Eur. J. Biochem. 29,642-662 (1972)

A Series of Specific Ribonucleoprotein Fragments from the 3 0 3 Subparticle of Escherichia coli Ribosomes

Joan MORGAN and Richard BRIMACOMBE National Institute for Medical Research, Mill Hill, London

(Received June 19,1972)

1. 30-5 ribosomal subparticles were hydrolysed with ribonuclease TI or pancreatic ribonuclease in the presence of 2 M urea. The RNA * protein fragments produced were separated on 501, polyacrylamide/0.5 ,Ilo agarose composite gels. Fractions from the composite gels were subjected to protein analysis on 17.5O/, periodate-soluble polyacrylamide gels run in the detergent sarkosyl, using the technique already published.

2. Several RNA protein fragments were obtained by this procedure, each containing equimolar amounts of a few specific ribosomal proteins. Strict criteria for specificity of the proteins in each fragment were applied. Proteins 58 and 515 were found reproducibly in a small RNA * protein fragment, with only trace amounts of the remaining ribosomal proteins. Six proteins (57, S9, 510, 513, 514, and S19) constitute another group, and fragments containing three, four, five, or all six of these proteins were observed. Protein S20 was occasionally associated with this group, or with 56, 516 or 517, and possibly 521. The latter (including 520) were also found in association with S8 and 515.

3. The individual proteins found in specific fragments are interpreted as being close neigh- bours in the 30-S particle. The fragment data can be incorporated into a re-arranged version of Nomura’s “assembly map”.

In our last paper [l] we described a method for the analysis of ribonucleoprotein (RNA * protein) fragments, which we obtained by mild nuclease digestion of Escherichia coli 30-5 ribosomes. The underlying objective of the work was that a precise analysis of small specific ribosomal fragments pro- vides a direct method for examining the arrangement of proteins and RNA within the particle. I n this paper, we apply the technique to a number of RNA .protein fragments, produced under a variety of different hydrolysis conditions, and discuss the results in relation to Nomura’s “assembly map” [2,31.

MATERIALS AND METHODS Preparation of Ribosomes

30-5 ribosomal subparticles were prepared from E. coli MRE 600 (obtained from MRE, Porton, U.K.) as described before [1,4], with the following modifica-

Abbreviations. RNA * protein, ribonucleoprotein; sarko- syl, N-lauryl sarcosine.

Enzymes. T, ribonuclease (EC 2.7.7.26); pancreatic ribonuclease (EC 2.7.7.16).

Definition. An A,,, unit is the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1-em path length cell.

tions. After grinding with alumina [a], the cell paste was extracted with 10 mM MgC1, rather than 0.1 mM, to maintain the ribosomes as 70-5 particles. These 70-5 particles were washed by spinning through 0.5 M NH4Cl, 10 mM MgCl,, 10 mM Tris-HC1 pH 7.6 (cf . [5]) , and were then dissociated into subparticles by resuspending in 50mM KC1, 0.3 mM MgCl,, 10mM Tris-HC1 pH 7.6. The subparticles were separated in a zonal rotor as before [I], and the 30-5 ribosomes precipitated with ethanol. The precipitate was dissolved in 0.3 mM magnesium acetate, 10 mM Tris-HC1 pH 7.6, and dialysed against this buffer, or against 1 mM magnesium acetate, 10 mM potassium phosphate buffer pH 7.2.

Ribosomes were labelled as before [l] with 14C-

labelled amino acids (CFB 104) and [3H]uridine (Radiochemical Centre, Amersham), except that isotope input was increased to give specific activities of approximately 450 counts x min-l x pg-l for 14C- labelled protein, and 2500 counts x min-l x pg-l for [SH]RNA.

Separation of RNA Protein Fragments Radioactive 30-5 ribosomes were hydrolysed

with ribonuclease T, or pancreatic ribonuclease (Sigma) for 4.5 h a t room temperature, in the

