was a generous gift from Dr C. Colson (Louvain-la-Neuve, Belgium). (b) CuZ2ure of E. coli in the presen.ce of nI,-ethionine

and naethylation

in vivo

Cells grown in complete medium (+ Met) were incubated in ethionine-containing medium, and t,hen returned t,o complete medium to allow methylation in wiwo of submethylated components as described previously (Alix & Hayes, lR74), except that during the final methylation step ilz viva rifampicin (0.3 mx) was added instead of chloramphenicol. The 3 steps of this process are summarized in Fig. 1. Differential labelling of the ribosomal proteins was realized by adding a 3H-labelled amino acid mixture (minus [3H]methionine; 4 &i/ml, Amersham, TRK 440) to the first (methionine) phase of the culture and a L-(U-I%)-labelled amino acid mixture (minus [‘*C]methionine; 1.25 &X/ml, Amersham, CFB 104) to the second (ethionine) phase. (c) Preparation

of S-150

enzymes, ribosomal ribosomal proteins

subunits,

LXX cores, and

Normal or ethionine-grown bacteria were ground with alumina 305(Alcoa) extract, made in buffer 1, was layered on a 0.5-ml 20% sucrose cushion,

and the S-30 containing 10

t The prefix M- designates normally mothylated ribosomes. E- designates the submethyleted ribosomes synthesized by bacteria in tho p~~r;enco of ethionine. ME- designates the mixture of normal (M) and submethylatsd (E) ribovomos present in bacteria after treatment with ethic&m (see Fig. 1).

ROLE

OF METHYLATION

IN

377

60 S RIBOSOMES

Second phase (ethionine)

First phase (methionine)

presence of rifampicin) 1

+2

c

I

.+.-4

--

+ 5

v

Time(h)

14C labelling

‘Ii labelllng t

ME- ribosomes

M-ribosomes

Methylated ME-ribosomes

Fm. 1. Preparation scheme for ethionine ribosomes. Itibosomes isolated from cells at the end of the first phase are normally methyleted (M). At the end of t,he second phase cells contain, in addition to the normally methylated ribosomes from the first phase, undermethylated ethionine-containing ribosomes (E). We call the mixed population of methylated and undermethylated ribosomes ME-ribosomes. In the t,hird phase ethionine is again replaced by methionine in the medium, and addition of rifampicin prevents de ~OVOsynthesis but allows methylation of the accumulated, undermethylated ribosomes. For details, see Materials and Methods.

mM-TrissHCl (pH 7.4), 500 mM-ammonium chloride, 10 m&f-magnesium acetate, 6 mM+ mercaptoethanol. After centrifugation for 17 h at 80,000 g, the layer above the ribosome pellet was discarded, and the pellet was dissolved in buffer 1. For separation of ribosomal subunits, an equal volume of a buffer containing 10 mu-Tris.HCl (pH 7*4), 60 mMammonium chloride, 10 mM-magnesium acetate, 6 mM-/?-mercaptoethanol and 800 mMsodium chloride was added, and the mixture w&s layered on sucrose gradients prepared in buffer 2 (SW27 or zonal rotor). Fractions containing 30 S and 50 S subunits were collected and the subunits were precipitated by low-speed centrifugation (30 min at, 10,000 g) after the addition of 10% (w/v) polyethyleneglycol 6000 (Expert-Bezanpon et al., 1974). The pelleted subunits were dissolved in buffer 1, and stored at - 70°C. Lithium chloride-cores and split proteins (SP) were prepared as previously described (Homann & Nierhaus, 1971). Individual ribosomal proteins were prepared, and their homogeneity verified by 2-dimensional electrophoresis a.3 described by Spillmann et al. (1977). (d) MethyZation in vitro The specific methyl group acceptor capacity (methyl groups per particle or protein molecule) of ribosomal subunits or ribosomal proteins was determined from the results of assays made at 3 to 4 concentrations of acceptor in a range in which the extent of methylation wss proportional to the amount used. Incubation mixtures (25 ~1) contained 80 mM60 mM-ammonium chloride, 6 mu-j3Tris*HCl (pH 7.8), 10 mM-magnesium acetate, mercaptoethanol, 20 ~M-[naethyZ-3H]-S-adenosyl-L-methionine (Amersham; 10 Ci/mmol), 10 ~1 of S-150 enzymes and, as indicated, up to 0.3 AaBOnm unit of crude extracts or protein solutions. Tubes were ribosomal subunits or up to 0.03 A230nm unit of ribosomal incubated for 1 h at 37”C, and the reaction wae stopped by addition of 2 ml of cold 5% trichloroacetic acid. Incorporation of [3H]methyl groups was measured in reaction samples filtered on nitrocellulose membranes (Sartorius) either directly (RNA + protein methylation) or after heating for 20 min at 95°C (protein methylation). The filters were washed, dried under an infrared lamp and counted. RNA methylation was calculated as the difference between total and protein methylation. For the oalculation, 1 Aseon,,, unit of 50 S subunit is equivalent to 30 pmol.

(e) Polyacrylamide (i) Non-labelled

gel electrophoresis of RNA

RNA

Integrity of RNA $ Maeba (1973).

in purified

30 S or 50 S particles

was chocked

by the method

of C&i

Tot+d RN.4 \va.s prepared by grinding ~vllolr cells \vitll ahlmina. rxtractitq tile wll past,{* \Vit,ll buffer I, and deproteinizing the (axtract \rith plwnol. A4ftt>r pwcipitation with c%lranol at -20°C in tlw presence of 100 mal-sodiuln cllloridc. tlw pelleted RNA IVHS dissolvcti ill (ct,s/tnirl per A,,,,, Iltrit) was rnwtsliretl. IO mwTris.HCI (pH 5.2) its 14(’ specific activit’y gel accorditlg to t,llcs and a samplr ~‘as snbmit,tcd tot~lcctlopllorrais in a, Z?.i’?,p 014-acrylamide the gel was rrlt int,o 2.mm slices. l)roccdnrr nf Weinberg et al. (I RBi). After electroplloresis \Vlriclr were elutjed overnigllt at, WY ill I ml of 600 mwsodium cllloridc. 60 rnM-sodiutn citrate (pH 7.2). Mcasnwrnc~nt of tlw absorptiotr of tile clnatrs at 260 tlm determined tlw positjions of migration of I6 8 and 23 S KNAs. To mea,sIw(x tlleir radioacti\,ity. 0.5.ml peaks wow samples of eluates corresponding to tlw tops of the Iii S and 23 S rll,NA applied to glass-fihrc filters, \I-hich were drird and counted. (f) Reconstitution

