Evidence that Escherichia coli 30 S ribosomal subunit populations are conformationally heterogeneous

Evidence that Escherichia coli 30 S ribosomal subunit populations are conformationally heterogeneous

J. Mol. Biol. (1979) 128, 561-575 Evidence that Escherichiu coli 30 S Ribosomal Subunit Populations are Conformationally Heterogeneous MARTNA K. T. L...

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J. Mol. Biol. (1979) 128, 561-575

Evidence that Escherichiu coli 30 S Ribosomal Subunit Populations are Conformationally Heterogeneous MARTNA K. T. LAM, Lr-MIXJ~ CHANGCIIIEN

AND GARY

It. CRAVEN

The Laboratory of Molecular Biology and the Department of Genetics Un~iversity

of Wisconsitr

1525 Linden Drive :k’adisotr, Wise. 53706, U.S.A. (Received

7 April 1978, and in

r~vissd fom,

15 Septentber

1978)

m-o have estimated the number of sites on eacll protein of the 30 S ribosome which are accessible to chemical iodination. First, the total number of iodinat)ablc sites was determined for the intact 30 S ribosome. The proteins were rtxtracted, separated and the relative distribution of iodine in vacll prokill d~~terminrd. This distribution of iodine divided into the t,otal sites pw ribosome gave an est,imate of tile number of sites per individual prokill.

Second. the iodinated proteins were purified and tlleir trypsin digestion products separated. The number of radioactive peptides \VHS taken as a measure of the number of sites on that protein open to the iodination reaction. Tllr ~llmlber of iodinatable sites for each protein was fomld to be radically different b> the two methods. In almost all cases, the number of unique, radioact’ively labeled prptides, derived from a &en 30 S protein, far exceeded the total incorporation illto that protein. Wr suggest that the best explanation for this unexpected discrepancy is that the 30 8 ribosome population WC used in these experiment’s is lwtcro~wwons in its t,opography. In addition we have compared the topography by the chemical iodinaLion procedure for ribosomes in two different conforma$ons: active and inactive (we Zamir et al., 1971). We have found \‘ery little change in the chemical reactivity of the proteins when tho ribosomex are in the two differwlt conformations. The most notable chanpes involve proteins 8 10, S 1s/S1 9 and especiall> Sl2]S13.

1. Introduction Bacterial

ribosomes

isolated

by conventional

procedures

have been found

to bc

heterogeneous in composition and function. The 30 S ribosome from Escherichia coli has been shown to contain less than one copy of some of the ribosomal proteins by Yoynow & Kurland (1971). It has been suggested that the compositional heterogeneity is in part, a consequence of the salt-washing procedures employed to remove contaminating cytoplasmic proteins (see Hardy, 1975). Both the 50 S and the 30 8 particles are commonly observed to have less than lOO’$/, activity (e.g. see Ginzburg of al., 1973: Skoultchi et al., 1969 ; Glukhova et al., 1975). This functional heterogeneity has not been adequately explained, although in one report 30 S ribosomes were found capable of 1OO0/Ubinding of transfer RNA (Bresler et al., 1975). These authors suggest that improved methods of preparation can yield functionally homogeneous ribosomes.

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In this report we present evidence t’hat bacterial 30 S ribosome populations are not only heterogeneous in function and protein composition, but also exkemely het,erogeneous in conformation. We have completed a quantitative analysis of the pattern of chemical iodination of the 30 S particle and found that at saturation t’he ribosome population can be divided into essentially three distinct classes of particles each having a different set of proteins accessible to chemical modification. Roughly 45% of the ribosomes appear to have only one protein accessible to chemical iodination. About 40% of t)he population have only four proteins exposed to iodine and approximately 15% are composed of between 10 and 14 proteins which become iodinated at saturation. Thus, it seems that nearly half of the 30 S ribosome population exists in some sort of extraordinarily compact form and the remaining half assumes a relatively open and accessible conformation. This result has implications which affect’ virt’ually all investigations of ribosome structure and function. In addition, using our quantitative techniques, we have examined t.he iodination patterns of 30 S ribosomes in the active and inacbive conformations according to the definitions of Zamir et al. (1971). We have found that, in agreement’ with Litman et al. (1976), most of the proteins do not change in their relative accessibility to iodine. However, in the minority class of riboaome conformation we present’ data to suggest that proteins SlO, SlSjSl9 and Sl2jSl3, undergo a change in structure when converted from the inact,ive form to the active configuration.

