Ribosomal proteins L7L12 of Escherichia coli localization and possible molecular mechanism in translation

J. Mol. BioE. (1983) 163, 553-573

Ribosomal Proteins L7/L12 of Escherichia Localization

and Possible Molecular

Mechanism

coli

in Translation

W. M~~I,I.)sK, P. I. f+'HKIEK, ,J. A. MAASSES. A. Z.~S’I’E:>~.\ E. &'HOP, H. RE:ISAI.I)A Department of Medical Biochemistry University oj Leiden, Sylvius Laboratories Wassenaarseweg 72. 2333 AL Leiden, The Netherlands

Department of Biochemistry University of Leiden, Wassenaarseweg 2333 AL Leiden, The Netherlands (Received

4 January

64

1982, and in revised form 16 August

1982)

Experiments were performed in order to determine the minimal requirement for the proteins L7/L12 in polyphenylalanine synthesis and elongation factor EF-Gdependent GTP hydrolysis. Via reconstitution, ribosomal particles were prepared containing variable amounts of Li’/L12. The L7/L12 content of these particles was carefull? determined by the use of 3H-labelled L7/L12 and by radioimmunoassay. The activity of the particles was determined as a function of the I,7/1,12 contrnt,. Our results show that only one dimer of L7/Ll2 is required for full activity in EFG-dependent GTP hydrolysis. On the other hand, two L7/L12 dimers are required for polyphenylalanine synthesis. In addition, we have determined the relation between the number of Li/LlS stalks, as observed by electron microscopy, and the L7/L12 content of the 50 S particles. Our interpretation of these results is that each ribosomal particle possesses two L7/L12 binding sites, each site being involved in binding one dimrr. Binding of L7/L12 dimer in one site gives rise to formation of the L7/L12 stalk. whrrcas binding in the 0tht.r site has no effert on thrt number of visiblr stalks.

1. Introduction The large subunit of ribosomes of Escherichia coli contains the acidic proteins L7/Ll2 with unique properties suggesting a special function in translation (Miiller, the two proteins are identical 1974: Weissbach $ Pestka, 1977). St ructurally except that the amino-terminal serine of L12 is acetylated in L7 (Terhorst et al.. 1973). Since 50s ribosomes have four copies of L7/Ll2 (Subramanian, 1975: Hardy, 1975) and protein L7/Ll2, detached from the ribosome, strongly dimerizes (M6ller et al., 1972 ; Wong & Paradies, 1974, asterberg et al., 1976), the functional 553

554

\v.

MO1 444 I PR F7' I .-II.1

(Init in protein synthesis is often assumed to act uirr two L7/Ll2 dimers. Support that the dimer is the active form of the protein on the ribosome is derived from c*lretuic~al moclitic*ation (Koteliansky it (11.. 1!)7X: (‘aldwell 11ft/l.. 1978) and crosslinking studies of the LS/Ll2 dimer (Maassen et al.. 1981). In solution two L7/Ll2 et al., 1977 : Gudkov et al., 1978; Pettersson & Liljas, dimers bind to LlO (osterberg 1979). t,he complex in turn being bound specifically to 23 S RNA (Pettersson, 1979 ; Dijk et ol.. 1979: Gudkov et nl.. 1980). There is also evidence that on the intact ribosome L7/L12 and LlO form a specific substructure (Schrier et al.. 1973: Highland & Howards, 1975: Stiiffler et al., 1974; Pettersson, 1979: Marquis & Fahnestock, 1980) with the carboxyl-t,erminal portion of L7/L12 pointing away from I,10 towards the cytosol (Van Agthoven et al., 1975: Marquis & Fahnestock, 19X0: Marquis et al.. 1981). Immune electron microscopy has revealed proteins L7/L12 to be present in a stalk extending from the large subunit] of procaryotic ribosomes (Strycharz et al., 1978: Marquis et nl., 1981 : Kastner et al.. 1982). Cross-linking studies on L7/Ll2 suggest a shifted parallel alignment of the two polypeptide chains of the dimer. with a free amino-terminal section of the L7/L12 dimer attached to protein LlO (Maassen it nl.. 1981). On t.he functional side. it looks as though the proteins L7/Ll2, LlO and Lll are involved in an important structural change in the ribosome at the expense of cleavage of GTP. As a result, ribosomes deprived of L7/L12 have a low activity in reactions that are dependent on the presence of initiation, elongation or termination factors (Miiller, 1974: Matheson et crl., 1980). Therefore it is of paramount importance to know whether the two dimers of L7/L12 are in an equivalent position on an active ribosome. In t,his paper we demonstrat,e that in contrast to uncoupled EF-G-t-dependent ~~‘l’l’asr. which reciuires the presence of one dimer of L7/L12. two dimers of L7/L12 art’ needed for efficient s,vnt,hesis of polyphen,vlalanine. In addition the number of rihosomal particles having a distinct, stalk in elec*tron micrographs varies as a func+ion of the degree of reconstitution so as to predicbt that. at least in the 50 S sllbunit,. one dimer is directed with its (‘-terminal part towards the cytosol, whereas the other dimer has t,his 1)art bent inwards to the main body of the ribosome.

2. Materials

and Methods

(a) (‘h~micds [y-“2P]W’P.

( 3H]WP.

13H]GMPP(‘P

and (3H]GMPPNP were products of the Radiochemical C‘entre. Amersham, I1.K. Non-radioactive GMPPCP and GMPPNP were pur(~hased from Koehringer. Mannheim. G.P.R. The purity of these products was checked on PEI-cellulose plates (Mackery and Nagel) developed with 1 M-potassium phosphate (pH 3.5 : RFvalues : GMPPCP, @5 : GMPPNP, 035). The non-radioactive GMPPCP was pure as shown by u.v. light. However, the non-radioactive GMPPNP was purified on DEAESephadex A25 according to Eckstein et al. (1971). Other nucleotides were obtained from Sigma, Saint Louis, U.S.A. Concentration of nucleotides was determined by measuring the a.bsorbance at 260 nm (6 = 11.8 x IO3 M-’ cm-‘).