Vo1.29, No.3,1972 J. MORGAN and R. BRIMACOMBE 543

buffer systems described below. The time allowed for hydrolysis did not appear to be critical (cf. [l]). Reaction mixtures contained 1000 units/ml of ribo- nuclease T, or 0.01 pg/ml of pancreatic ribonuclease (except where specifically indicated), and 20-30 A,,, units/ml of 30-5 ribosomes. The enzyme hydrolysates (0.2-0.4 ml), together with controls (minus enzyme), were loaded into 2-cm slots of 5O/, polyacrylamidel 0.5O/, agarose composite gel slabs [6] as described previously [l], but with modified buffer systems as indicated below. Electrophoresis was continued for 18 h at 6 “C with bromophenol blue as a dye marker. Current and voltage (I0 to 30 mA, 50 to IOOV) were adjusted in each experiment so that the dye marker ran 10 to 15 cm in this period. Two buffer systems were used for the electrophoresis : in the “phosphate system” [l], gel and reservoir buffer were both 1 mM magnesium acetate, 10 mM potassium phosphate pH 7.2 ; in the “Tris system”, the gel buffer was 25mM Tris-HC1 pH 7.8 and the reservoir buffer was 25 mM Tris-citric acid pH 8.8, with 20 mM KC1 and 1 mM or 5 mM magnesium acetate in both gel and reservoir.

I n the various experiments, the following com- binations of hydrolysis buffer and electrophoretic system were used:

a) Hydrolysis in 1 mM magnesium acetate, 10 mM potassium phosphate pH 7.2 [l]; electro- phoresis in the “phosphate system” (see above).

b) As for (a), but with 2 M urea added to the hydrolysis buffer.

c) As for (a), but with 2 mM EDTA added to the hydrolysis buffer.

d) Hydrolysis in 20 mM KC1, 1 mM magnesium acetate, 10 mM Tris-HC1 pH 7.8; electrophoresis in the “Tris system”, with 1 mM magnesium acetate throughout.

e) As for (d), but with 2 M urea added to the hydrolysis buffer.

f ) As for (e), but with 5 mM magnesium acetate in the electrophoresis buffers.

g) As for (e), but with 5 mM magnesium acetate in both hydrolysis and electrophoresis buffers.

After electrophoresis, the gels were sliced and analysed as described previously [ 11, taking one 1.3-cm and one 0.5-cm longitudinal strip from each 2-cm gel slot. The 1.3-cm strip was kept for protein analysis, and the 0.5-cm strip was assayed for radio- activity, after cutting both into 0.17-cm slices (cf. Fig. 1 of [I]).

Xeparation of Proteins The ribosomal proteins were separared as before [I]

on gels containing 0.50/, sarkosyl (Geigy [U.K.] Ltd) [7], and 1.5O/, N,N’-diallyl tartardiamide [8], but the acrylamide concentration was increased from 1 5 O / , to 17.5O/,. Since the enzyme hydrolysate

was applied to the RNA-protein gel in narrower slots than before (2 cm), single slices only from the latter (1.3 x 0.17 cm) were loaded into each slot of the protein gel, together with unlabelled 30-5 subparticle and pancreatic ribonuclease [I]. (Pre- viously, 2 or 3 slices were applied to each slot of the protein gel, see Fig. 1 of [l]). After electrophoresis, the gels were stained and analysed exactly as described before [I].

Examination of RNA Slices from RNA * protein gels were loaded into

1.3-cm slots of a 3 O / , polya~rylamide/0.5~/~ agarose composite gel, side by side with 2343, 1643, 5-S and 4-5 RNA markers. Electrophoresis was in the “Tris system” (see above), but with O.O5O/, sodium dodecylsulphate in place of KC1 and magnesium acetate (cf. [el). Electrophoresis was a t 300 V, 30 mA for 3 h. The gel was sliced and assayed for radioactivity, after homogenizing the slices [ 11. Peaks of [,H]RNA were clearly distinguishable from the peak of dissociated 14C-labelled protein com- plexed with dodecylsidphate. The marker RNA species showed the expected linear relationship between mobility and log molecular weight, from which the molecular weights of the RNA components of the RNA * protein fragments could be estimated.

RESULTS AND DISCUSSION Our method of analysis of RNA * protein frag-

ments has already been described in detail [l]. To summarize it very briefly, the radioactive fragments are separated on a polyacrylamide/agarose composite gel, and individual fractions of this gel are analysed for protein by loading the gel slices directly onto a second polyacrylamide gel. The second gel contains the detergent sarkosyl[7], and the periodate-sen- sitive gel cross-linker N,N’-diallyl tartardiamide [8]. The detergent dissociates protein from RNA, and separates the ribosomal proteins on the basis of molecular weight [7]. An aliquot of intact unlabelled 30-S ribosome is added to each sample to give a stainable pattern of total 3023 protein. The gel is analysed by cutting out each stained protein band, dissolving it in periodic acid, and assaying for radioactivity. In these experiments, the acrylamide concentration of the sarkosyl gels was increased to 17.5O/,, and the improved pattern of separation of the proteins is illustrated schematically in Fig. 1. Three new bands, labelled E,, F, and G,, can be seen (cf. [l]). Resolution between bands E, and E,, F, and F,, and G, and G, was occasionally poor; in such cases the two badly-resolved bands were cut out together as a single band. Fig.1 also shows the ideni,ification of the proteins with their literature counterparts [9]. It should be noted that bands F, and G, each contain two proteins, and that proteins