procedures awl f wnctional tests

The preparation of RNA and t,otal proteins (TP 50) of 50 S subunits. and total reconstitution experiments were done as previously described (Dohme & Nicrhaus, 1976). The specific activities of the reconstituted particles were determined in the fragment assa) a.nd in poly(U)-directed 1“C]polyphel~~lalarlit~e synthesis: all assays were carried out in a range of ribosomr concentrat,ion itI M.hiclI ttlcrtl is a linear dependencr of measured activity OII the amount of 50 R subunits. Whrrl ttrr prptidyl t,ransferase activit.\of ribosomcs cotltaining [3H]mettlyl groups introduced by methylation in r6w or ire &ro was assayed. separation of the 3H-1abelled product of ttle fragment assay (Ac-Len-puromycin) from the “H-labelled ribosomes required the following modification of the standard experimental procedure: the reaction, performed in a volume of 0.225 ml, was stopped by the additiotl the of 0.1 ml of a solution of chloramphenicot (I mg/ml) in WV0 ethanol. After pelleting ribosomes (3 min at) 10,000 g). 0.25 ml of eactl supernatant \vas takrtr. clxt,ract,rd \vit)ll ethyl acetate, and thcl extracted radioactk-it,y countpti.

3. Results (a) Sedimentation behaviour and isolation of the large ribosomal subparticle (E-56’S) formed during incubation of E. coli in the presence qf ethionine Preliminary analyses of the sedimentation behaviour of ethionine ribosomal subunits (Cheng $ Harbman. 1968: Beaud & Hayes, 1971a,b) led to the conclusion t,hat they were indistinguishable from normal subunits in the presence of 10 mMmagnesium but possessed lower sediment)ation coefficients than the lat,ter in a medium containing 0.1 mM-magnesium. A more detailed study of the sedimentation characteristics of E-50s subunits has shown that they behave differently from normal of magnesium and has provided 50 8 subunits at, both high and low concentrations t,he basis for their isolation on a preparative scale. TWO procedures have been used to reveal t)he sedimentation properties of E-50s subunits in ME-mixtures: differential labelling using a 3H-labelled amino acid 14C-labelled amino acid mixture mixture during the first phase of cell growth and a during the ethionine phase (see Fig. 1). or specific detection of non-labelled E-50s subunits by methylation in vitro. In this case, the sedimentation behaviour is accurately represented by the distribution of specific methyl group acceptor capacity (nwthyL3H cts/min incorporat’ed per =I 260nm unit of ribosomes) in sucrose gradients and not by t,he distribution of total methyl group acceptor activity. The calculation of the .s value was made according to MacEwen (1967). Both met,hods showed that E-50s particles in the presence of 10 mM-magnesium possess a significant’ly lower nedimentation coefficient (44 S) than normal 50 S ribosomal subunks. As shown in Figure 2, the sedimentation properties of E-50S particles are more sensitive than those of normal 50 S subunits to variations in magnesium concentration.

ROLE

OF

METHYLATION

IN

50

S RIBOSOMES

379

In these experiments normal 50 S subunits were compared with ME-50s particles isolated from ethionine-treated cells and both types of particles were detected by their ultraviolet absorption. At 0.4 mM-magnesium (Fig. 2(c) and (d)) the E-50s particle appears as a peak at about 40 S with a shoulder at 30 S, and in the presence of 0.1 mM-magnesium (Fig. 2(e) and (f)) as a peak at 30 S wit.h a shoulder in the 21 S region. Furt’her analyses carried out at intermediat,e concentrations of magnesium failed t,o reveal additional components and sho\+.rd t’hat the proportions of 50 S, 40 S. 30 8 and 21 S species found in sucrose gradients depend on the magnesium concentration used. Therefore. zonal cent8rifugation in the presence of O-1 nmmagnesium was used as an isolation procedure : particles sediment,ing at, 50 S (normal 50 S subunits) and 30s (E-50s particles) were collected and t)heirspecific in zitro 13H )methyl group acceptor capaciGrs were found to be 0% and 4.i 1.respc*ctivrly, whereas t hat groups per particle. This result reveals of the original ME-50s particles was 1.8 methyl an excellent separation of normal 50 S subunit,s and E-5OS particles. and shons that, by this procedure large amounts of the latter can readily be prepared in a highly purified state. However, when its integrit,y was testrd by t,he met.hod of C&i & Maeba (1973), 23 8 RNA isolated from purified E-50s particles was found to contain numerous hidden breaks? although the same test applied to 23 S RNA isolat#ed from the original ME-50s subunits showed it to consist txssentially of intact, 23 S molecules (results not shown). Extensive nucleolytic cleavage of their 23 S RNA therefore takes place during t,he purification of t,he E-50s partic& This makes their 23 S R,?;,I

Direction of sedimentation 2. Sedimentation of &lE-ribosomes at various magnesium concentrations. Normal 50 S subunits ((a), (c) and (e)) or ME-SOS subunits ((b), (d) and (f)) were dialyzed against a 600-fold volume of a buffer containing 10 miw ((a) and (b)), 0.4 rniw ((c) and (d)) or 0.1 rnM ((e) and (f)) magnesium as indicated in the Figure (other ions were as in buffer 1). Dialyzed units of ME-SOS or 1.5 Azeonrn units of normal samples (3 A,,,,, 50 R subunits, respectively) were applied to 12.ml linear sucrose gradients (6% to 20%) of the same constitution as the dialysis buffer, which were then centrifuged for 15 h at 50.000 g,, in an SW40 rotor. FIG.

380

*J.-H.

ALIX,

D.

HAYES

AND

K.

H.