L7/L12

PROTEINS

355

[3H]- and [‘4C]formaldehyde and [3H]NA3H, were purchased from New England Nuclear, Boston, U.S.A. Total tRNA or tRNAPhe (Boehringer) both of Escherichti coli was charged with [ 3H]phenylalanine (Radiochemical (:entre, Amersham). essent*ially according t,o Ravel & Shorey (1971), lyophilized and stored dry at - 30°C. Poly(U) was obtained from Roehringer. Fusidic acid was a generous gift from Leo Pharmaceutics, thiostrepton from Squibb, Princeton. V.S.A. Nonidet P40 was a gift from Servo B\‘. Delden. The Netherlands. (b) Preparation of ribosomal cores and split proteins 70 S ribosomes from E. coli MRE 600 were prepared according to Gesteland (1966). in standard buff& (20 rnM-Tris-H(‘I (pH 7.1). 10 mM-Mg(~~AL\c),, 50 rnM-r\‘H,(‘l and 6 rn~~-$ mercaptoethanol) and washed through a I.1 M-sucrose cushion in standard buffer made III) to 1 M-NH,CI in case 70 S ribosomes were used in a functional assay. Ribosomal subunits were separated as described earlier (Miiller et al.. 1970), except that the buffer used in zonal centrifugation was replaced by standard buffer containing 1 rnM >lg(OAr),. Ribosomes and ribosomal subunit,s \vere stored as pellets or as a solut,ion in standard buffer at, -30°C. The concentration of ribosomes was determined by measuring the absorbance at 260 nm. One =1260 unit was taken to represent 25 pmol of 70 S, 39 pmol of 50 S or 69 pmol of 30 S particles. 50 S core particles deprived of L7/Ll2 were prepared according to Hamel et al. (1972). The various 50 8 core preparations used are defined as follows. P,, cores, 50 S ribosomes extracted twice with NH,(‘l and ethanol at 0°C: P, -30 cores, P, cores ext,racted twice at 30°C’: P,. 3, cores. P, rores extracted twice at 37°C: P,, cores, 50 S ribosomes extracted twice at 37°C’. The concentrations were determined by measuring the absorbance at 260 nm. The proteins split off during the ammonium chloride!ethanol wash procedure are defined as follows: SP,. the protein split off during the preparation of the P, cores: SP,,, the protein split off during the preparation of the P,, cores. Split protein fractions were concentrated 4 times by evaporation at ambient temperature under reduced pressure. The split proteins wt’re then dialyzed against ().I?,, formir acid, 6 mM-2-mercaptoethallol. Ipophilized and stored at -30°C. Just before use, the fractions were dissolved in 1% acetic acid. Cores were dissolved in a small volume of standard buffer, dialysed overnight against standard buffer and after addition of glycerol to P5Q0 (by volume) stored in portions at -3O’(‘. PO cortbs anti cores were subjected to 2-dimensional polyacrylamide gel electrophoresis as described PO-30 by Kaltschmidt & Wittmann (1970). Judging by the electropherogram and the sensitivity of t)he (‘oomassie blue staining, protein L7/Ll2 was absent in the various P-cores. A more reliable procedure in the form of a radioimmunoassay gave a maximlrm estimate of (1.1 equivalent of L7/L12 in the P, and P,- J0 cores. (c) Preparation

of nor-rdioactive

proteins

The proteins L7 and L12 were extracted from 70 8 ribosomes and purified as described by Miiller et al. (1972). with the exception that the original extraction of A-protein was replaced by the ammonium chloride/ethanol extraction of SPO proteins as described by Hamel et al. (1972) and that 6 mM+mercaptoethanol and (bl rnM-El)TX were present throughout all stages of preparation and during storage. Desalting after DEAE chromatography was done in I”/, acetic acid/6 m&r-2-mercaptoethanol. Before separating L7 from L12, the protein was dissolved in standard buffer containing 5 mM-dithioerythritol and kept at, 30°C for IO min t,o reduce oxidized methionine (Gudkov & Behlke. 1978; Caldwell et al., 1978). In earlier work fl-mercaptoethanol was not always present during the fractionation. desalt iny and storage. resulting in possible partial oxidation of’ methioninc residues. Sll(*tl preparations are indicated in the text as L7/Ll2 “not reduced” and gave maximally 2 to 2.5 copies of L7/1,12 bound to P, cores. Unless indicated otherwise. the experiments refer to L7/Ll2 samples that were kept reduced and bind 4 copies as described. The amino-terminal fragment I-73 and the carboxyl-terminal fragment 74-120 of L7/L12 were prepared as

556

w. Miil,LER

E7’ dl,

described by Van Agthoven et nl. (1975). Anti-l@ molecules of I,7 or I,12 were prepared according to Stiiffler & Wittmann (1971). The protein L7 or L12 gave one band on pH 3.5 gel electrophoresis, sodium dodecyl sulphate/gel electrophoresis and isoelectric focussing in the pH range 3 to 10 (MGller et al.. 1972). Two-dimensional gel electrophoresis according to Kaltschmidt & Wittmann (1970) also gave one spot. Protein concentrations were determined according to Lowry it ul. (1951), using insulin as a standard. The concentration of the standard solutions of L7 for the radioimmunoassay was also determined by amino acid analysis using norleucine and n-amino-fl-guanidino-propionic acid as internal standards for calibration (Beckman Instruments). The concentration thus found agreed with the value found with the Lowry method. (d) Preparation

of radioactive

riboaowml

proteins

L7, L12 and their fragments were labelled essentially as described by Amons & M6ller (1974). [ ‘H]L7 : 14.2 Ci/mol (containing 1 methyl group per 7 molecules of protein); [ 14C]L12: 7 Ci/mol (containing 1 methyl group per 9 molecules of protein); [3H]L7NTF: 85 Ci/mol (containing 1 methyl group per 12 molecules of protein); ( 14C]L7CTF: 13.4 Ci/mol (containing 1 methyl group per 1 molrc*~~lrs): and 1“H jLl0: 35 (‘i/mol (csontaining 1 methyl group per 3 molecules of prot&). [“H ]I,7 \vit)h high spew. act. for the radioimmunoassay was labellrd as described above using 25 m(‘i[ 3H ]I%aBH, (l(b 5 (‘i / mot) and nori-radioactivc formaldehyde. The radioac+ve prot,eins were stored in 5 rnbl-H(‘I at) - 30”(‘ or in 20 mMTris.H(‘l (pH 7%). IO mu-Mg(‘l,. 60 m&l-NH,(‘I, 6 m~~-~-~nc~~~~al~tocthanoland (bl mhlEDTA. In the case of protein LlO. 6 M-urea was also present. (e) Elongation

factors

Elongation factors EF-G. EF-Tu and EF-Ts were prepared according to Arai et nl. (1972) and were pure according to analysis on polyacrylamide gels containing SDS. Purified factors were stored as a solution in 20 mw-Tris. HCI (pH 75). 10 mM-Mg(OAc),, 6 miv-2-mercaptoethanol and 25% glycerol at -30°C. (f) Assay for the binding

of radioactive protein

to ribosomal

cores

325 pmol of P,, cores, 650 pmol of 30 S ribosomes and 3H or 14C’-labelled L7 or L12 were inrubated for 5 min at 37°C in 325 ~1 of 2 x concentrated assay buffer (100 mxl-Tris. HCl (pH 74). 20 m~-Mg(0A~~),. l(H) mmNH,(‘l. and 11 mM-2.mcr~al,toethanol). A portion of 25 pl was t,akrn off t,o assay for I-3-t :-tfrpendmt (:Tl’asr. To the remaining 300 ~1 an quaI volumc~ of water was added and thcb mixture was c*entrifuged for 1 h at 40.000 revs/min at, lO”(’ in a Beckman SW 50.1 rotor equipped with 0% ml adaptors. The bottom of the cellulose nitrate tube was cut off and the pellet dissolved in 05 ml of 2 mw-Tris. HCl (pH 75), @5% SDS and counted in 10 ml of Instagel (Packard Instruments). The recovery of A,,, units (usually 85%) was determined from the Az6,, prior to addition of Instagel. The amounts of protein bound to the cores as given in the Figures and Tables have been corrected for the amount of protein bound to 30 S ribosomes in isolation. (R) Jktprrrtirtntiort

of tbu nmorrnt

of IZ/J,J2

it/ recor/utitutud

cores by rodioinLrntrnoflsscc?ls~s(l?~

0.5 nmol of cores and proteins as indicated in the legend of Fig. 2(b) were incubated in 05 ml of 2 x concentrated assay buffer (50 mM-Tris HCl (pH 7.4), 10 mM-Mg(OAc),, 50 mi%-NH,Cl and 7 mM-2-mercaptoethanol) for 5 min at 37°C. After adding @5 ml of water, the mixtures were layered on 4 ml of 10% (w/v) sucrose in assay buffer and centrifuged for 15 h at 35,000 revs/min at 2°C in a Beckman 50Ti rotor. The pellets were dissolved in assay