2. Materials (a)

and Methods Buffers

(1) Tris/Mg buffer. 0.01 M-Tris.HCl (pH 8), 0.01 M-M~(OAC)~. (2) Tris/K/Mg buffer. 0.01 M-Tris.HCl (pH 8), 0.05 M-KCl, 5x 1O-4 M-Mg(OAc),. (3) Activation bu#er. 0.02 MTrisaHCl (pH 7.2), 0.02 M-Mg(OAc),, 0.20 M-NH&I, 2 mM-dithiothreitol. (4) Inactivation. 0.1 M-NH,Cl, 1 rnx-/3-mercaptobuffer. 0.01 IT-Tris*HCl (pH 7.2), 0.5 mm-Mg(OAc),, ethanol. (5) Binding buffer. 0.05 M-TriseHCl (pH 7.2), 0.02 M-Mg(OAc)a, 0.15 M-NH&~. (6) Wash buffer. 0.01 M-Tris.HCl (pH 7.2), 0.01 M-Mg(OAc)z, 0.05 X-NH,Cl. (b)

Preparation

of ribosomes

Ribosomes were isolated from frozen E. coli MREGOO cells according to the method of Kurland (1966). Frozen cells, thawed in Tris/Mg buffer, were disrupted in a French press at approx. 6000 to 8000 lb/in2. 500 pg DNase was added to digest DNA in suspension for 5 min in ice and the cell debris was removed by 2 successive centrifugations at 17,000 revs/min for 30 min each in a Sorvall SS34 rotor. The crude ribosomes in extracts were recovered by centrifugation through 1 M-ammonium sulfate solution. This process was repeated 3 times. The final ammonium sulfate pellet, dissolved in Tris/Mg buffer containing 7.8% (w/v) of ammonium sulfate, was purified by high-speed centrifugation at 29,000 revs/min for 16 h in a 30 rotor. The pellet consisted of mainly 70 S ribosomes and was dissolved in Tris/Mg buffer. Debris was removed by a low-speed centrifugation in a Sorvall centrifuge and was purified again by centrifugation at 29,000 rovs/min for 16 11. After the second centrifugation, the 70 S ribosomes were dissolved in Tris/Mg buffer, dialyzed overnight against the same buffer and stored at -70°C. (c) Separation

70 S ribosomes

of 30 S and

50 S sdunits

were dialyzed for 24 11 against Tris/K/Mg buffer to dissociate into 30 S and 50 S subunits. They were then separated by zonal centrifugation through a 59/o to 30% sucrose gradient containing Tris/K/Mg in a BIV rotor (Beckman Instrmnents). Fractions containing 30 S subunits were pooled and diluted with 3 vol. of Tris/Mg buffer and were concentrated by a hollow fiber concentrator (Amicon). 30 S ribosomes were t,hen dialyzed against Tris/Mg buffer and st,ored in small samples at - 70°C.

30

S RIHOSOME (tl)

CONFORMATIOSAL and

lrtactivation

HETEROGESETTY

activation

.X3

of 30 8 rihosomes

‘l’hc> prepamtioll of inactive ribosomes was carried ollt by dialysis of ribosomes agaitkst, ilractivation buffer overnight according to the procedure of Zamir et a/. ( 1971). Activation of rihosomes was accomplished by diluting the ribosomes wit,11 an appropriate buffer so Illat the final concentration of the salt solution was that of tllc activation buf?‘er. After Irlcubation for 25 nlin at 4O”C, the ribosomes were chilled. Activity was tested by t,akiny sarnplw of ribosomes (0.1 A,,, unit) into 50 ~1 of binding buffer containing 5 ~1 of poly(L) ( IO m~jml) phls 10 ~1 of [14C] Phe-tRNA (280 AzGO units/ml). The react,ion was allowed to take placr at O’C for 20 min and was ttren stopped by adding 1 ml of cold wash buffer. f’c~llowrd 1)~ filtjerirrg through a nitrocrllulose filter (Millipore Corp. type HA). The rhoc~c~llulose filt,car \vas \vashed 3 times with 3 ml of wash buf%‘cr each time and was thelk dissol~~:tl itr IO 1111 of scintillation fluid containing Biosolv BBS-3 (90 nil/l) (Beckman Inc.) t\tltl PPO (2,5-dipllengloxazonr) (5 g/l). Radioa&vity was counted in a Beckman LR-250 licllG(l srintillatiori collnter. (e) lodinal%on Ribosomes Lvere dialyzed against their appropriate butlers without fi-mercaptoettrat~ol or dithiothreitol prior to iodination. Iodination was carried out by the addition of a mixture of W-5 nz-RI and 0.05 ~-1~ according t,o the method of Covelli & Wolff (1966). The anlo trt of lz51 r~sed for iodination in individual experimentjs varied between IO7 and IO9 ct,s/min per pm01 of iodine. The volumes used were limited to 100 to 200 ~1. The rc~act,ion was carried out at 0°C for 5 min and was terminated with 100 ~1 0.02 11.socliuul I triosrllfatje. Proteins were extracted with 667; acetic acid and M$+ as described I, Hardy et al. (1969). Excess iodide was removed by dialysis. ‘I’lrcb specific activity of t,he iodine solutions was determined by dirt& titration of t11tl 1, 1 stock solution using a standard solution of sodium tlliosulfat,t~. The amount of radioactiI,ity was determined by direct counting in a liquid scintillation counter (Beckmarl I,S-300) using a system previously described by Changchien & Craven (1977). The specific itcf ivity was also determined by reacting wit’h known amounts of lysozyme in 8 M-Ilrwl. Using ttte Sam61 conditions as described by Corelli & Wolff (1966) we found 6.48 t,o 6.75 Iv-at,oms of I/molecule of lysozyme. This compares favorably with tile maximum found 1,) ;tl(ss(, allttrors of 7.0 corresponding to 3 diiodotyrosines and 1 rnorloiidollist,idille.