544 RNA Protein Fragments from Escherichia coli 30-S Ribosomes Eur. J. Biochem.

Gel origin Hydrolysate

S

D1[ 19 700 Dz[ 18 100

[S12)------ (Sl1)-

(521)-

15 500) 14 900) 14 700) 13 900) 13 700) 13 300) 12900) 12500) 12300) 1 1 800) 11 600)

Fig. 1. Schematic representation of the separation of 30-8 ribosomal proteins on 17.5°/0 plyacryEamide-sarko.sy1 gels. Our nomenclature [i] is shown on the right of the diagram, and the corresponding international nomenclature [9] on the left. Molecular weights given were measured from mobility in the sarkosyl gel (cf. [i]). (Faint aggregate bands in the upper part of the gel are not included in the diagram)

52, S11, S12 and 521 have not yet been identified. The positions of these four proteins in Fig.1 have been allocated on the basis of their published molec- ular weights [lo], as compared with the molecular weight values estimated from mobility in the sarkosyl gel (cf. [l]). We tentatively suggest that 821 corres- ponds to G,, for which no counterpart has so far been found. There are also faint bands in the sarko- syl gel in the positions which we have assigned to S2, Sl1 and S12. However, until all 21 proteins have been positively identified in the sarkosyl system, we retain our nomenclature for the description of the experimental results. 513 (“PlOa” [2]) corres- ponds to F,, not G, as previously reported [l].

In the hydrolysis conditions of our previous experiments, the yield of the spec& fragment “Band 111” [l] was variable, and there was always a considerable quantity of apparently unhydrolysed 30-5 material. In order to obtain more complete breakdown, and a wider spectrum of fragments, there was a clear need to modify the hydrolysis con- ditions so as to allow both greater accessibility to the nuclease, and a readier release of the fragments after the RNA has been broken. This of course must be achieved specifically, and without dissociation of the fragments into RNA and protein. Our first

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Fig.2. Electrophoretic profile of RNA . protein fragments from a ribonuclease-Tl hydrolysate in 2 M urea (condition f ) . The fractions are 0.17-cm slices from a 5% composite gel, and the direction of electrophoresis is from left to right. The bromophenol blue marker ran to to fraction 60. ----, Radioactivity in 14C-Iabelled protein; ~ , radioactivity

in [3H]RNA

attempts to do this involved simply lowering the magnesium ion concentration to below 0.1 mM, to allow the 30-S particle to ‘‘unfold’’ (cf. [4]). However, while it was easy to find conditions which led to complete breakdown of the 30-5 particle into a range of small RNA - protein fragments, there was no discernible protein specificity in the products. We therefore had to resort to other ways of increasing the extent of hydrolysis, the most successful of which so far has been the inclusion of 2 M urea in the hydrolysis buffer. Both pancreatic and T, ribo- nucleases have been used, and the precise composi- tions of the reaction mixtures and electrophoretic buffers are listed under Methods.

A typical electrophoretic profile from a hydrolysis carried out in 2 M urea is shown in Fig.2. This hydrolysis was made under condition (f) (see Meth- ods), using ribonuclease T,. The hydrolysate contains

Vo1.29, No.3,1972 J. MORGAN and R. BRIM

little or no 30-5 material when compared with the control, but has a large aggregate peak a t the origin. The breakdown products comprise three well- defined peaks, labelled I, II and I11 in Fig. 2. Peak I is broad and has a complex protein composition of a type which will be dealt with later. Peak I11 con- sists of small fragments of RNA moving ahead of the dye marker. This peak does contain some l4C-labe1, which may be due in part to small amounts of protein attached to the RNA fragments, but mainly arises from the incorporation of some 14C-label into the RNA itself. (This is unavoidable when a mixture of 14C-labelled amino acids is used for labelling). Peak I1 is a small fragment of RNA * protein. It is formed reproducibly by the action of ribonuclease T, under hydrolysis conditions (f) or (e) (see Methods), and it can be seen from Fig.2 that is has an RNA/protein ratio similar to that of the 30-5 control.