L7/Lld

--:x,,

PROTEINS

buffer and after protein extraction of a certain number of AZ60 units, the Li’/Ll2 was determined by radioimmunoassay. (1-l) h’xtrrrction

of protein

from

ribosomes

for

content

the rtrdioivvvvvv vrnoo,s.sv~!y

To 50 ~1 of a solution of 1 to 10 A,,, units of ribosomes or ribosomal particles, 25 ~1 of extraction buffer (02 M-methylamine.HCl (pH 7), 8 M-urea and 4 M-LiCl) were added. After adding 5 ~1 of a solution of pancreatic RNase (2 mg/ml). the samples were incubated for 5 min at 37°C. Then another 25 ~1 of extraction buffer was added and the samples were left for at least 1 h at 2°C. The L7/L12 content of 1 to 4 ~1 of this protein extract was assayed b) radioimmunoassay, adding an equal volume of 2 x diluted extraction buffer to the standard L7 sxm pies. (i) Radioimmunoassay Reaction mixtures of 100 ~1 contained @l M-Tris.HCl (pH 8), @12 M-NaCl. 2y0 Se&x NNP 10 (corresponding to Nonidet P40) or Triton X, 250 pg serum albumin, 0.01 to 0.15 PL# of L7 or 1~12,til pg [3H]L7 (4.2 lo6 disints/min per pg) and 3 rg of anti-L7 IgG (enough to precipitate 50”/: of the amount of [3H]L7), which was precipitated with an excess of goat anti-rabbit IgG. Before adding the antibody, the mixtures were thoroughly mixed and left for IO min at 37°C’. Then, in succession. 20 kg of carrier rabbit non-immune y-globulin (Calbiochem) and an excess (1.5 unit) of goat antiserum to rabbit IgG (Calbiochem). dissolved in @l M-Tris.HCl (pH 8), @12 M-NaCl, were added and the resulting react,ion mixtures (135 ~1) were incubated for another 30 min at 37°C. After cooling to 0°C. samples of 100 ~1 were layered on @5 ml of an ice-cold buffer containing @l M-Tris. HCl (pH 8). 012 MNaCl. 1 M-sucrose and 1% Triton X-100 in Eppendorff tubes. After 4 min centrifugation the supernatants were carefully sucked off and the pellets were dissolved in 200 ~‘1 of 0.25 w NaOH, 0596 SDS. After neutralization with 100 ~1 of @5 M-HOAc, samples of 200 ~1 were counted in Instagel (Packard Instruments). A blank obtained by chasing the radioactive protein with a large excess of non-radioactive protein, was subtracted from all values. (j) Recovrstitutiovr

Prior to each functional assay, a preincubation step was performed to reconstitute the ribosomal cores and proteins. Reaction mixtures of 25 ~1 contained 100 mM-Tris. H(‘l (pH 5.4). 20 mM-Mg(OAc),. 100 mu-r-SH,(‘l and I4 mm2-mercapt,oethanol (2 x (WIcentrated assay buffer). ribosomes or 50 S ribosomal cores. The mixtures were incubated for 5 min at 37°C. After cooling to 0°C the components for the appropriate assay were added and the end volume was adjusted to 50 ~1. Next the assay for EF-G-dependent (+TPase or polyphenylalanine synthesis was performed as described below-. (k) EF-G-depmdmt WPasc Preincubation was carried out as described under reconstitution. Reaction mixtures of 50 ~1 contained 50 mw-Tris HCI (pH 7.4), 10 mM-Mg(OAc),, 50 mM-NH,Cl and 7 mM-%mercaptoethanol (assay buffer), ribosomal articles and proteins as indicated in the legend of Fig. 1. 10 pg of EF-G and 100 nmol of (y- 8 P]GTP (1 Ci/mol). The reaction was started by addition of GTP, and after incubation for 7 min at 37°C. terminated by addition of 100 ~1 of I ~H1’10,. Inorganic phosphate was determined as described by Schrier et al. (lBi3). All values were corrected for a blank containing assay buffer, 30 H ribosomes, EF-G and GTP. I’suallg the blank value was about 3% of the added amount of radioactivity, most,ly dur to inorganic phosphate present in the GTP preparation. (I) I’olyphenylalanine

synthesis

Preincubation was carried out as described under reconstitution. Reaction mixtures of 50 ~1 rontained assay buffer. 1.6 pmol of 50 S P,, cores, 3.2 pmol of 30 S particles. L7/L12 BS

558

W. MiiLLER

E’I’AL.

indicated in the Figures, @l mM-GTP, 5 pg of poly(U), 3 P”Qof EF-Tu, 2 pg of EF-Ts, 1 pg of EF-G and 50 pg of tRNA, precharged with 40 pmol of [ Hlphenylalanine (1 Ci/mol). The reactions were started by addition of GTP and after keeping them at 37°C for 15 min they were stopped by cooling the reaction mixtures to 0°C. After addition of 1OOpg of carrier bovine serum albumin. 1 ml of 5 y0 trichloroacetic acid was added and the samples were heated for 15 min at 90°C. After cooling, the reaction mixtures were filtered through Whatman GF/C filters, which were washed twice with an additional amount of 3 ml of 5% trichloroacetic acid, twice with 2 ml of ethanol, dried and counted in In&a fluor (Packard Instruments). (m) Electron microscopy A Philips electron microscope, EM 300, was used. The specimens for electron microscopy were prepared by the sandwich technique (Valentine et al., 1968) and were negatively stained by 1% uranylacetate. Electron optical magnification was about 40,000 x Care was taken to expose the fields of ribosomal particles to be imaged, with a minimum number of electrons. (n) A’edimentution equilibrium The meniscus depletion sedimentat)ion eyuilibrium as described by Yphantis (1964) was used with interference optics; partial specific volumes were estimated from the amino acid composition of the proteins. The proteins were dissolved in 10 mm-sodium phosphate (pH 7.4), 150 mM-KC1 ; the temperature was 5 to lO”C, dependent on the experiment and the duration of the runs (24 to 48 h). Sedimentation equilibrium time was shortened by exceeding the equilibrium speed for a certain period before reaching equilibrium. (0) Stntistics

(i) The two binding-nites

of L7/L18

binditty

to I’, COTP.~

of I’, cows how equal nrui indeputldrnt

nfjnity

to an L7/1,12 dime,,

In the simplest case we are concerned with the problem of calculating the distribution of particles having 1 or 2 dimers bound at different ratios of ribosomal core: L7/L12 dimer (in a non-co-operative way). Putting the degree of available dimer sites occupied on the ribosome at r, one can write for the fraction of particles having one dimer, 22( 1 - 2) ; for the fraction having 2 dimers, x2, and for the fraction having no dimer, (1 -z)~. Using these expressions, the percentages of particles having 1, 2, or at least 1 dimer can be easily calculated as a function of the average number of dimers bound per PO core particle. (ii) The two binding-sites ON P,, CY~I’YS how different but independent alfinity to nrl LI”/I,I% dintrr Imagine a particle containing 2 sites reacting rapidly at only 1 of them, e.g. Pa/P, = 10 where P, and Pb represent the affinity of the particle at site a and b. This ratio is closely dependent on the precise model for the system as, for instance. on the relative orientation of the 2 sites, their proximity to the surface or exposure to the solvent. In view of the high binding constants of L7/L12 to P,-cores (see Discussion), we first assume that the on-rates of LT/Ll2 to PO cores determine the affinities I’, and P,. the off-rates being small. Hubsrquently considering t,hr occupation of each of the 2 sites as a simple bimolecular reackion, we can write: dx L=cP,(l-Zs) dt and

dx, = cP,(l -zb).