(f) Polyacrylamide

gel electrophoresis

The polyacrylamide gel electrophoresis procedure described by Voynow & K~uland (19il) was used. Three Azso units of 30 S ribosomes or the equivalent amount of protein \VNY: applied to eactl gel. Proteins extracted with acetic acid as described above w’erc \.acuctrn-dried in a desiccator. The dry proteins were redissolved in 150 ~1 8 M-urea COILtainitlp 50 ~1 /3-mercaptoethanol. Proteins were allowed to stand at room temperature fol 20 to 30 min to ensure reduction before being applied to 13$4 acrylamide gels (13.3”,, (by/v) acrylamide crosslinked with 1 y/o (w/v) methylenebisacrylamide and a short spacer of 0.625?& methylenebisacrylamide crosslinked with 2.57; acrylamide to condense ttle protrGns). Electrophoresis was carried out at 4°C: in p-alanine buffer (pH 4.5) using m&lyltsrle green as a tracking dye. Preparative gels were 20 cm in length and electrophoresis ~~1s for 30 h at 2 mA/gel. Short analytical gels of 7 cm were electrophoresrd for 6 h at 4 t~lA/gel. (:els were stained with 0.1 ‘$6 Coomassie brilliant blue in 12.5?, tricllloroacctic* arid at, 37°C’. Radioactive gels were crushed hy a Uilsoti gel cruslirr wit11 I m*n pcht’ ft~actrotL.

(g)

Trypsin

digestion

Radioactive ribosomal proteins were purified by preparative gel elrctrophorrsia. were fractionated and 400 ~1 8 M-urea were added to eactl vial. After incubation +tation 50 ~1 were taken from each fraction for counting to obtain a radioactive l+artions from t,tt(l middle portion of eacll peak were pooled and dialyzed against

(:els wit11 profik,. 3 to 4 I

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The protein-gel suspension was adof water for 2 days with frequent changes of water. justed to 0.2 M-ammonium bicarbonate. 100 ~1 trypsin (Calbiochem; 10 mg/ml) were added to each protein suspension. Digestion took place in a waterbath at 3’7°C with constant agitation. After 5 h of incubation, another 100 ~1 trypsin were added. Incubation for 20 h resulted in complete digestion of the proteins. Protein suspensions were filt,ered through Whatman no. 1 paper. The filtrate containing the peptides was lyophilized and was redissolved in 1 ml of pyridine/acetic acid (pH 3.1) buffer. They were stored at - 20°C. 10.~1 portions of each sample were counted to estimate the total radioactivity applied to the Dowex 50 column for peptide analysis. Recovery from tile Dowex 50 column was usually between 85% and 95%. (11) Peptide

analysis

The radioactive peptides were separated by ion-exchange chromatography essentially as described by Craven et al. (1969). The peptides in pyridine/acetate buffer (pH 3.1) were adsorbed to a Dowox 50X-8 resin column equilibrated wit,h the same buffer. The column was maintained at 56°C and was eluted with a linear gradient of pyridine/act%ate from 0.2 t)o 2.0 M and from pH 3.1 to 5.0. The eluate was collected in 2.ml fractions, a total of 250 fractions for each analysis. The fractions were then dried under vacuum. Peptides in each fraction were solubilized with 100 ~1 water before the addition of scint)illation fluid for counting.

3. Results Using the method of Covelli & Wolff (1966) (see Materials and Methods) we find that 30 S ribosomal subunits incorporate radioactive iodine very rapidly, reaching saturation within one to two minutes (data not shown). Figure 1 illustrates the incorporation of 1251 by the 30 S particle using different molar ratios of iodine. tn these experiments three different solvent conditions, known to alter ribosome structure, were used. When 30 S ribosomes are suspended in either activation buffer or inactivation buffer (Zamir et al., 1971), the incorporation of iodine increases linearly, relative to the molar excess of iodine to ribosomes, reaching a saturation plateau at approximately 100 moles iodine per mole ribosomes. Beyond this no significant increase in incorporation was observed even up to a 200 molar excess of reagent. In contrast the incorporation in 8 M-urea was linea,r over a far greater range reaching saturation at a 350 molar excess of iodine. These data indicate that the 30 S ribosome suspended in activation or inactivation buffer is relatively compact and inaccessible to the rea.gent having only 15 total sites available for reaction. In contrast: 8 M-Urea dramatically disrupts this structure revealing approximately 95 reactive sites. The iodinated proteins were extracted from both inact.ivated and activated 30 S particles (modified with a loo-fold molar excess of iodine) and separated by polyacrylamide gel electrophoresis (Fig. 2). The patterns of iodine incorporation of the individual ribosomal proteins superficially appear to be t,he same for ribosomes in the active and inactive conformation. Furthermore, the profile of iodine modification is very nearly identical to one previously published for ribosomes suspended in reconstitution buffer (Craven et al., 1974). The incorporation of iodine into the 30 S ribosomal protein moiety, as seen in Figure 2, is remarkably uneven in distribution. Several proteins (Sl, S3, S7, S9jSll and SlSjSl9) take up substantial amounts of iodine, whereas many of the ot,hcr ribosomal proteins appear to be modified at very low levels. Finally there is a group of proteins (SS, 520, 521, and some members of the peak occupied by S14, S15, S16, and 517) which completely fail t’o become iodinated. The large difference in the amount of iodine incorporation among the three classes of proteins suggests that the