Slices from the RNA 'protein gel containing peak I1 (Fig.2) were subjected to protein analysis on 17.5O/, polyacrylamide-sarkosyl gels (see Methods and [l]). In most cases, only the radioactivity from the stained protein bands was measured; however, for about every sixth RNA * protein gel slice, the whole protein gel strip was cut up and analysed, in order to check that the radioactivity did lie within the stained bands. This procedure also provides a check on the absence of the unidentified proteins 52, 511 and 512 (Fig.l), as well as on any protein aggrega- tion which may be occurring. Results from these experiments were plotted as histograms, examples of which are shown in Fig.3, from peak I1 (Fig.2) and the corresponding 30-5 control. It is clear that peak I1 contains just two proteins, and that there is virtually no radioactivity anywhere else in the pro- tein gel (Fig.3A). The histogram of the 30-5 control (Fig.3B) shows that there is no preferential loss of protein as a result of the urea treatment, although the urea-treated particles usually showed a slight decrease in protein/RNA ratio on electrophoresis. The proteins contained in peak I1 are E, or E,, and G, or G, (see Fig. 1). As has been already noted, both these pairs of bands are poorly resolved on the aarkosyl gels and it was usually not possible to cut out the individual bands within each pair without some cross-contamination. For clarity, the radio- activities of these bands are plotted in Fig.3A as the sums of E, and E,, and of G, and G,.

In our previous paper [l], we showed that the molarity of the proteins contained in an RNA pro- tein fragment could be measured by dividing the radioactivity found in each protein band by the appropriate protein molecular weight. This measure- ment is made for each protein in every gel slice analysed, and the results plotted using a Calcomp plotter. Fig.4 shows the result of such an analysis made on gel slices from peak I1 (Fig.2), using the 36 Eur. J. Biochem.. Vol. 29

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Fig.3. Histograms of proteins in RNA protein fragments. Radioactive RNA 7 protein gel slices together with unlabelled 30-5 ribosomes were run on sarkosyl gels and analysed as described in the text and [l]. Cross-hatched areas indicate stained protein bands. (A) Proteins from peak I1 (Fig.2).

(B) Proteins from a 30-S control

molecular weights of Fig.1. As in Fig.3A, we plot the sum of bands E, and E,, and the sum of G, and G,, because of the cross-contamination between these pairs of bands. However, in all the experiments, the radioactivity in band E, exceeded that in band E, (usually by a factor of two or more), and the radio- activity in G, similarly exceeded that in G,. Further, it is clear from Fig.4 that E, + E, and G, + G, contribute equimolar amounts of protein, and that no other proteins are present in significant amounts. The mobility of peak I1 in the RNA-protein gel precludes the possibility that all four proteins (E2, E,, G,, G,) are present as a single fragment (see Fig.8 below); we therefore conclude that peak I1 contains only the two predominating proteins E, (58) and G, (515). It is interesting to note that when the hydrolysis was carried out under condi- tion (b) instead of (e) or (f) (see Methods), then a sub- stantial loss of protein G, occurred, leaving an almost pure single-protein fragment containing E, (58).

RNA*protein gel slices containing peak I1 (Fig.2) were also examined for their RNA content,

646 RNA . Protein Fragments from Escherichia coli 30-S Ribosomes Eur. J. Biochem.

32 34 36 38 40 Fraction number

Fig.4. Calcomp plot of proteins in peak 11 (Pig.2). Radio- activity in each protein from every RNA . protein gel slice is shown, after division by molecular weight (see text and

111). Fraction numbers correspond to those in Fig.2

by running them in 3 o/io polyacrylamide/agarose composite gels containing dodecylsulphate (see Methods, and cf. [S]). One RNA peak of approx- imately 110 to 120 nucleotides was obtained repro- ducibly from these experiments, together with some smaller fragments. Assuming that the 30-5 ribosome contains 1600 nucleotides of RNA and 300000 dal- tons of protein [lo, 111, then a fragment with the same RNAIprotein ratio consisting of proteins 58 and 515 (27200 daltons of protein, Fig. 1) should contain 145 nucleotides. Allowing for the fact that the RNAlprotein ratio of peak I1 was always slightly greater than that of the 30-5 control (prior to urea treatment), then peak I1 should contain about 170 nucleotides of RNA in all. This suggests that the smaller RNA fragments which were observed were not all merely breakdown products of the 120-nucleotide fragment ; however, the 120- nucleotide fragment accounts for about 70 of the total length of RNA expected from peak 11. I n similar experiments, our original RNA - protein fragment consisting of D,, El, P, and P, (“Band 111” [I]) reproducibly yielded an RNA species which was 330 to 350 nucleotides long; this agrees well with the predicted length of 320 nucleotides [I]. As we pointed out in a previous paper [12], it is