-

dt

where 2, and xb are the fraction of sites on site a or b, respectively,

(2) occupied by a dimer.

L7/Llf

PROTEISS

559

Integration of equations (1) and (2) gives: In (l-z,)

= P,/P,(l -xb).

6%)

Using small increments of x,, series of pairs of 2, and zb were calculated for different ratios of I’JP, between za = 0 and 5, = 1, on the basis of equation (3) (as soon as za approaches one. occupation of site b is linear). (4)

(5)

3. Results (a) In&ewe

of L7/L12 on factor-dependent GTPase and polyphenylalanine

synthesis

Figure l(a) and (b), taken from Schrier (1977), shows the effect of addition of protein L7 or L12 to PO cores on the uncoupled EF-G and coupled EF-T-dependent GTPase activities. In agreement with Brot et al. (1973) and Schrier et al. (1973) it was found that L7 is just as active as L12 in the reconstitutions of the two GTPase activities: in both cases optimal reconstitution is obtained when four to five ecjuivalents

of L7/L12

are added.

Inspection of Figure l(a) shows that close to 75% of the EF-G-dependent GTPase activity of control 50 S particles is obtained on addition of two equivalents of L7/L12. Earlier results of Schrier et al. (1973) and Schrier (1977) showed that two moles of purified L7 or L12 bound to one mole of cores are needed to obtain particles that in EF-G-dependent GTPase are as active as intact ribosomes (see Table 1, in the case of P, cores +L7 “not reduced”). The same conclusion was reached when SP3, split protein, containing two equivalents of LT/L12 as determined by radioimmunoassay, was added to P,--37 cores. Since an excess of SP, split proteins always yielded four to five copies of L7/L12 bound to the PO cores (Table 1, line 3), these early results were interpreted as one dimer being sufficient for EF-G-dependent GTPase (Schrier, 1977). The EF-G profile (Fig. l(a)) is curved while the background observed in the absence of L7/L12 amounts to 5% of the complete effect. Since the PO cores contain 0.1 equivalent of L7/L12 as determined by radioimmunoassay (Table l), the background GTPase could well be due to some residual L7/L12 present in the P, cores. Figure l(c) illustrates the effect of readdition of L7 to P, cores on the synthesis of polyphenylalanine. Again the background is about 5% of the total effect and therefore explainable in terms of residual L7/L12 protein in the PO cores. The most striking feature of Figure 1(c) is its sigmoidal response to L7, as opposed to the hyperbolic plot of EF-G against L7/L12. Addition of four to five equivalents of L7 is required to regain full activity of protein synthesis. The same is true for EF-T. but here the background of 30% presents a more serious problem, due to the rela,tively small amount of GTP hydrolysed in coupled versus uncoupled GTPase (compare the scales of the ordinates of Fig. l(a) and (b)). A closer analysis of Figure l(b) is therefore abandoned.

560

w.

I

I

I

I

MiiLLlCR

ET

AL.

/

0 2 4 6 Moles of L7 or Ll2 added /mole of POcore

0

(0)

4 CL b Moles of L7 or Ll2 added /mole of POcore ib)

8

J

lo-OE” 8.0. ” EB B :

4.0.

z $

2.0.

0

6.0.

0

L70/ /

Polyphenylolanine synfhesls

/O

2 4 6 Moles of I-7 added/mole (c)

8 I( of POcore

IQ<:. 1. (a) and (b). Effect of L7 and Ll2 on (:Tl’ase

of PO WI’W dependent on elongation factor EF-(~ or mixtures of 50 ~1 contained, in addition to 30 pmol of P,, CORS,30 pmol of 30 S, and increasing amounts of L7 (0) or LIZ2 (A). 6 pg of EF-G and 50 nmol of GTP (a) or 1 erg of EF-T, 20 pmol of Phe-tRNAPhe, 1Org of poly(ll) and 1OOpmol of GTP (b). The GTPase activities of native 708 ribosomrs correspond to 3.3 nmol and 1.7 pmol of GTP hydrolysed/min in the case of EF-G and EF-T. respectively. (c) Effect of L7 on 13H]Phe incorporation. For details see Materials and Methods, section (k). EF-T.

Reaction

The combined data of the reconstitution experiments confirm that protein L7/L12 is required to carry out EF-G-dependent hydrolysis of GTP and polyphenylalanine synthesis (Kischa et al., 1971; Hamel et al., 1972; Brot et al., 1973 ; Sander et ai., 1972 ; Petterson & Kurland, 1980) and support the notion that this protein is an essential element in protein synthesis (Miiller, 1974). Since conclusions on the basis of added amounts of protein to cores require knowledge of both the protein fraction that is active in binding and the percentage of core particles, inactive in binding, the binding of L7 and L12 to PO cores was studied (Fig. 2). These binding profiles were obtained after pelleting reconstituted particles directly from solution or via a 10% (w/ v ) sucrose cushion to remove any free or weakly bound L7/L12. It should be mentioned that the binding curves are similar if separation of

L7/Ll2

PROTEINS TABLE

Dependence

Experimental

of GTPase

1 on L7/Ll2

proteins

Equivalents of L7/Ll2 bound

conditions

Percentage EF-(:dependent GTPase

P, cores P 030 cores P, cores+SPo split protein P 030 cores+SP,, split protein P, cores+L7 not reduced P 030 cores+ L7

0.1 0.1

7

4.6 4.4 2.4 0.3

97 96 93

30 s

0.1

50 s

3.8 3.9

70 s 70 S + 10 equivalents

6

18 100

of 70 8

split proteins

4.5

From Schrier (1977). The amounts of L7 or L12 in the core particles and reconstituted particles were determined by radioimmunoausay (see Materials and Methods, sections (g), (h), (i)). The EF-G-dependent GTPase was measured in a 50 ~1 volume parallel experiment. The low value of 2.4 copies L7/particle refers to an LT preparation, purified under conditions where the sample L7 not reduced was not kept in 6 mM+mercaptoethanol during the preparation. For details on EF-G GTPase see Materials and Methods. section (j).

L3ti]Me

L7 blnding

RlA I

z 42 s r” 3‘i ‘0, z EYI *-

0

0

/

/

u > w” 50

d

I-

/ Q0

4I 6 2, Equivalents of [3ti]Me t.7 odded (0)

0

0q%77Eqwvalents

of L12 added

(b)

FK:. 2. Binding of L7 or L12 to PO cores. (a) Binding of [‘HIMe (%methylated) L7 to 50 Y P, cores. Step 1: preincubation for 5 min at 37°C of 325 pmol of PO cores, 650 pmol of 30 S ribosomes and 1 to 8 equivalents of [3H]Me L7 in 325 ~1 of 2 x cone. assay buffer (Materials and Methods, section (i)). Step 2: centrifugation in Beckman SW 50 rotor after 1 : 1 dilution with water: determination of [‘HIMe Li in pellet. (b) Binding of L12 to 50 8 PO cores. Step 1: preincubation for 5 min at 37°C of 500 pmol of PO cores, 1 nmol of 30 S ribosomes and 1 to 6 equivalents of Ll2 in 2 x cont. a&say buffer in 500 ~1. Step 2: centrifugation in Beckman 50 Ti rotor after 1 : 1 dilution through 4 ml 10% (w/v) sucrose in assay buffer. Determination of L7jLl2 content in pellets via radioimmunoassay (RIA). Par details of espwimth. see under Materials and Methods, sections (f) to (h).

mc: 3(a)(h)

I+:. 3. Electron micrographs of 50 S subunits eauivalents of L7 (Ccl and Cd)).