NIERHAUE

unsuitable for use in reconstitution experiments. Nonetheless, t)he rase with which normal and E-50s subunits can bc separated by sedimentation in O-1 mM-magnesium makes this a valuable method for their preparation. (b) The protein wmposition

of E-50&’ particlex

Two-dimensional electrophoretic analyses (Fig. 3) of the protein complement of purified E-50s particles prepared by sedimentation in the presence of 0.1 mMmagnesium (section (a), above) revealed the complete absence of L16, the presence of traces only of L6, of reduced amounts of L25, L27, L28, L30, L32 and L33, and of normal amounts of all other 50 S proteins; the presence of normal amounts of L7/L12 and of reduced amounts of L33, proteins which are not present’ in Figure 3, waH

;(b)

-(a);

FIG. 3. Protein pattern of E-60s subunits Proteins extracted from 10 Aaeon,,, units of phoresis (Roth t Nierhaus, 1976). (a) ME-60s subunits; (b) M-SOS subunits; units. M-SOS are the particles collected in the purify E-SOS subunits.

$I)

purified particles

in the presence were subjected

of 0.1 mM-magnesium. to 2-dimensional electro-

(c) E-60s subunits; (d) mutant prm-1 60 8 sub60 S region of the zonal sucrose gradient used to

ROLE

OF METHYLATION

IN

60 S RIBOSOMES

381

verified in similar analyses, in which the duration of migration in the first dimension of electrophoresis was reduced. It may be noted that contaminating 30 S proteinst, in particular S9jSl1, which migrates to a position just below that occupied by L16, and 87. which is situated immediately to the right of L6, are present’ in larger amounts in the purified E-50s particles than in the ME-50s subunit preparation from which they were purified (compare Fig. 3(a) and (c)). The absence of L16 from E-50s particles isolated in the presence of 0.1 mM-magnesium was confirmed by comparative CM-cellulose chromatography of proteins released from these particles and from prm-1 50 S subunits by washing with high concentrations of lithium chloride. Under the conditions used (see legend t)o Fig. 4), proteins Lll and L16 and some others are expect,ed to be present in the split fraction (Homann & Nierhaus, 1971). As can be seen in Figure 4(a), proteins released from t’he E-508 particles do not contain a peak

0.2

0.15 ;

0.1

z

0.05

: x i :

0.3

b) Ll6

0,2 0-I

Conductivity

(mmho)

Fro. 4. CM-cellulose chromatography elution profiles of ribosomel protein fractions. Ypht proteins were obtained from purified (0.1 mM-magnesium) E-50s subunits and 60 8 subunits of mutant prm-1 by incubation with 0.8 M and 1.3 M-lithium chloride, respectively, as described elsewhere (Homann & Nierhaus, 1971). RNA was removed from the split proteins by extraction with acetic acid and t,he proteins were equilibrated against buffer containing 10 mMTris .HCl (pH 7.5, O”C), 10 mix-KC1 and 6 M-urea, and applied to a CM-cellulose column (Whatman CM52; 1 cm x 16 cm) equilibrated with the same buffer and eluted with a linear 10 mM to 200 rnM gradient of KC1 in Tris . HCI (pH 7.5, O”C), 6 M-ur08. The gradient covered a conductivity range from 0.7 to 7 mmho and the absorbance at 230 nm (-o-O-) of eluate fractions is plotted against their conductivity. Ethionine-Lll was detected by its [3H]methyl group acceptor capacity using 10 ~1 of each fraction for in vitro methylation assays, taking advantage of the fact that this assay, as described in Materials and Methods, section (d), is not greatly inhibited by final urea concentrations as large as 2.4 M (Alix & Hayes, 1974). Duplicate determinations were made for each fraction, and the average of the values w&s taken (-•--•-), (a) SP 0.8 from 1800 A,,,,, units of E-60s subunits; (b) SP 1.3 from 4300 A,,,,, units of SOS subunits of E. co.%prm-1. ldentity of proteins was obtained by 2-dimensional gel electrophoresis analyses. t ME-SOS preparations are slightly assays of polyphenylelanine synthesis with the purified E-SOS particles.

contaminated by ME-30s subunits (2% estimated by without added 30 S subunits) and these are recovered

3X:!

J.-H.

ALIS.

Il. HAYES

ANI,

Ii. H. XIEKHAI'S

at’ the position expected for Llci. whereas the suhmethylated form of Lll is found at thtl expected position (2.3 mmho) by test’ing t,he metshy group acceptor capacity of’ column eluate fractions. Analysis by t\vo-dimensional gel electrophoresis of t,ho peaks in t)he neighbourhood of t,he absent I,16 rc>vcaleti tht> presence of L15, S9 and 1,“. but no trace of L16. The observation of reduced amounts of certain 50 8 proteins in E-50s particles prepared in 0.1 mM-magnesium could be an artefact associated wit,h the unfolding of these part’icles that takes place under these conditions. For t’his reason, similar twodimensional electrophoretic analyses were carried out on prot’ein samples prepared from purified E-50S particles isolated in the presence of 10 mM-magnesium (where they sediment at 44 S). The results obtained (not, shown) were identical t’o t,hostl observed with proteins of particles prepared in a medium containing 0.1 mnlmagnesium. VTe conclude that the absence of Ll6 and the partial absence of other prot8eins from E-5OS particles is not, an artefact of t’heir unfolding in t’he presence ot low concentrations of magnesium. In order to determine the protein composition of E-50s particles without submitting t,hem to any purification procedure. an experiment of the t,ype summarized in Figure 1 was carried out. Bacteria were labelled wit)h a 3H-labelled amino acid mixt,urc during the first and with a 14C-laballed amino acid mixture during the second stage of t,he experiment’. ME-50s particles were prepared and their proteins separated by t\\,o-dimensional gel electrophoresis. Protein Sp(JtS were cut out) of t,he stained gels. their contents of 3H and “C’ radioactivity wcrc measured and the ratio 3H/14CI was calculated for each spot,. The results (Table 1) show bhat four prot’eins. L6. L16. I,27 and L30. are present in greatly reduced amounts in the E-50S particles (or are present in modified form. migrat’e to abnormal positions and are therefore not) detected). The prot,ein composition of unpurified E-SOS particles determined in this \\‘ay xvas in good agreement \\.ith that, found by direct’ analyses of the protein complement of highly purified particles (SW above). (c) Activities