20

S RI

HOSOME

CONFORMATIONAI~

Molar

rotlo

HETEROGENEITY

of lodme

x5

to rlbsome

FIG. 1. Iodine incorporation into the 30 S ribosome suspended in 3 different, solvents. Iodination \vas carried out as described in Materials and Methods. The reaction was terminated and the samples exhaustively dialyzed followed by analysis for radioactivity and RNA content. Thn number of sit.es iodinated per ribosome was estimated from the known specific activity of 0x1 mixture of lz51 and I,. Active ribosomes were prepared by dialysis against activation buffet followwl by heat,ing to 42°C. Inactive ribosomes were prepared by dialysis agair& inactivat’ion tmffer. Activit,y studies using poly(U)-directed [lV]Phe-tRNA binding at 4°C showcd that t,h(b active part,icles bound 0.154 pmol Phe-tRNA/mol 30 S ribosome and under t,he same conditions inactive preparations bound 0.017 pmol. -e-o--, 30 8 ribosomes in 8 &r-urea; -- .2-2 --. actiw 30 S ribosomex; -- x --x --, inactive 30 S ribosome.3.

30 S ribosome population is heterogeneous, containing some particles more accessible t’o the reagent than others. This conclusion is strongly support’ed b,y a quantitJabive analysis of the iodinat.ion pattern as described below.

(a)

Betermination

of the wumher

of iodination

sites for each riboaomal

protein

WC’~have estimated the degree of incorporation of iodine into most of the individual proteins by two completely different methods. One method involves the isolation ot t,he individual iodinated proteins from the polyacrylamide gel followed by complete proteolytic digestion and separation of the resultant peptides. Figures 3 and 4 shot\, the peptide separations of iodinated proteins extracted from eight regions of the polyacrylamide gel electrophoresis pattern shown in Figure 2 (cross-hatched areas). In general, the radioact.ive peptides are resolved into several distinct entities on the Dowrx-50 column. For example, in the case of protein S3 (Fig. 3, iii), the radioactivtb iodine is distributed in a qualitatively equal way among four unique tryptic peptides. suggesting t’hat this protein has four sites equally accessible to bhe reagent in the intact ribosome. Similar estimates of iodine reactivity for most of the other individual proteins can be made on the basis of their peptide patterns. Our second method for determining the relative number of iodination sites of t,he 30 S proteins ut,ilizes t,he data of Figures 1 and 2 directly. In Figure 1 we estimat,e tha,t.

566

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c

(a)

,-

I-

(b) , -

I-

i-

SI

;;dl 53

>-

$2 % s4

l-

50

100 Fmctian

no.

s2

150

FIG. 2. Polyacrylamide gel electrophoresis analysis of iodinated (lz51) 30 S ribosomal proteins. Ribosomes were iodinated with a loo-fold molar excess of reagent. The protein component was extracted and applied to polyacrylamide gels. The gels were fractionated and samples taken for estimation of radioactivity. The cross-hatched areas represent pooled fractions used in later peptide analyses. (a) Inactive 30 S ribosomes; (b) act,ive 30 S ribosomes (see legend to Fig. 1).

on the basis of the known specific activity of the iodine solution, approximately 15 atoms of iodine become bound to each 30 S ribosome at saturation. Assuming that there are no selective losses of individual proteins in the polyacrylamide gel electrophoresis procedure, we can estimate the relative percentage of these iodination sites which can be accounted for by the total counts incorporated into each protein. Thus, the percentage of total counts recovered from the gel for each protein, divided by the total counts recovered from the entire gel should be proportional to the percentage of total iodination sites. These data are summarized in Table 1 and compared to the estimated number of iodination sites derived from the peptide map studies outlined above. In Table 1 the first column lists the percentage of the total counts recovered from the polyacryalmide gel present in each individual protein. In column 2 this percentage of total counts is converted to the number of iodination sites per protein by multiplying it by the total number of accessible sites per 30 S particle (i.e. 15). This should be compared to the total number of iodination sites found for each protein

30

S RIHOSOME

CONFORMSTIOSA4L

HETEROGENEITY

z KA -w

,E 2 : a

5ti5

15

2 IC

15

IO

5

5

0

-

0

IE,I

l&B

4

IC , 5

C

50

100

150

200

250 Fraction

0

50

100

150

200

250

no

&I:. 3. Radioactive peptide patterns of purified proteins extracted from ‘“51-iodinated 30 S with trypsin as described ribosomes. Pooled protein fractions as indicated in Fig. 1 were digwted in Materials and Methods. The digests were separated by ion-exchange chromatography using Dowox 50. The reproducibility of this chromatographic separation has been previously documented by Changchien & Craven (1977). (a) From inactive 30 8 particles; (Is) from active 30 8 particles: (i) from protein Sl ; (ii) from prot,ein R2; (iii) from protein S3 ; (iv) from prokin S7.