(a). P, cores (b) and P, KWS. reconstituted

wit.h 1 and 4

504

IV. MOLLER

BT z-l/,.

reconstituted ribosomes from L7/L12 was performed by chromatography on LSephadex G200. It is clear that the binding profiles reflect a very tight binding process where, in the range of up to four copies of L7/L12, the amount of added 1,7/L12 equals that bound. In view of the presence of four copies in normal 50 S particles (Subramanian, 1975: Hardy, 1975; Table 1 line 7, t,his paper), t,he fraction of P, cores unable to rebind L7/Ll2 seems small. Table I also shows t)hat 50 S and 70 S ribosomes may lose a few per cent of t’heir L7/L12 protein during isolation and that the reconstituted particles contain a certain excess of L7/1,12. possibly aspecifically bound. (b) Electron microscopy of P, cores with different amounts of L7 Figure 3 shows electron micrographs of fields of 50 S ribosomal subunits and L7/L12-deficient 50 S core particles. Analysing the fields of 50 S subunits (Pig.. 3(a)) reveals that 50% of particles have a stalk. The preferential orientation of the 50 8 ribosomal subunits on the carbon support is the so-called quasi-symmetric projection (Lake, 1976). On removal of the protein L7/L12 from the 50 S subunit by treatment with ethanol/ammonium chloride according to Hamel et al. (1972), the stripped ribosomal subunits no longer show any arm (see Fig. 3(b)). These results fully confirm the original observations of Boublik et aZ. (1976) and Strycharz et al. (1978). In order to follow the reappearance of the stalk as a function of addition of lJ7/IJ1P. reconstitution experiments were performed with P, core particles and varying amounts of L7. The number of copies of L7/L12 bound was determined by means of radioimmunoassay after pelleting the reconstituted particles through a loo/, sucrose cushion as described in the legend to Figure 2. The results of these reconstitution experiments are represented in the electron micrographs of Figure 3(c) and (d). The electron micrographs show that the number of particles with a distinct elongated appendage regularly increases with the number of L7/L12 molecules inclorporated in bhe 50 S subunit. For statistical analysis of each reconstitution experiment a large number of particles (order 250) were analysed. The percentage of particles possessing a distinct arm is presented in Table 2. There is a linear rela,tionship between the number of bound L7/L12 molecules and the T.-\RI,E 2 In$?uence of protein L7 on the number of arms observed in electron reconstituted 5&S proteins Reconstitution mixture Ratio of copies of L7 to P, cores 0 I 2 3 4 8 50 s

Number of particles counted

Number of particles with arm

250

0

228 239 229 290 279 250

33 55 x3 132 120

125

microscopy

Percentage particles with arm 0 IRf2 23+4 3Bf5 46&S 444+4 50+4

of

L7/L12

0

4 2 Equvalents of L7 or LI2 bound

(a)

5lL-i

PROTEIXS

Equwolents

of L7 or Ll2 bound (bi

FK:. 4. Reconstitution of ribosome activity from ethanol/NH,Cl-treated 50 S PO cores and L7 or LlP. (a) Percentage of EF-G-dependent GTPase as a function of the number of equivalents of L7 bound. (0) L7 content.determined vin radioimmunoassay: ( x ) I,7 content determined virt binding of 13H]Me 1,;. (1~) Pewentage of polyphenylalanine synthesis as a function of the number of equivalents of Li/Ll2 bound : for conditions see section (k) of Materials and Methods (0) Li content determint~d vitr ~adioimmunoassay : (V) idem. from separate reconstitution wperiment ; (0) L7 cwntrnt drtrrmirwd rsicrbinding of [ 3H IMeL7/1,12. Lines drawn in (a) and (b) represmt comptrd profiles. ((8) Ptwrntagv of 50 S partkles on electron microscope grid having a stalk as a function of t,ht, number of dimcrs boanti. Erwr bars represent the standard deviation from several replicate determinations made on different regions of the grid. (‘ontrol 50 S ribosomes: prcrntagr of stalks 50: 50 S I’, cww+ 8 cxcluivalents of 1.; added pwwntagr of stalks 11. For details SW Table I

percent,agr of particles with an arm with up to four col)ics twund (Table 2 :in:j Fig. 1(c)). Incorporation of four copies of L7, corresponding to two dimers of L7, yielded an assembly of particles that morphologically and with respect to the number of arms was indistinguishable from untreated, active 50 S particles. Since 50 S ribosomes reconstitute from PO cores and protein L7/L12 via a route in which at half saturation most particles carry only one dimer (Schrier, 1977 ; Lee et al., 1981; this paper), the observed linear dependency of the frequency of arms as a function of the average number of L7/L12 dimers bound, may tell how the dimers are arranged in the arm and whether all arms are alike in containing one or two dimers of L7/L12.

5%

W'. Mi5LLER ETA/. (c) Interpretation

of tk functional

dependence of ribosomes on L7/L12

Figure 4 illustrates the background-corrected results of a number of titration experiments of P, cores with L7/L12 using the capacity to cleave GTP (Fig. 4(a)), synthesize polyphenylalanine (Fig. 4(b)) or form a stalk (Fig. 4(c)). It is obvious that especially the first two types of activity follow a drastically different course with respect to their dependence on protein L7/L12. Assuming a random distribution of L7/L12 dimers over the two binding sites on the ribosome (Lee et al., 1981), the percentage of particles having zero, one or two dimers bound at different averages of dimers bound, has been computed (Materials and Methods section (0)). As seen from Figure 4(a), the experimental points for EF-G-dependent GTPase nicely follow the computed profile of the sum of the percentage of particles with one or two randomly bound dimers of L7/L12. On the other hand, the polyphenylalanine activity obeys a theoretical profile corresponding to the percentage of particles with two randomly bound dimers of L7/L12. Therefore the EF-G-dependent GTPase (Fig. 4(a)) indicates that a single dimer of L7/L12 is sufficient to trigger full activity, whereas our minimal requirement for protein synthesis is that there are two dimers of L7/L12 on the ribosome (Fig. 4(b)). With respect to the EF-G-dependent GTPase. it is immaterial which of the two dimer sites is filled to trigger activity. If there was only one, and the two sites fill randomly, the percentage of GTPase should have fallen on the diagonal; halfway on the curves of Figure -i(a) and (b). In the case of a strong, positively co-operative binding of L7/L12 to PO cores, one would also expect a linear dependence instead of the convex profile of Figure 4(a). Moreover, one would then expect to see 5O”/b GTPase, and not 75% as observed by us and Lee et al. (1981), when one dimer, on average, is bound. So far the results were interpreted on the premise of a random filling of the two sites. We are of the opinion that this assumption is correct, at least under the conditions of the functional assays used here. In order to get an impression of the situation in which the intrinsic affinit,ies of L7/L12 for the two dimer sit’es would differ from each other, t,he frequency distribution of t)he different’ types of partic,les was computed using a ratio of the intrinsic affinity I’ for the a and b sit*r of I, 10 and co (see Materials and Methods. section (0)). As seen from Figure 5. the experimental results of the EF-G-dependent GTPase indicate that the ratio ofaffinity Pa//‘b is ofthe order often at the most. On the other hand. a sample of L7/1,12 “not reduced” (Table 1) binds only two copies maximally and gave. as expected. a GTPase profile caorresponding to I’,/Y, = cc;. However, it seems likely that, during reconstitution. four copies of “not reduced” L7 were bound to the ribosomal particle. As a result of centrifugation, two copies (one dimer) of not reduced L7 were removed, leaving a population of ribosomes in which each single ribosomal particle contained one dimer, resulting in full GTPase activity after centrifugation (see also Zantema et al., 1982). The interpretation of the electron microscopy data of Figure 3(c) is based on a choice of four different models. each of which allows a prediction about the relative frequency of arms as a function of the degree of reconstitution of ribosomes with L7/L12. The premise of model I (Fig. 6) is that no reconstitution intermediates are formed, implying strong co-operation in binding between the two dimers. The