of ethionine ribosowal

subunits in vitro in vivo

before nrd rkfter methylatiott

The capacity of normal and ethionine ribosomal subunits to form 70 S ribosomes can easily be compared in zdro by test’ing the interaction of a mixture of the differrnt#ially labelled normal and E-5OS subunits with normal. non-radioactive 30 S subunits. Figure 5 present’s the result’s of experiments of this kind. Figure 5(b) shows that E-50s particles associate very poorly with normal 30 S subunits in the presence of 10 mM-magnesium. although a small amount sediment at about 60 8, forming a heavy shoulder on the leading edge of the 50 S peak. However. their interaction wit,h 30 S subunits is strongly enhanced at higher concentrations of magnesium (20 rnbt, Fig. 5(d): 30 InM, Fig. 5(e)) and is considerably stimulated by meth.vlation in, eivo (Fig. 5(c), 10 mM-magnesium). E-50s particles possess no peptidyltransferase activity after purification. As pointed out in Results. section (a), this could be due to the hidden breaks introduced into their 23 S RNA during their purification: the activities of unfractionated mixtures of normal and ethionine subunits isolated from ethionine-treated cells were determined (23 S rRNA of ME-ROS particles is mainly intact. see section (a), above). At various times during the incubation of cells in the presence of ethionine (phase 2. Fig. 1) samples were taken, ME-50s subunits were isolat,ed, their specific activities ill poly(U)-directed polyphenylalanine synthesis and in the fragment assay, and their

ROLE

OF

METHPLATION

IX’

50

383

S RTBOROMES

TABLE 1 Protein

3H

covnplement

of the large

ethioni)ae

ribosovnal

.subunit

‘4C

5

15

20

A sample containing 2 mg of unlabelled 50 8 ribosomal proteins and doubly was ribosomal proteins (465 x lo6 3H cts/min and 1.95 x 10 s 14c’ cts/min)

labelled

ME-508

submitted 1970).

to %dimensional polyacrylamide gel electrophoresis (Kaltschmidt & Wittmann, In this preparation, 3H and 14C labels were present in normal and ethionine proteins, respectively. Prot,eins were detected after electrophoresis by staining the gel slab with amide black. Samples were cut from stainod and neighbouring non-stained regions of the gel slab. Each sample was kept in loo-potassium-containing glass scintillation vials. in t,he prwence of 2 ml of Soluene 350 (Packard) for 3 tlavs at room temperature. After addition of 8 ml of scintillation fluid, samples were left, for 2 days at room temperature and counted (10 min). Corrections for the spillover of 14c’ and 3H ratlioact ivitiw WCPP applied and thr ratio of the isotopes was calculated for each protein.

specific i,r d-o methyl group acceptor capacities \vere measured. As can be seen in Figure 6(a). after t#hree hours of incubat,ion in the presence of ethionine their activity in the two functional assays has decreased by about 500,;. while their specific methyl group acceptor capacit,p has increased from 0.2 (zero t,ime) to about 24 residues per ME-subunit,. To deduce the specific activities of ribosomal E-subunits from those of the MEqubunit*s test,ed in bhese experiments it was necessary to determine the proportions of normal

and othionine

particles

in these

mixtures.

Two

approaches

have

beeu usetl to

obtain this information. In the first the decrease in the specific activity of [14C]uracillabelled 23 6 RNA synthesized during bacterial growth in the presence of methionine (phase 1. see Fig. 1) is measured during subsequent incubation of labelled cells in non-radioactive, ethionine-cont)aining medium (phase 2).

(,-01

X UIUI/S~3)

~+lAl(3OOlPDJ

3,,

ROLE

OF METHYLATION

IN

50 S RIBOSOMES

385

Figure 6(a) shows that half of the 23 S rRNA isolated from ethionine-treated cells is synthesized during their incubation in the presence of this analogue. In the second method, the methionine and ethionine contents of the same amounts of normal and ME-50s subunits were compared, after hydrolysis of the proteins and separation of the two amino acids on a Durrum amino acid analyser. It. was found that about one additional ethionine residue appeared in the ME-50s subunits for each methionine residue that disappeared (results not shown). These observations show that half of

I

Second phase

Ethionine (0)

Thtrd phase Methlonlne (b)

Fro. 6. Synthesis and activities of 60 S subunits during ethionine treatment of E. coli (a), and subsequent methylation in viva (b). (a) The spectic activities of ME-60S~particles~in~the sH-labelled fragment assay and in the poly(U)-directed [‘*C]polyphenylalanine synthesis (in the presence of normal 30 S subunits) were determined. At zero time (time of addition of DL-ethionine) they were 4.7 x lo3 3H cts/min per unit, respectively (lOOoh normal 60 S). At subA ssODmunit and 6x IO5 14C cts/min per A,,,., sequent times the measured specific activities are expressed as percentages of these dues. The two curves are superposed (-O-O--). During the first phase (M) of the culture (see Fig. I), RNA was labellsd with [‘4CJuracil (42 mCi/mmol; 0.1 pCi/ml) but no radioactive precursor w&9 present during the second (E) and third phases. At zero time (time of addition of DL-ethionine) the specific activity of 23 S RNA was 2.62 x 10s [‘%]uracil cts/min per A,,,,, unit (100% normal 60 S). At subsequent times the measured specific activities are expressed as percentages of this value (-o-a-). (b) The absence of differential degradation of E-SOS particles and M-SOS particles during methyl&ion in tiwo is shown (-•-a-). In addition, data from the poly(U)/polyphenylalanine synthesis experiment in the 4th column of Table 2A are presented (-O-O--), the specific activities being expressed as percentages of the value measured for normal 60 S subunits. FIQ. 6. 70 S couple-forming aotivity of ethionine ribosomal subunits. Bacteria were labelled with [S-sH]uridine during the methionine phase and with [U-14C]uridine during the ethionine phase of a standard growth experiment. The ethionine treatment was followed by a third stage of methylation in wiwo in the presence of unlabelled methionine for 30 min (see Fig. 1). 60 S ME-subunits isolated before and after the in viva methylation stage (6 AaBonm units in each case) were supplemented with a slight excess (4 A,,,,, units) of normal non-radioactive 30 S subunits, the magnesium concentrations of the mixtures were adjusted to those indicated in the Figure, and they were incubated at 37°C for 30 min and applied to 6-ml linear sucrose gradients (6% to 20%) prepared in buffer 1 containing the indicated magnesium concentrations. After centrifugation (2 h at 14O,OOOg.,; rotor SW60), the absorbance at 260 nm ( A (--e--e--) of the collected the 3H radioactivity (-O-O--) and the 14C radioactivity fractions were determined. Correctionv were applied for incomplete separation of 14C and sH radioactivities. (a) MX-606; (b), (d) and (e) ME-SOS + M-30& concentrations of magnesium 10 mM (b). 20 rnM (d), 30 rnM (e); (c) ME-SOS after in viva methylation + M-30& 10 mM-magnesium.