by peptide analysis (column 3). As can be seen in most] cases the number of actual unique groups on t,he protein which become iodinated at saturat’ion is significantl! greater t)han predicted from the relative distribution of radioactivit,y as reflected in column 2. One possible explanation for this discrepancy bet,ween t#hr number of iodinatabh sites estimated by the two different methods might be traced to the ribosome preparation itself, which is heterogeneous in protein composition (Voynow & Kurland, 1971). Thus, a protein which has four unique radioactive peptides but incorporates onl!, two hits per ribosome might be present in only 0.5 of a copy per ribosome. On this basis we can calculate the relative stoichiometry each protein would need to account, for the observed differences. These calculated stoichiometries are listed in column 1 of Table 1 along with the published values for salt-washed ribosomes (Voynow & Kurland, 1971) in column 5. Clearly only in a few cases, proteins Sl and S7, do the

568

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0 x c

T.

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CHANGCHIEN

AND

30

15

20

IO

IO

5

0

0

30

15

20

IO

IO

Y 0

5

0

: E

0

G.

R.

CRAVEN

15 IO -

,

3 Fraction

no

FICJ. 4. Radioactive peptide patterns of purified proteins extracted ribosomes. Conditions are the same as described in the legend to Fig. (ii) from proteins S9jSll; (iii) from proteins S12/S13; (iv) from proteins

from lz51-iodinated 3. (i) From protein SlSjSl9.

30 S SlO;

stoichiometry values calculated from the discrepancies in iodination sites come close to values found in the literature. A better explanation of this phenomenon would appear to be that the ribosome population is heterogeneous in its accessibility to the iodination reaction. That is, not all ribosomes in the population have the same proteins exposed to the reagent. The actual percentage of the population having any one protein reactive to iodine can be computed by dividing the calculated stoichiometry (column 4) by the value from the literature (column 5). This percentage figure, then, corrects for the possibility that some of the proteins are present in the population in amounts of less than one copy per ribosome. As can be seen, the resultant percentage of the 30 S ribosome population having any given protein accessible to iodination is far less than 100% except for proteins Sl and X7. Proteins 52, S3 and S9jSll are exposed in approximately 30 to 60% of the particles which have these proteins as components, and proteins S5/S6, SlO, S12/S13, SlSjSl9, and probably 54 are reactive in less than 17% of the ribosomes containing these proteins. The conclusion seems inescapable that the 30 S ribosome preparation used in these

dctlve

Sl s2 S3 s4 S5/HF Si SlO S9jSll S12/813 s14-SK s1sjs1n

s 1 S% 83 s4 s.Lii/s(i s7 SlO S9jSII SlZ/Sl3 814-817 SlS/Sl9

Inactive

l’rotein

30

s

30 S

1” 5 3 5

15 4 14 6 5 29

12 4 13 5 5 34 3 1” 5 4 6

‘jO Total cts/min

14.86

2.25 0.60 2.10 0.90 0.75 4-36 0.15 1.80 0.75 0.45 0.75

14.93

1.75 0.63 1.74 0.78 0.70 5.11 0.42 1.77 o-77 0.55 O-81

Yredictrd no. of sites

0.10

O*%O 0.50 0.75 0.88 1.60 WHO 0.75 1.40 1.50

1 *30

0.23 1.28 0.13 0.44 0.10

Vahcs from literature for etoichiometry

0.20 0.50 0,75 0.88 1.60 0.90 0.75 1.40 1.50

0.44 O-28 o-35

(‘alcdateti .+toichiometry

> 101) 60 56

8

14 3> 100 17 3 I 7

>- 100 56 47

‘lo Kibosomcs iudinatetl

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experiments is composed of a population of particles heterogeneous in their accessibility to protein modification. We suggest that this is due to a heterogeneity in particle topography. (b) The active to inactive

transition

involves a conformational 30 X proteins

change in several

The peptide patterns shown in Figures 3 and 4 include a comparison between t.he proteins extracted from 30 S particles iodinated in two different buffer conditions. Ribosome suspensions were equilibrated by dialysis with the buffers prescribed by Zamir et al. (1971), which induce a conformational shift in ribosome structure (Ginzburg et al., 1973). The two conformational forms are referred t’o as the active and inactive configurations. The active form also requires a short period of pre-heating (see Materials and Methods). Inspection of the peptide patterns in Figures 3 and 4 reveals that most of the corresponding proteins from the two different 30 S ribosome preparations have nearly identical profiles. Thus, in general, the protein component of the 30 S particle does not change its accessibility to iodine as a consequence of the active to inactive transition. This conclusion was also derived previously from the enzymatic iodination studies of Litman et al. (1976). However, several proteins are exceptions to this generalization, showing distinctly different peptide profiles. Protein SlO and proteins S18/S19 are more exposed in the 30 S ribosomes suspended in inactivation buffer, whereas proteins S12/S13 appear to be extensively rearranged showing dramatically different peptide patterns due to the change from inactive to act’ive conformation. It is difficult to assess the significance of these alterations in protein structure as related to protein function, but the fact that several lines of investigation have implicated both proteins S12 and S13 as important in ribosome activity should not be overlooked (e.g. see Cantrell & Craven, 1977; Chang & Craven, 1977; Pongs et al., 1975; Zengel et al., 1977). (c) The observed conformational heterogeneity ribosome preparation