L7jLl2

PROTEINS

q

IOO-

i Dimers L?/L I2 bound FK:. 5. Comparison between experimental and computed EF-O-dependent GTPase as a function of the number of L12 dimers bound. Experimental: (0) GTPase using L12 reduced (max. binding 4 ~yiex): (0) (~‘l’l’asr~ using L12 partially oxidized (max. binding 2.1 copies). (‘omputrd : curve 1’. predicted on basis of 1 dimer being sufficient for GTPasr and 1’,/1’, = 1: curve 2’. idem and l’r/l’b = 10: curve 3’, idem and P,/P, = 03 ; curve 1, predicted on basis of minimal 2 dimers being sufficient for GTPase and Pa/P, = 1: curve 2, idem and Pa/P, = 10; curve 3, idem and PJP, = co. P,, Affinity of a dimer of L12 to site A on P, core. P,, Affinity of a dimer of L12 to site B on PO core. The curves 1.2 and 3 should also btk c~m~pared with the p~~l~l~hen~lalatri,Ir results of Fig. 4(b). (‘urvt~ 4: predicted (~‘l’t’as+~ on t,hr basis of a minimum of one ,$xed dimer bring suffkient for activity.

prediction therefore is that the percentage of particles with an arm would imrease linearly with the number of L7/L12 dimers bound. Model I. although consistent with electron microscopy data, is not preferred because the functional studies point to a random occupation of the two independent binding sites under the conditions of reconstitution used. Studies in which fluorescent L7/L12 preparations used for recaonstitution of PO cores are in full agreement with a model in which the two binding sites for L7/L12 are randomly filled (Zantema et al., 1982). Hence these studies also argue against model I. With respect to model II, it should be noted that the relative frequency of arms is significantly beyond that expected if the minimal requirement for a morphologically observable arm was two dimers (cf. the experimental points of Fig. 4(c) with the computed curve of Fig. 4(b)). If the requirement is reduced to one dimer. model II would predict the theoretical curve of Figure 4(a), which is well above the observed frequency of stalks. The electron microscopy data therefore seem inconsistent with model II. Two other models remain, the quintessence of which is that on electron miCroSC(Jpy grids, particles having two dimers are scored about twice as frequent’ly as particles carrying one dimer as concerns a morphologically observable arm.

568 RECONSTITUTION

MODEL I

No IntermedIate one- dlmer particle

INTERMEDIATES

MODEL II

OF 50s

RIBOSOMES

MODEL IU

MODEL Ip

One-and two - dlmer One-dlmer portlcle portlcle shore has one, two-dimer some stalk portlcle has two stalks

Estimated Average number of L7/L12 dimers bound per particle

Model I

Model II

0 0.5 1.0 15 2.0

0 25 50 75 loo

0 43.75 75 93.75 100

One-dlmer portlcle has, on average m half of the portlcles. one stalk, all two dlmer portlcles hove one stalk

percentage with arm

Model III 0 25 50 75 loo

Model IV 0 25 50 7.5 100

Observed percentage with arm 0 15 23.0 36.0 460

Control 50 S rihosomes: 50”/0 of particles on grid contain visible arm. FIG. 6. Predicted

percentage of stalk particles using different

reconstitution

models for L7/L12

Models (e.g. model II) in which the two dimers are lined up in the ribosome side by side (Marquis et al., 1981; Leijonmarck et al., 1981) offer no easy explanation for such greater statistical weight of the two-dimer particle. Physical separation of the two dimers, however, automatically leads to a factor of two in the frequency distribution function of two- versus one-dimer particles. With respect to model III, Strycharz et al. (1978) have reported that they never observed particles having two antibodies bound on opposite sites of the central protuberance but only on one site, the stalk region. Besides, the formation of two stalks on the ribosome as a result of reconstitution of P,, cores with L7/L12 has never been observed. This makes model III highly unlikely, although Boublik et aE. (1976) did find ribosomeantibody aggregates suggesting L7/L12 reactive sites on both sites of the central protuberance. In view of the low occurrence of opposite site labelling versus double labelling of the stalk (Strycharz et al., 1978) we prefer model IV, which shows L7/Ll2 dime1 located in a single appendage of the large subunit while the ot,her L7/L12 dimer is bent towards the main body of the 50 S subunit.

L7/Ll2

PROTEINS

Sri!)

(d) One NTF dimer blocks one L7lL12 binding site and leaves the other intact for EF-O CTPase Earlier work showed that L7 and L12 can be cleaved at the arginine residue number 73 into an amino-terminal fragment of L7/L12 (NTF 1-13) and a carboxylterminal fragment of L7/L12 (CTF 74-120) (Van Agthoven et al., 1975). Evidence that the ribosome contains two sites, each for binding a dimer of L7/L12, with one site filled being sufficient for EF-G-dependent GTPase, was obtained in the following manner. The L7 amino-terminal fragment was 3H reductively methylated and tested for its ability to bind to 50 S P, cores (NTF l-73). The NTF fragment of L7 used was dimeric. The average molecular weight. was 164+0.1 x 103, which was in agreement with the chemical estimation of NTF dimer, being 150 x 103. It has been shown earlier that preincubation of PO cores wit,h L7 NTF markedly impairs the functional reconstitution by intact L7 (Van Agthoven et al., 1975). The PO cores were incubated in the following reaction. First, PO cores were incubated with variable amounts of NTF. Second, an excess of L7 was added to each preincubation mixture. Next the amounts of NTF and L7 per particle were measured after centrifugation in a 10% sucrose gradient, together with the EF-G-dependent GTPase of a portion before centrifugation. Figure 7 shows that P, core particles can bind up to two dimers of NTF. Without NTF the cores bind two 1~7 dimers. With two NTF dimers bound, there is little L7 binding and GTPase activity left. The EF-G-dependent GTPase activity is hardly influenced by NTF binding up to one dimer of NTF bound; titration of the second binding site with another NTF dimer causes a sharp decline of the EF-G-dependent GTPase t,o thcl level of t,hr core particle alone. The experiment of Figure 7 ~)rovitlrs

0

2 Equivalents

5 of NTFadded

Fw. 7. Effect of binding of N-terminal fragment (NTF) on subsequent binding of L7 and EF-Gdependent GTP hydrolysis. In the first experiment, Pc cores were incubated with variable amounts of %labelled NTF, and the amount of NTF bound was determined (Materials and Methods, section (f)) (m-m). Addition of an excess of cold L7, in the second step, does not lower the 3H-labelled NTF binding. In the second experiment, P, cores were incubated with variable amounts of non-radioactive NTF, followed by incubation with 8 equivalents of 3H-labelled L7, and the amount of 3H-labelled L7 bound was determined (A -A). The activity of these particles in EF-G-dependent GTP hydrolysis was also measured (0 - 0).