Wi

.1.-H.

.\J,lS.

I).

H.\YKS

.1X1)

I- (set Results, section (a)). After various periods of methylation in viw in the ~JI'~WWY~ of 1n/rlh!/l-3H]met,hionine. t,hc slower moving peak of E-5oS part,icles (30 S pwk in Fig. 7(b)) diminished propwssi\-elv (Fig. 7(c) and (d)) awl significant amounts of “H appeared in the 50 S region. indicating a shift of th(b lC5OS particles from t ht. 30 S t)c, the 50 S region of gradients (normal 50 S partic1c.s do not incorporate nwtlryl groups, as show1 in Tabh 2.B). If the: amounts of I”H]mct,h~4 group s incorporatc~tl into the RNA and prottailw of E-50s particles ww~~ determined srparatcly. methylation of protein \vas found to prrced(~ that’ of RSA : Figure 7((a) and (f) show that after ten minutes of methylation in vi?:o 3H radioactivity pwwnt in t,ht%50 S region was found only in ribosomal proteins but, that after 30 minut)es of mrthylation it/. /+VO both protein and R,NA itI thr, 50 S region \verc metllylated. Although extewive meth~lation-depentlrrlt recovery of activity bg EGOS particlcs occurs during th(>ir met’hplation itt viva. reactivation of purified E-50s part’icles b\. methylation itr hv has not bwn achieved. in spite of numerous attempts under a variety of conditions (methylation in the presence of S-adenosyl-r,-methionine? S-150 c’nzymes and ATP regenerat,ing syst’em, 50 S prot’ein Ll6: incubation of crude extracts of ethionine-treat)ed bacteria under conditiorls permitting methylation i,n. vitro and so on. result!s not shown). Several explanations for the failure of at’tempts to reactivat)? E-5OS part,iclcs by methylation in vitro are considered in the Discussion, section (d). Reactivation of E-50s particles in vitro was therefore studied using material reconstituted from normal rRNA and prot’eins of the E-5OS subunit,. (cl) ‘I’otnl

recomtitutio7t.

of 50 S .whuGt.s in vitro ftvtn

rornpotr~fwt8

of notmnl

and

E-5OS submits Particles reconstituted in vitro from normal 23 S + 5 8 RNA and the protein complement of E-50S particles were completely inactive in polyphenylalanine synthesis hut could be activated by addition of prot)ein L16 (filled symbols, Fig. 8(a)). but note by addition of any of a series of ot,her 50 S proteins (Ll, L7/L12, LS/L9. L13. 1%. L32. L33. normal or submethylated I,1 1 : open circles, Fig. 8(a)) to

388

3000

2oocJ

1000

-

Fraction

no. -

FIG. 7. Sucrose gradient sedimentation of ME-SOS subunits in the presence of 0.1 maa-magnesium before and after methylation in wivo. units of normal 60 S subunits prepared from cells taken at the end of the methionine 1.6 &mu phase (a) and 3 ~~~~~~ units of ME-60s subunits from cells taken at the end of the ethionine phase (b), after 10 min ((c) and (e)) and 30 min ((d) and (f)) of methylation in viva in the experiment described in Table 2, were dialysed against buffer 3 in the conditions described in the legend to Fig. 2, and sedimented on 12-ml 6% to 20% linear sucrose gradients prepared in buffer 3. Sucrose gradients were centrifuged at 4°C for 4.26 hat 160,000 g,, in an SW40 rotor and collected (-O--O--, in 30 fractions whose A,,,,, (--O-O-, (a) to (d)) and total sH radioactivity (c) and (d)) were determined. Their cold and hot acid-insoluble radioaotivities were then measured as described in Materials and Methods, section (d), using 200 ~1 of eaoh fraction. Protein methyl&ion (-A-A-) was measured as hot acid-insoluble radioactivity and RNA methyl&ion (--O--O--) was calculated 88 the difference between cold and hot acid-insoluble radioaotivities ((4 and (f)).

ROLE

OF

METHYLATION

I_

IN

~-1 o-o I

Isolated

022

50

S RIBOSCMES

389

0%

protem(s) odded/reconstitutm (A Lsc,nm units)

FIG. 8. Specific polyphenylalanine synthesizing activity of various reconstituted particles. Total reconstitution of 50 S subunits was performed (see Msterials and Methods, section (f)) with normal (23 S + 5 S) rRNA and E-TPBO prepared from purified E-50s subunits. Portions of the reconstitution mixtures were tested for poly(U)-directed polyphenylalanine synthesis. (a) The effects of addition to reconstitution mixtures of increasing amounts (Ass,,,,,,, units) of L16 (-•--@-), of one of the following proteins Ll, L7/L12, L8/L9, normalL11, undermethylo f mixtures of L16 and normal Lll (-n-m-), ated Lll, L13, LZO, L32 and L33 (-o-O-), and L16 and undermethylated Lll derived from the prm-1 strain (-- :\--a--). For the latter two curves abscissae refer to the amount of each of the two proteins added to reconstitution mixtures and not to their sum. All preparations of individual normal ribosomal proteins used in these experiments, in particular those of Lll and L16, were homogeneous as judged by 2. dimensional electrophoresis. The preparation of Lll of mutant prm-1 was contaminated with Ll and L6. (b) The effect of addition of increasing amounts (Az3enm unit’s) of normal Lll (--~3~-~--) to a reconstitution mixture containing normal 5 S + 23 S rRNA, ethionine TPSO, and a limiting amount of L16. The effects of addition of L16 (-a-e-) or of increasing amounts of a mixture of normal Lll and L16 (-D--Cl--) are also shown. As before for the curve Lll + L16 in (a), abscissae refer to the amounts of each of the two proteins added to reconstitution mixtures. Two independent preparations of normal (5 S + 23 S) rRNA were used in the experiments described in (a) and (b). Control reconstitutions with these RNA preparations and normal TP50 yielded 50 S particles with specific activities of (a) 3250 and (b) 28000 i4C cts/min per A,,,,, unit. 16

390

J.-H.

ALIX,

1). HAYES

:l?JI)

ti.