is not due to the method of

The 30 S ribosomes used in the above experiments were prepared by the (NH&SO, washing procedure devised by Kurland (1966). This procedure normally involves three consecutive washing steps with high concentrations of salt. It is known that these salt-washing steps cause partial detachment of some ribosomal proteins (Hardy, 1975). We have found that although a single washing with the high salt does not remove all contaminating supernatant proteins, it does yield 30 S particles with greater relative amounts of the fractional proteins (data not shown). We examined the possibility that this preparation of 30 S ribosomes is less heterogeneous than ribosomes composed of lower amounts of the fractional proteins. Figure 5 shows the gel electrophoresis profiles of iodinated proteins extracted from the two preparations. The two gel patterns are essentially identical suggesting that the observed heterogeneity in iodine modification is not due to the method of ribosome preparation.

4. Discussion Chemical modification has been widely used to probe the function of ribosomes (Noller et al., 1971; Thomas et al., 1975: Shimizu & Craven, 1976; Chang & Craven,

s7

16-(o) 14 12 IO 8x

SI

6x

4-

c (b)

14

20

57

40

60

80

100 Fraction

I20 no

140

160

180 ZCC

PIG. 5. I’olyacrylamide gel electrophoresis analysis of ‘251-iodinated 30 8 riboaomal proteins obtained from 2 different salt-washing procedures. 30 S ribosomes were separated from 70 S ribosomes which had been washed either once or 3 consecutive times with 7.8% (w/v) ammonium tinlfate. (a) One salt wash; (b) 3 salt washes.

1977 ; Cantrell & Craven, 1977). In addition, a number of investigators have ut,ilized a variety of chemical reagent,s to probe ribosome topography as reflected by relative chemical reactivity (Benkov & Delihas, 1974; Moore & Crichton, 1974: Moore, 1971; Craven & Gupta, 1970; Chang, 1973; Kahan & Kaltschmidt, 1972: Miller & Sypherd, 1973). Without exception these studies have shown that, regardless of t’he chemical reagent employed, there is a wide variation in relative chemical reactivity among the 30 S ribosomal proteins. Thus, some of the proteins have consistently been found to be highly reactive, a second class of proteins usually has been seen to be moderately reactive and a third group consistently has been shown t’o btl c&her completely unreactive or only slightly reactive. The pop&r explanation for the existence of these three classes of reactivity has been vaguely attributed to sonn sort of relative degree of macromolecular association. That is, those proteins which are unreactive are ones which are most involved in protein-protein and protein-RNA interactions making their amino acid functional groups essentially inaccessible. Cominuing this argument, the greater the reactivity of a pa,rticular protein to a variety of chemical reagents, the less the protein is involved in inter-molecular interactions. Although this is an attractive hypothesis on the surface, there is ver>r little hard evidence to support it,. If this simple hypothesis is correct,, a protein which incorporates. for example, four times as much of a chemical reagent as anot)her protein, should have four sites of‘

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reaction for every one site in the other protein. The results we report here effectively t,est this hypothesis by quantitating the pattern of iodine incorporatjion of the 30 S subunit followed by peptide map analyses t’o reduce the actual number of reactive sites per protein. The results of this investigation as summarized in Table 1 are startling. In fact, there is no correlation between t,he ext,ent of iodine incorporation and the number of iodine reactive sites per protein. For example, prot’ein S7 incorporates approximately 30% of bhe total iodine bound to the 30 S particle, whereas protein SlO incorporates between 1 and 3%. However. the peptide map analysis shows that protein S7 has only four reactive sites as compared to three sites for protein SlO. The conclusion seems inescapable that, the ribosome population is composed of only a minor fraction, approximately 150/,, which have protein SlO situated so that it is accessible to iodination. Careful inspection of the data presented in Table 1 reveals roughly three distinct categories of protein reactivity in our ribosome preparations. All ribosomes containing Sl and 57 have these proteins positioned in such a way that they are accessible to iodine. A second class of 30 S ribosomes has, in addition t’o protein 57 (and Sl, if present), proteins S2, S3 and S9jSll exposed to the iodination reaction. This class composes roughly 30% to 50% of the populat’ion. A final category (class III), representing between 5:/, and 150/ of the subunit population, has almost all proteins available for iodine modification (the exceptions are 88, S20, S21, and probably some components of the S14, S15, S16, S17 peak). The proposed t,hree classes of ribosomes are schematically summarized in Figure 6. We propose, on the basis of these results, that 30 S ribosomes, as they are routinely prepared, can assume at least three different conformat,ions and that any given preparation is composed of a distribution of these mixed forms within the population. Furthermore, at least half of the 30 S particles in the population exist in an extremely compact form having only protein X7 (and Sl, if present) exposed to iodination. This is a surprising conclusion in itself, but when contrasted with class III ribosomes becomes even more unexpected. Class III particles have between 10 and 14 proteins approximately equally accessible to chemical modification. This suggests a dramatically “loose” and open topography for this class with between 30 to 40 iodine reactive sites as opposed to class I subunits with only four exposed sites.