W. M~LLEK.

570

E7' AL.

independent evidence t,hat the ribosome contains two sites. each for binding a dimer of I,7 or its amino-terminal dimeric fragment,. Binding of one dimer of 1~7is sufficient for full EF-G-dependent GTPase activit,y. 4. Discussion (a) EF-(I-dependent

GTPase

needs

dimrs

one

and

polyphenylalnnine

needs

two

L7/1,1%

on thr ribosom

The strong dimerization of L7/L12 type proteins in procaryotes and eucaryotes (Mijller et al., 1972; Matheson et al., 1980) suggests that the functional unit on the ribosome contains two dimers of L7/L12 attached to LlO (Leijonmarck et al., 1981). The assembly of L7/L12 takes place at two more or less independent sites, each binding a dimer of this protein. Uncoupled EF-G-dependent GTPase activity is fully restored at half-site saturation with L7/L12, which means that only one of the ribosome sites has to be filled for full EF-G-dependent GTPase activity. The experimental set-up of our GTPase studies was such that L7/L12 dimers were distributed evenly among the t,wo sites. although in other studies (Zantema et wl.. 1982) conditions were such that one dimer could be selectively removed from the ribosome. Nevertheless one dimer of L7/1,12 remained sufficient for full EF-(:-dependent GTPase activity. A similar effect of the loss of one of the two dimers of L7/Ll2 has been seen in the case of the reconstituted complex of 1,7/1,12-L10-23 8 RXA (Dijk et al., 1979). Oxidation of methionine in the amino-terminal part of L7/LlP is known to cause poor binding (Koteliansky et nZ., 1978). Another. more straight,forward explanation is that the on-rates for t’he binding of L7/L12 dimers to both sites are equal while the off-rates differ (Zantema et al.. 1982). The experiments described here were done under conditions in which off-rates were negligible: even after pelleting of the rec~onst~it~ut~erl particdlrs four copies were bound. Independent support that the ribosome contains two sites. each for binding a dimer of L7 and one being sufficient for GTPase, came from studies using an aminoterminal fragment of L7/L12 (NTF l-73). 0 ne NTF dimer blocks one L7/L12 binding site but leaves the other intact for binding an LS/L12 dimer, thereby triggering full EF-C&dependent GTPase activit’y. The same conclusion has been drawn by Lee et al. (1981) using mixed reconstit,utes of active and inact,ive 1,7/1,12. An important clue towards the function of L7/L12 comes from our finding that for efficient translation polyphenylalanine synthesis requires two dimers rather than one. To our surprise Lee et al. (1981) found no such requirement and reported that one dimer was sufficient for full synthesis. The reason for this is not clear and it may be due to a difference in experimental conditions for protein synthesis. Lee et al. (1981) used a rather crude cell-free system (Nirenberg, 1963) with the 100,OOOg supernatant fraction as a source of elongation factor, whereas we used a highly purified system of separated elongation factors, transfer RNAs and ribosomes. It should, however, be noted that Brot et al. (1973) also found no sigmoidal curves for polyphenylalanine synthesis as a function of L7/L12 added to P, cores in a highly

L7/L12

5il

PROTEINS

purified cell-free system. Unfortunately the amounts of L7/L12 left in the cores and the number of L7/L12 copies bound were not mentioned. (t)) Lyon-eqtrimleme

of the positions

of the two dimrrs

of L7/Ll%

in the rihosoltrr

Our electron microscopy study was designed to find how the four copies of L7/1,12 are internally arranged in the ribosome. This expectation has been partially fulfilled using electron microscopy of ammonium chloride/ethanol washes of 50 S ribosomes to which varying amounts of L7/L12 were added. The electron microscopy data were interpreted as indicating that one dimer of L7/Ll2 is positioned along the stalk of the large subunit, whereas the other is bent inwards to the main body of the ribosome. Such an arrangement is not necessarily inconsistent with observations that antibodies against L7/L12 react with a single region on the large subunit, characterized as stalk (Strycharz et al., 197X). and that there is in the ribosome a pentameric structure LlO(L7/L12)4 with LlO at the base and the two dimers extending from it (Strycharz et al., 1978; Leijonmarck et al., 1981; Marquis et al.. 1981). It has been proposed that all four copies of L7/L12 are aligned side-bpside over their total length and that the carboxyl terminals of both dimers ext,end into the solvent (Leijonmarck et al., 1981; Marquis et al., 1981; Tokimatsu et d., 1981). However, Tokimatsu et al. (1981) do not exclude a division of the four copies between two symmetrical sites in the stalk region. Moreover, Strycharz et al. (1978). Lake (1980) and Marquis et al. (1981) mention that the globular region of the stalk may come closer to the body of the ribosome. Such an arrangement would not, explain why removal of the carboxyl-terminal parts of one dimer leaves the GTPase dependent on EF-G intact, whereas removal of these parts from both dimers results in complete loss of enzymatic activity (Fig. 7 ; Van Agthoven, 1975 : Marquis & Fahnestock, 1980; Marquis et al., 1981). However, if one dimer of L7/L12 has its carboxyl-terminal part turned inwards in the direction of the GTPase centre (the interface region where Lll and EF-G are located) while the other dimer retains a stalk-like conformation, the GTPase fragment studies could be explained better (Fig. 8). Such an arrangement would fit the mapped localization of the EF-G binding centre at the base of the rod-like appendage (Girshovich et al.. 19X1 : Maassen & Miiller. 1981) and the known involvement of L7/L12 in factor binding and hydrolysis of GTP (Mijller, 1974). That crosslinks of LlO, Lll, L2,L4. 1~14and

(0)

(b)

(cl

FIG:. X. l’osail~lr orientations of the 2 L7/Ll2 dimers in the 50 S subunit oxirntationn. (a) Both dimrw (c) other dimer tlwnf4 aligned side-by-side (IAjonmarck et al.. 1981): (h) one dimer turned inwards: inwards

572

w. MiiI,l,EK.

BT /IL.

L6 to proteins L7/L12 occur (Traut et aE., 1980) would also be consistent least part of the L7/L12 stalk being turned towards the subunit interface

with at region.

Proton magnetic resonance studies indicate an exceptional mobility of L7/L12 in the ribosome (Gudkov et al., 1982) and support the proposal of L7/L12 being a dimeric motile protein (Kischa et al., 1971). (c) Concluding