H.

NIERHAITF

reconstitmion mixtures. This confirms t,hat LIL is not, directly involved in t,he peptidyItransferase activity (Howard & Cordon, 1974; Ballest,a & Vazqurz, 1974: Bernabeu el ul., 1976) and that there was no contaminat,ion of Lll hy LI6 in t,he preparationused. However, maximum fixationof Ll6 is not reached even with a 3.%-fold Cxcess of Ll6 over all other components. and activat’ion of reconstituted particles by L16 was found to be increased about twofold by simultaneous addition of normal Lll to reconstitution mixtures (open squares, Pig. S(a)). This observation cannot, be explained by an incomplete complement of submethylated Lll in E-50s particles for the following reasons : (1) Table 1 shows that the M-5OS and E-50S components contain equal amounts of Lll.

of ME-5OS

subunits

(2) Absence of loss of Lll during purification of E-50s particles is demonstrated qualitatively by comparison of the t.wo-dimensional electrophoretic analyses of t,he proteins of ME-5OS, M-50s and purified E-50s particles (Fig. 3(a), (b) and (c)), and quantitatively by the values of their specific in vitro metfhyl group acceptor capacities (1.8. 0.6 and 4.0 methyl groups per particle. respectively : see Results. section (a)). This observation shows that, normal Lll competes with and functions more eficiently than E-L11 during reconstitution of 50 S subunits irr. ,uitro. Furthermore. it suggests that integration of L16 into the 50 S subunit structure may be influenced by the state of methylation of Lll. However. it can be seen that Lll mediates more effective integration of L16 only when this protein is present, in limiting amounts. In Figure 8(a). when the concentrations of Ll6 and Ll 1 are increased co-ordinately, the stimulatory effect, decreases in spite of t,he increased ratio of M-L11 to E-Lll. The same situation is seen in Figure 8(b) w h ere, for saturat’ing amounts of L16, the effect. of Lll is low and rapidly falls to zero. This agrees with results showing that a tenfold excess of L16 in a reconstitution assay with core particles lacking Lll and L16 can overcome the Lll stimulation (Schulz?, Hampl & Nierhaus, unpublished results).

4. Discussion (a) YropoytiorL of normal und ethionine subunits i?e ethionine-treated

E. coli ~310

Interpretation of many of the experimental results described here requires accurate estimates of percentages of normal and ethionine ribosomes in ethionine-treated bacteria. We have used two independent methods to obtain this information: both procedures show that after two to four hours of ethionine treatment bacteria contain normal 50 S and E-50s ribosomal subunits in equal amounts. Earlier estimates (Table 1; Alix & Hayes, 1974) were based on the separation of subunits in sucrose gradients containing 0.1 IUM-IXXLgneSiUnL As we show here, this method overestimates the amount of E-30s particles and underestimates that of E-50s particles, because the latter sediment at 30 S in the presence of 0.1 mM-magnesium (see Results, section (a)). (b) Isolation

of E-SOS particles

1968 ; Beaud & Hayes, 1971a,b) have desPrevious reports (Cheng & Hartman, cribed properties of E-50s particles, upon which it seemed possible to base procedures for their isolation ; namely (1) lack of association of E-60s particles with normal 30 S

ROLE

OF METH.YLATION

IN

60 8 RIBOSOMES

30 1

subunits (Fig. 5) under conditions optimal for 70 S ribosome formation, (2) sediment#ation of E-50s particles at 30 S in the presence of 0.1 mM-magnesium (Fig 2) and (3) sedimentation of these particles at 44 S in the presence of 10 mM-magnesium (results not shown). The use of these properties for the purification of E-50s particles has been t,ested on a preparative scale, separation of normal and E-50s subunits being c&mated in each case by measuring the increase in the specific irl. &TO methyl group of purified particles. For undetermined reasons. attempts to accept~or capacity purify E-5OS particles based on their inability to associate wibh normal 30 S subunits were unsuccessful. However, complete purification, as judged by a slightly greater t.han twofold increase in the specific methyl acceptor capacity of isolated E-5@ particles, was achieved by differential sedimentation of normal and E-50S subunns in the presence of 0.1 mM or 10 mM-magnesium (data not shown). This is the maximum expected increase for material initially containing equal amount,s of normal and cthionine subunits. Xlt hough pure E-50s particles and their proteins can be prepared in large amount,s by t~hesr methods, it has invariably been found tha,t the isolated particles contain onl!, fragmented 23 S RNA. DegradaGon of this RNA takes place during the isolation OI the E-50s particles, since the 23 S RNA of the ME-AOS particles used contains main]?indact RNA. The cause of the extreme sensitivity of the 23 S RNA of these particles to RNase attack is not known but may be related t.o the lack of ribose Y-OH methylation in this RNA4 (Brown 8: Todd. 1955). to the flexible structure of the E-56s subunit,, or to its lack of L16. Further attempts t,o isolate this RNA in the presence of 10 mw-magnesium or by t,he use of more stringent condit’ions (sterility. RNascs inhibitors, etc.) during E-50s subunit purification are in progress. However, thta protein composit.ion of E-50X particles i s not’ significantly influenced by the degrecb of int,t*prit.y of their 23 S RNA. as discussed in the following section.

(c) Protek

cornposition

of ethimine

particles

Table 1 shows the results of analyses of the protein compositions of unpurified E-508 particles which contain, as noted above, intact E-23s RNA. These particles possess a much smaller content of L16 and significantly lower contents of proteins L6, L27. L28 and L30 than normal M-50s subunits. These results were confirmed and extended by analysis of the protein composition of purified E-60s particIes in which prot,ein L16 was found to be completely absent. proteins LA, L25, L27, L28, L30, L32 and L33 to be present in reduced amounts, and all other proteins including L7/Ll2 and Ll 1 t*o be present in normal amounts (Fig. 3). It, can therefore be concluded that 23 S rRNA fragmentation during isolation of E-50s particles does not lead to protein loss (with the possible exception of L25, L32 and L33) and that the presence of reduced amounts of certain prot,eins in these particles is due to a defect in the 50 S subunit, assembly process. Similar analyses described in a previous report (Fig. 1, Alix 8: Hayes. 1974) revealed partial absence of proteins L6 and L30 but did not detect absence of Ll6. L27 or L28. This result was probably caused by the use of ribosomal proteins of relatively low specific radioactivity for electrophoretic analyses. The normal and ethionine forms of Lll are not significantly separated during standard two-dimensional electrophoret’ic analyses of ribosomal proteins (e.g. see Fig. 3 and Table 1). Their separation (Fig. 1: Alix & Hayes. 1974) was achieved previously by great,ly increasing the resolving power of t.he first dimension of elect,rophoresis.