Class II

FIG. 6. Schematic illustration have distinguishable protein It could be a member of either metry in a given preparation

of the 3 different chemical reactivities. of the other 2 classes of particles.

Class III

topographical 81 has been of ribosomes

classes arbitrarily depending

of 30 S ribosomes which placed in class III. on its actual stoichio-

30 S RIBOSOME

CONFORMATIONAL

HETEROGENEITY

.??I1

There are at least two possible explanations of the class III type of ribosomc conformation. First, ribosomes may become partially degraded, denatured and unfolded during preparation opening up many new sites t’o the chemical reagent. Alternatively, it could be that ribosomes of the class III conformation are actually t,hose involved in biological functions which require an open and flexible structure. The ribosomes used in these investigations were usually between 57;) and 15% active for poly(U)-directed Phe-tRNA binding. This percentage corresponds well with the percentage of ribosomes in the form most accessible to iodination. More important. recently Shimizu & Craven (1976) demonstrated that iodination of 30 S ribosome:: results in a complete loss of poly(U)-directed Phe-tRNA binding. Since reconstitut’ion of these inactivated particles in the presence of unmodified prot#eins rest)ored the activity, it, was possible to identify the proteins at the site of inact’ivation. The proteins identified were Sl, S2, S3, S14 and S19. All of these proteins are members ot class II and class III ribosomes. The two prot,eins implicated as most important, in Phe-tRNA binding were S14 and S19 from class III. This supports the notion that funct,ionally active particles have a more flexible or accessible conformat’ion than inactive particles. However, a counter-argument to this proposal could be made on the basis of the comparision of iodination patterns between act’ive and inactive ribowomes. ,4s we have shown, and as was also shown by Litman et al. (1976), there is really very little change in iodine reactivity when t’he 30 S part,icle undergoes the transition from inactive to the active form. Thus, the question as to whether t ht. cla,ss III conformation represents the active members of the population or a disrupted component cannot be answered by the data presented here. Such speculations could be tested with preparations of 30 S ribosomes which are lOOo/o active. Our proposal that 30 S ribosome preparations from E. coli are composed of a conformationally heterogeneous population of particles is not a new idea. In fact, data supporting this concept have been published by several laboratories. For example, Ginzburg & Zamir (1975) quantitatively examined t’he incorporat.ion of fl-etJhyl maleimide int,o individual 30 S ribosomal proteins. In one experiment they found that proteins S2 and S4, both of which have a single cysteine residue, incorporated 2431 counts and 710 counts, respectively. If the amount of maleimide bound t,o protein 82 represent,s a single site per ribosome for all the ribosomes in the population, then only about 30% of these ribosomes also contain S4 in a configuration exposed to this reagent. These data may mean that a minority of t’he ribosomes under these conditions have a conformation in which the cysteine of S4 is exposed and the majorit>assume a structlure which blocks the modification of the S4 sulfhydryl group h! malrimide. More convincing evidence of conformational heterogeneity in 30 S particles can 1)~ seen in the data of Michalski & Sells (1974) and Litman et al. (1976). These authors report the quant’itative analysis of enzymatic iodination of the 30 S ribosome. Thesca t’wo independent laboratories found that the amount of iodine bound by prot#ein S7 was: for example, at least 30 times the amount taken up by proteins S6, S15 and Siti. As all of t’hese proteins contain some tyrosine, one is forced to conclude that only a minor proportion of theribosome population has S6,815 and S16 exposed to enzymatic iodination. The antibody studies of StXler et al. (1973) should also be cited as possible evidence for conformational heterogeneity. These investigators used antibodies specific fol ckach 30 S rihosomal protein t>o determine the acccssibilit,y of each protein in t,ht:

674

M.

K.

T.

LAM,

L.-M.

CHSNGCHIEN

AND

G.

R.

CRAVEN

intact 30 S subunit. They used three different techniques to arrive at quasiquantitative data regarding the extent of antibody reaction at saturation. They found, for example, by one technique, that 100% of the ribosomes contain exposed antigenic sites for proteins S9 and X12, but only approximately 30% have available antigenic sites for proteins Sl, S8, SlO, S13, S15, S16, S17 and S19. Once again, the most logical and direct conclusion from these data is that the ribosome population is composed of particles having significantly different topographies. Our proposition that 30 S ribosomes are conformationally heterogeneous has many important ramifications for all investigations involving structure-function relationships. For example, the immuno-electron microscopy work of Tischendorf et al. (1975) and that of Lake & Kahan (1975) involves the determination of antigenic sites for specific proteins on the surface of the particle. Their techniques necessarily examine a minor proportion of the ribosome population. Thus, the model of the 30 S ribosome developed by these workers could reflect a minor conformation of the particle. A similar indictment can be made about most of the investigations of proteinprotein relationships in the 30 S ribosome by the use of cross-linking reagents. These reagents have been used by a number of laboratories to determine near-neighbor relationships of the proteins; however, in virtually all of the experiments so far reported, the yields of specific cross-linked products is extremely low (e.g. see Lutter et al., 1972 ; Sun et al., 1974 ; Shih & Craven, 1973 ; Sommer & Traut, 1976). Recently, for example, Expert-Bezancon et al. (1977) showed that the commonly used crosslinking reagent dimethyl suberimidate produces cross-linked pairs of proteins in yields of above 20% in only four cases.All other cross-link products were obtained in 3% or less of the ribosomes. Over 30 pairs of proteins have been implicated to be situated close to one another through the use of these reagents and, in general, the yields of most of these cross-links are extremely low. One possible reason for the low yield of some of these products is that they are derived from a minority conformation of the particle. This idea is now even more plausible in light of our data supporting the hypothesis that 30 S ribosome populations are heterogeneous in conformation. We are indebted to Dr B. Rigby for his participation in preliminary studies on iodination of the 30 S ribosome. This work was supported by the Graduate School and the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by research grant GM15422 from the National Institutes of Health. We also acknowledge use of the Biochemistry Department pilot plant, directed by Dr John Garver and supported by United States Public Health Service grant Fr-00214.