remarks

The main problem of L7/L12 is to interrelate the different aspects of the protein, such as GTP hydrolysis, elongated dimer structure, and resemblance to motile proteins, in a coherent picture. The core of the results of this paper, the required presence of two dimers in polyphenylalanine synthesis and of one dimer in GTP hydrolysis, together with the electron microscopy results, may be consistent with an alternating, surface arm model that is steered by hydrolysis of GTP (Fig. 8). As a result, ordered entry and exit of factor-associated transfer RNAs through the ribosome may be greatly facilitated. Quantitative work on distances of proteins L7/L12 to LIO, by fluorescent probes at fixed portions of the protein chain will be reported elsewhere (Zantema et nl.. 1982). This investigation was supported by the Netherlands Organization for the Advancement of Pure Research (ZWO) and the Netherlands Foundation of Chemical Research (SON). REFERENCES Amons, R. & Moller, W. (1974). Eur. J. Biochem. 44, 97-103. Arai, K., Kawagita, M. & Kaziro, Y. (1972). J. BioE. Chem. 247, 7029-7037. Boublik, M., Hellmann, W. & Roth, H. E. (1976). J. Mol. Biol. 107, 479-490. Brot, N., Marcel, R., Yamasaki, E. & Weissbach, H. (1973). J. BioE. Chem. 248, 69524956. Caldwell, P., Luk, D. C.. Weissbach, H. & Brot, N. (1978). Proc. Nat. Acad. Sci., U.S.A. 75, 53495352. Dijk, J., Garrett, R. A. & Miiller, R. (1979). Nucl. Acids Rea. 6, 2717-2730. Eckstein, F., Kettler, M. & Parmeggiani, A. (1971). B&hem. Biophys. Res. Commun. 45, 1151-115s. Gesteland, R. F. (1966). J. Mol. Biol. 18, 356-368. Girshovich, A. S., Kurtskhalia, T. V., Ovchinnicov, Y. A. & Vasiliev, V. D. (1981). FEBS Letters, 130, 54-59. Gudkov, A. T. & Behlke, *J. (1978). Eur. J. Biochem. 90, 399-312. Gudkov, A. T., Tumanova, L. G., Venyaminov. S. Yu & Khechinashvili, N. N. (1978). FEBS Letters, 93, 215218. Gudkov, A. T., Tumanova, L. G., Gongadze, G. M. & Bushuev, V. N. (1980). FEBS Letters, 109, 34-38. Gudkov, A. T., Gongadze. 0. M.. Bushuev, V. N. & Okon, M. S. (1982). FEBS IJetters, 138, 229232. Hamel, E., Koka, M. & Nakamoto, T. (1972). J. Biol. Chem. 247, 805814. Hardy, S. J. S. (1975). Mol. Gen. Genet. 140, 253-274. Highland, J. H. & Howards, G. A. (1975). J. Biol. C&m. 256, X31-834. Kaltschmidt, E. & Wittmann, H. G. (1970). Anal. Biochem. 36, 401-412. Kastner, B., Stoffler-Meilicke, M. &, Stiiffler, G. (1982). Proc. Nat. Acad. Sci., U.S.A. 78, 66526656. Kischa, K., Miiller, W. & Stoffler, G. (1971). Nature New Biol. 238. 6243. Koteliansky. 1’. E., Domogatsky, S. P. & Gudkov. A. T. (1978). E?rr. .I. Hiochem. 90, 319323.

1,7/L14 PROTEINS

.i;:(

Lake. J. A. (1980). In Ribosomes: Structure. Function and Grnrtica ((‘hambliss. G.. (‘raven. (:. R.. Davies. J., Davis, K., Kahan. I,. & Nomura. M.. eds), pp. 207-236. ITni\-ersit!. I’ark Press. Baltimore, Maryland. Lee, (1. C., Cantor, C. R. & Wittmann-Liebold, B. (1981). J. Biol. Chem. 256, 41-48. Leijonmarck. M., Pettersson, I. & Liljas, A. (1981). In Structural Aspects of Recognition and Assembly in Biological Macromolecules (Balaban, M., Sussman. *J. L., Traub. W. & Yonath. .A.. eds). pp. 761-777. ISS, Rehovot and Philadelphia. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1953). J. Biol. Ph~em. 193. 265275. Maassen, ,J. A. & Mailer, W. (1981). Eur. J. B&hem. 115, 279-285. Maassen. J. A., Schop, E. N. & Miiller, W. (1981). Biochemistry, 20, 102+1025. Marquis. 1). M. h Fahnestock, S. R. (1980). J. Mol. Biol. 142, 161-179. Marquis, D. M., Fahnestock, S. R., Henderson, E., Woo. I>.. Schivinge, R.. Clark. M. W. &! Lake, ?J. A. (1981). J. Mol. Biol. 150, 121-132. Matheson, A. T., Mijller, W., Amons, R. & Yaguchi, M. (1980). In Rihosomes: Struct~rcrr. Function and Genetics (Chambliss, G., Craven, G. R., Davies, ,J.. Davis, K., Kahan. L. & Nomura, M.. eds), pp. 297-332, University Park Press, Baltimore, Maryland. MGller. W. (1974). In Ribosomes (Nomura, M., TissiBres, ,4. Br Lengyel, P.. eds), pp. 711-731. (‘old Spring Harbor Laboratory Press, New York. MGllrr. W., Castleman, H. & Terhorst, C. (1970). FEBS Letters, 8. 192-196. MFller. W.. Groene, A., Terhorst, C. & Amons, R. (1972). Eur. J. Biochem. 25. 5~-12. Nirenberg, M. W. (1963). Methods Enzymol. 6, 17-23. ijsterberg. K., Sjiiberg, 13.. Liljas, A. & Pettersson. I. (1976). FEBS IAm. 66. 4X-51. iist)rrherg. K.. Sjiikwrg. K., I'c~ttersson, I.. Liljas. A. & Kurland. (‘. (:. (1977). FElZS /Afro. 73, 22-24. Pettirsson, I. (1979). Nucl. Acids Res. 6, 2637-2646. Pettrrsson. I. & Liljas, A. (1979). FEBS Letters, 98. 139-144. Pettcbrsson. I. & Kurland. (‘. (:. (1980). l’roc. So/. ddcnd. SC;.. I~.,S..~.. 77. a()()7 4010. Itavc>l. .I. M. & Shoreg, J. (1971). Methods Enzymol. 29. 30&316. Santlt>r. (:.. ,Ilarsh. R. (‘. & Parmegpiani. A. (1972). Hiochrnt. Bj(~$/!y,s. Kps. (‘o,,/n/,,,,. 47, XBCt873. Schrier. P. 1. (1977). Doctoral Dissertation, Leiden University, Krips Repro Meppel. Schrier. P. I., Maassen,
Stijffler. G. & Wittmann, H. G. (1971). Proc. Nat. Acud. Sci., U.S.A. 68, 2283-2287. StGfflrr. G.. Hasenbank, R.. Bodley, J. W. & Highland, J. H. (1974). J. MoZ. Biol. 86. 17 I174. Strycharz. W. A.. Nomura, M. & Lake, J. A. (1978). J. Mol. Biol. 126, 123-140. Subramanian, A. R. (1975). J. Mol. Biol. 95, 1-12. Terhorst, (‘.. Miiller, W., Laursen, R. & Wittmann-Liebold. B. (1973). Eur. J. Biochem. 34. 13%152. Tokimatsu, H., Strychalz, W. A. & Dahlberg, A. E. (1981). J. Mol. Bid. 152, 397. Traut. R. R.. Lambert, J. M., Boileau, G. & Kenny, J. W. (1980). In Ribosomes: Structure, Function and Genetics (Chambliss, G., Craven, G. R., Davies, J., Davis, K., Kahan, L. & Nomura, M., eds), pp. 81+110, University Park Press, Baltimore, Maryland. 7, 2143-2152. Valentine. R. C.. Shapiro, B. M. & Stadtman, E. R. (1968). Bio&misty. Van Agthoven. A. J., Maassen, J, A.. Schrier. P. I. & Miiller, W. (1975). Biochem. Biophya. Res. Com,mun. 64. 1184-1191. Weissbach. H. & Pestka, S. (1977). Molecular Mechnni.sn/,s (d f’rotuin Bioqnthusis. ~1. 720. Academic Press, New York, San Francisco and London. Wong, K. P. & Paradies, H. H. (1974). Rio&em. Biophys. Res. Ccwnmvn. 61. 378-184. Yphantis. I). A. (1964). Jliochrmi.stry, 3, 297-317. Zantema. A.. Maassen, J. A.. Kriek, J. & Miiller. W. (1982). Biochemistry. 3(H%k.308~. Edited

by H. E. Huxley