392

.1.-H.

ALIS,

I).

(d) Biological

HAYES

ANI)

activities

Ii.

H.

qf E-5&‘+

NIlr:RHAllS

particles

(i) Bbsenee of L16 Three trivial

explanations

for the inactivity

of E-50s particles

can be excluded

:

(1) The presence of ethionine in 50 S proteins : E-30s particles possess considerable residual activity (data not shown) ; active 50 S subunits can be reconstituted in vitro from normal rRNA and total proteins of EGOS particles supplemented with L16 (Fig. 8); when ethionine-treated bacteria (E. coli EAl) are incubated in methioninecontaining medium their ethionine part’icles recover normal ribosomal properties without loss of ethionine (Beaud & Hayes, 1971~). (2) The presence of immature (precursor) forms of 23 S rRNA and 5 S rRNA or of an abnormal amount of the latter. The T, RNase fingerprints of E-23s and E-5S rRNAs contain normal amounts of the 3’-OH, and 5’-P terminal oligonucleotides of mature 23 S and 5 S rRNAs (unpublished results). Maturation of these rRNAs is therefore independent of their methylation. Determination of the 5 S rRNA content of unpurified E-50s particles of E. coli EAl and EA2 gave normal values (Beaud & Hayes, 1971a). (3) The presence of breaks in the 23 S rRNA of E-50s particles. Breaks present in the 23 S rRNA of isolated E-50s particles are introduced during their purification (see Results, section (a)). ME-50s particles contain mainly intact 23 S rRNA but their E-50s component lacks L16 (see Results, section (a) and Table 1). In addition, it has been shown (Kuechler et al., 1972) that the high molecular weight RNAs of E. co.2 ribosomes can be extensively degraded by pancreatic ribonuclease treatment of 70 S particles without significant loss of peptidyltransferase or polyphenylalanine synthesizing activity and hence a fortiori loss of L16. The absence of L16 in E-50s particles (Table 1; Fig. 3) explains their functional inactivity, since this protein is part of the peptidyltransferase site of the 50 S subunit (Dietrich et al., 1974; Moore et al., 1975). Other 50 S subunit properties unrelated to peptidyltransferase activity, e.g. association with 30 S subunits, and structural rigidity are altered in E-50s particles but it is not yet known whether these alterations are direct results of the submethylation of E-50s proteins and 23 S rRNA, or indirect results related to the absence of L16. It has been reported (Kazemie, 1975) that L16 is important for 30 S-50 S association and the results shown in Figure 5(b) support this idea. However, it should be noted that the results shownin Figure5(d) and (e) demonstrate that efficient association of E-50s particles which lack L16 is observed at high concentrations of magnesium (20 to 30 mM), suggesting that this protein has a role in the overall structure of the particle. Ultimately the absence of L16 (and that of L6. and the partial absence of several other proteins) in E-50s particles must be explained in order to understand the relationship between ribosomal protein and RNA methylation and the assembly of functional 50 S subunits. Some deductions concerning the importance of methylation of 50 S protein Lll, which contains nine of the 12 methyl groups so far detected in E. coEi 50 S ribosomal proteins can be made by comparing the protein complements and properties of E-50s particles and 50 S subunits of E. coli mutant prm-1. The latter, which is functionally active, contains a full complement of 50 S proteins (Fig. 3(d)), including the normal methylated form of Ll6 (Brosius, unpublished result) whose N-terminal amino acid is N-monomethylmethionine (Brosius & Chen,

ROLEOF

METHYLATIONINEiOS

RIBOISOMES

393

1976 ; Chen et al., 1977) ; but it also contains t#henon-methylated form of Lll. Assuming that protein Lll of mutant prm-1 is completely unmethylated, it can be concluded that methylation of Lll is unnecessary for the integration of L16 into the 50 S subunit structure in viva and for the peptidyltransferase activity of the complete particle when all other 50 S components are normal. The presence of unmethylated Lll in E-50s particles cannot’ therefore by itself account for the absence of L16. However, the reconstitution experiments presented in Figure 8 suggest that the integration of L16 in vitro is not completely independent of the state of methylation of Lll. Thus one effect of Lll methylation could be optimization of assembly of L16, but comparison of properties of E-50s and prm-1 50 S subunits suggests that the defect in 50 S ribosome assembly in ethionine-treated E. coli is also related to the absence of methylation of another ribosomal component (23 S RNA and/or perhaps t,hat, of L16 itself). (ii) EJ%ctR of methyl&ion in vivo on the properties

of E-50s particles

As well as leading to recovery of activity, methylation of E-50s particles in vivo counteracts their susceptibility to unfolding in the presence of low concentrations of magnesium (compare the “30 S” and 50 S peaks in Figs 7(b), (c) and (d)). Figure 7(e) and (f) shows that methylation of the proteins and 23 S RNA of E-50s is not simultaneous. The fact that methylation of E-23s RNA in E-50s particles takes place before this susceptibility is lost suggests that methylation of 23 S RNA is required for conformational stability of the 50 S subunit. (iii) E’ailure to reactivate E-50s particles in vitro Alt*hough ethionine particles are converted to active ribosomal subunits during methylation in vivo, attempts to simulate this conversion in vitro by methylation of ME-5OS particles combined with other treatments such as incubation under reconstitution conditions in the presence of L16, of normal TP50, etc., have been unsuccessful. Possible explanations for the failure of such experiments are: (1) the labilit’y of E-23s RNA in ME-50s particles, since the conditions used for methylation of ME-50s subunits in vitro (incubation in the presence of an S-150 extract for 1 h at 37°C) are likely to lead to nucleolytic attack of exposed regions of 23 S RNA in E-50S particles. The use of partially purified ribosomal RNA and ribosomal protein methylases in methylation studies in vitro should provide a solution to this problem. (2) Loss or inactivation of ribosomal RNAmethylases during S-150 preparation. This possibility is at present being investigated. (3) Lower efficiency of methylation of E-50s particles in, vitro than in viva: there is no methylation of 23 S RNA of E-50s particles in vitro (result not shown). Quantitative comparison of both procedures suggests accumulation of an inhibitor(s), possibly S-adenosgl-L-homocysteine. during methylation reactions in vitro.