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:I0

S ItIB080ME

CONFORMATIONAL

HETEROGENEITY

.-I; 5

(‘ravrktr. G. K., K&by, H. & Changchien, L.-M. (1974). In Kibosomes (Norn~~ru, M., TissiBres , A. & Lengycl, P., eds), pp. 559-571, Cold Spring Harbor Laboratory. Ne\v I’ork. 7 bxpel~t.Hrzanco~r, A., Rarritault, D.? Milet, M.. Gnernin. hI.-F. $ Hayes, D. H. (197;). ./. Mol. Rio/. 112, 603-629. (:itlzburp. 1. bi Zarnir. A. (1975). J. Mol. Biol. 93, 465-476. (:inzburp. I., Miskin, R. & Zamir, A. (1973). J. ,lilol. Biol. 79, 481 -494. (:lukllova. M. A.. Relitsina. N. V. & Spirin, A. S. (1975). Eur. .I. Biochem. 52, l!jim202. Hardy, 8. .J. S. (1976). Viol. Gen. Genet. 140, 253-274. Hard),, S. .J. S., Kurland, C. G., Voynow, P. & Mora, G. (1969). Biochemistry, 8. 2897 2905. liahatl. I,. & Kaltschmidt, E. (1972). Biochemistry. 11, 2691 2698. Kurland. C. G. (1966). J. ;Wol. Biol. 18, 90-108. I,akr. J. A. & K&an, L. (1975). J. ilrcol. Biol. 99, 63lG644. I,itmarl, D. J.. Reekman, A. L Cantor, C. R. (1976). Arch. Biochem. Biophys. 174. 523 -531. I,ntt.ctr. I,. (‘.. Zeichardt, H., Kurland, C. G. & Sttiffler, G. (1!)72). ~Wol. Gen.. G&et. 119, Xii- 366. Jlichalskl. (1. .I. & Sells. B. H. (1974). Eur. ./. Biochem. 49. 361-367. Miller, K. V. & Sypllerd, P. S. (1973). J. Mol. Biol. 78, 539~5.70. Moorc~. (:. &, (‘richton. R. R. (1974). Biochem. J. 143, 607 612. Moorcx, P. B. (1971). ,I. Xol. Biol. 60. 169-184. ?joll(~r. H. ld’.. Cilatlg. C.. Thomas. G. & Aldridgo, J. (1971). ./. ‘Mol. Bid. 61, 669 679. I’OII~S, O., St,ijfflrr, (:. & Lanka, E. (1975). .I. Xol. Biol. 99, 301-315. Shill. C. Y. T. dz CraveI), G. R. (1973). J. &!ol. Biol. 78, 651-663. Sllimlzll. AM. & Craven, (:. R. (1976). Eur. J. Biochem. 61, 307-315. Skoultjca)li, A.. Ono. Y., Waterson, J. & Lengpel. P. (1969). noltl ,Ypring Harbor ,qymp. Qctatct. Hid. 34, 437 -454. Somnx:r, A. Jt. Trallt. R. It. (1976). J. ~Uol. Biol. 106, 995-1015. Stiifflf~r. G.. Hasellbank, K., Liitgehaus, M., Maschler, K., Morrison, (‘. A., Zeichardt, H. k Garrett, K. A. (1973). &Iol. Gen. Genet. 127, 89~ 110. SIIII, I’-‘~.. Hollcn, A., Kahn, L. & Traut, 1%. R. (1974). Biochemistry, 13, 2334--2340. ‘I’llonus. (i., S\verrrcy, It., Chung, C. & Noller, H. F. (1975). J. Mol. Biol. 95, 91&102. Tischrndorf. G. IV.. Zeicllardt, H. & Rtaffler, (:. (l!175). I’roc. :Vaf. .4carl. Sci.. T7.S.d. 72, 4820 4824. \:oynow, 1’. 8.x Kurland, C. C:. (1971). Biochemistry, 10, 517~-524. Zamir, A., Miskin, K. 8: Elson, D. (1971). J. Mol. Biol. 60, 347-364. Zc*rlg(~I. .I. M., Yo11ng, IX., Dennis, P. P. & Nomura, M. ( 1977). J. Bacterial. 129, 13261329.