Methylation of certain components is required for the correct assembly and normal functional activity of the 50 S subunit. Lack of methylation causes failure to complete a late step or steps in the 50 S assembly process and leads to formation of incomplete inactive particles which lack L36, contain reduced amounts of several other proteins. and possess structures in which 23 S rRNA is very accessible to

,l..H.

394

ALlX,

1). HAYES

ANI.)

h:. H.

KIERHAIIS

nucleolytic degradation. The absence of I,16 in thescb E-50?? part,icles oxplains t,ht!ir inactivity but cannot yet be accounted for in other t,han general terms. In part.icular, t#he roles of the methylation of 23 S rRNA, and of prot,eina Ll 1 and L16 itself. in t.hr insertion of this protein into the 50 S subunit structure, are not Yet understood. :It present only four E. coZi mutants are known in which tnet)hylation of 50 S ribosomal caomponents is modified. These are mutant prm-1: referred t#o earlier. which contaitls the unmethylated form of protein Lll (Colson & Smith, 1977): tnutjantS prm-2. in which protein L3 lacks an N5-methylglutamine residue present in wild type L3 (Lhoest & Colson, 1977); and two K12 mutants lBl0 and tB14. whose 23 S rRNAs lack, respectively, the single mlG and the single m5C residues normally present in this RNA (Bjiirk & Tsaksson. 1970; Bjark t Kjellin-Str%by, 1978). These mutants display normal growth rates and presumably possess normally active 50 S ribosomal subunits. The methyl groups lacking in their 50 S components are apparent.ly not essential for the assembly or activity of the large ribosomal subunit. Together t.hestx 12 methyl groups (protein Lll normally contains nine methyl groups. six in t,wo NEtrimethyllysine residues (Dognin & Wittmann-Liebold, 1977) and three in a singIt% iv-terminal trimethylalanine residue (Lederer et al.. 1977)) makeup nearlyhalf of tht. t,otal of 28 methyl groups so far det’ected in the RNA and proteins of E. coli 50 S ribosomal subunits (16 in 23 S rRNA, Fellner. 1969: ten in proteins L3 and Lll, see above; and one each in proteins L16 (Brosius & Chen 1976: Chen et al.. 1977) ; and L33 (Wittmann-Liebold $ Pannenbecker. 1976 : Chang et al.. 1976 ; Chen et aE., 1977)). Hence. t,wo explanations for the defective asxembl.vof 50 S subunits in ethionine-treated E. coli are possible : (1) A combination rRNA.

of effects of submethylation

(2) Absence of one or more essential methyl

of L3, TJl 1, Ll6.

L33 and 23 S

groups in L16, L33 and 23 S rRNA.

A 50 S ribosomal subunit precursor which accumulates in a mutant of E. coli has been found to possess properties very similar to those of E-50s particles: a sedimentation coefficient of 47 S, greater sensitivity of its hydrodynamic properties to magnesium concentrations than those of normal 50 S subunits, presence of the mature and not the precursor form of 23 S RNA, lack of proteins L16, L28 and L33 and of peptidyltransferase activity (Butler et al., 1978). Although preliminary studies suggest that the 23 S rRNA of this particle may be slightly undermethylated (MarkeJ et al., 1976), the degree of methylation of its components has not been extensively studied. This work forms part of a doctoral thesis (Doctorat 6s Sciences) to he submitted b), one of us (J. H, A.). We thank Dr H. G. Wittmann for criticisms and discussions and Drs J. Brosius, H. Teraoka and H. Schulze for help and advice. One author (J. H. A.) thanks the European Molecular Biology Organization, the Deutsche Forschungsgemeinschaft. and the Centre National de la Rechercho Scientifique (CNRS) for fellowship support during this work. Research in the Laboratoire de Chimie Cellulaire (Paris) was supported by grants from the CNRS (E.R.lO1) and the DBlPlgation GBn&ale $. la Recherchra Scientifique et Technique (ACC no. 7570199). REFERENCES Alix, J.-H. & Hayes, D. H. (1974). J. Mol. Bid. 86, 139-159. Beaud, G. & Hayes, D. H. (197la). Eur. J. Biochem. 19, 323-339. Beaud, G. I% Hayes, D. H. (1971b). Eur. J. Bhchem. 20, 525-534.

ROLE

OF METHYLATION

IN

50 S RIBOSOMER

39.5

Ball&a, J. P. G. & VBzquez, D. (1974). FEBS Letters, 48, 26tC270. Bernabeu, C., VLizquez, D. 8: Ballet&a, J. P. G. (1976). Eur. J. Biochem. 69, 233-241. Bjijrk, G. R. & Isaksson, L. A. (1970). J. Mol. Biol. 51, 83-100. BjGrk, G. R. & Kjellin-St&by, K. (1978). J. Bacterial. 133, 499-507. Brauer, D. & Wittmann-Liebold, B. (1977). FEBS Lettera, 79, 269- 275. Brosius. J. & Chen, R. (1976). FEBS Letters, 68, 105.-109. Brow), D. M. $ Todd, A. R. (1955). NucZ. Acids, 1. 409. Hrowr~. .I. L. (1973). Rio&m. Hiophys. Acta, 294, 527 -529. Butler. P. I)., Sims, P. F. G. & Wild, D. G. (1978). Biochem. J. 172. 503-508. C&i, H. & Maeba. P. Y. (1973). R&him. Biophys. Acta, 312, 337.-348. C’hany. C’. N. & Chang, F. N. (1974). Nature (Lon,don), 251. 731 -733. Chang. (:. R’. & (‘hang, F. N. (1975). Biochemistry, 14. 468-477. C’hanp. C’. N., Schwartz, M. k C%arq. F‘. X. (1976). Hiochem. Rioph,yls. Rex. Gommuu. 73. 233-23!). Chng. F. S., Chang, P. N. & hk. W. K. (1974). J. kzcteriol. 120, 651-656. Chen, K. & Chen-Schmeisser. T:. (I 977). Proc. lvat. dcud. Scl:., c:.S.A. 74, 4905-4908. C’lwn. I