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Fig.5. Electrophoretic profile of RNA . protein fragments from a pancreatic ribonuclease hydrolysate in 2 M urea (con- dition e, see Methods and cf. Fig. 2 ) . Electrophoresis is from left to right, and the bromophenol blue marker ran to frac- tion 60. The peak of the corresponding 30-S control was in fraction 3 (arrowed). The [14C]protein peak at fraction 15 (arrowed) indicates the position of protein Z (Sl). ----, Radioactivity in 14C-labelled protein; ~ , radioactivity

in [SHIRNA

not a necessary criterion for specificity that an RNA - protein fragment should contain only a single piece of RNA. Nevertheless, if the greater part of the RNA in a fragment is a single piece, then the possibility is eliminated that the fragment concerned was in reality a mixture of independent smaller fragments, moving together in the RNA * protein gel. This will be discussed later (see Fig.8, below).

The great advantage of the method of analysis exemplified by Fig.4 is that it enables us to detect specific fragments which are not clearly resolved in the RNA - protein gel. Peak I (Fig.2) is one instance of a broad RNA - protein peak consisting of a series of overlapping RNA - protein fragments. In fact, the majority of fragments which we have found fall into this category. We take as an example a hydro- lysis by pancreatic ribonulease in 2 M urea, under condition (e) (see Methods). The elution prose of this hydrolysate is shown in Fig.5. I n this instance, there is very little material a t the origin, and the main peak (fraction 6) is not quite coincident with the 30-5 control peak, the position of which is marked on the diagram. Further, there is no peak corre- sponding to peak I1 (Big.2), nor is there any sign%-

Vol.29, N0.3, 1972 J. MORQAN and R. BRIMACOMBE 547

I I , I , , , , I , , , , , , , , ,

8 12 16 20 24 28 Fraction number

Fig. 6. Calcomp plot of proteins from the pamreatic ribonuclease hydrolysate of Pig. 5 . Radioactivity of each protein was divided by molecular weight, as in Fig.4. Fraction numbers correspond to those in Fig.5

cant amount of free low-molecular-weight RNA. The hydrolysis products appear as a broad multiple peak, which was not resolved into individual peaks by increasing the running time of the electro- phoresis. Again, the RNAlprotein ratio of the hydrolysis products was similar to that observed for the 30-5 control (cf. Fig. 2). The whole RNA * pro- tein gel was subjected to protein analysis in the manner described above, and the Calcomp plot which was obtained is shown in Fig.6. It is clear that there are two distinct RNA * protein frag- ments, with peaks a t fractions 12 and 21, respectively, and that the two fragments have entirely different protein compositions. Fractions to the left of frac- tion 8 (Fig.5) showed no discernible protein specific- ity, and are therefore not included in Fig.6. The slower-moving fragment contains E,, G,, G, and G, in equimolar amounts, but with a significant level 36.

of background “noise” contributed by the other proteins. I n this particular experiment the resolution of the protein gels was sufficiently good for cross- contamination of E, with E,, and of G, with G,, to be negligible; the contributions of E, and G, lie among the other “background” proteins. The mobili- t y of this slower-moving fragment in the RNA - pro- tein gel is rather anomalous, and will be discussed later. The faster of the two fragments contains D,, El, F, and proteins from the F,, F, region. I n this case, the background “noise” is much less. F, and F, were not clearly resolved in these protein gels, and were therefore treated together (cf. E, and E,, G, and G,, Fig.4). However, the radioactivity in F, + F, (after dividing by the protein molecular weight) was greater than the corresponding radio- activities from D,, El and F, by a factor of 3; in Fig.6, the contribution of F, + F, has been divided

548 RNA - Protein Fragments from Escherichia coli 30-S Ribosomes Eur. J. Biochem.

by 3 to bring it into coincidence with the other proteins. It can be seen from Fig.1 that there are in fact three proteins in the F,, F, region, and we conclude that all three are present. The composition of this fragment is therefore S7 (DJ, S9 (El), SIO (F,) and S13, S14, 819 (F,, F,), i .e . it is similar to our original four-protein fragment “Band 111” [I], but with two extra proteins. The same fragment was also found in a pancreatic ribonuclease hydrolysate in the absence of 2 M urea (condition d of Methods). The composition of the slower-moving fragment in Fig.6 is correspondingly S6 (E,), S20 (GI), S16 or S17 (G,) and, tentatively, S21 (G,).

It should be noted that protein S1 (Z, Fig. 1) has not been included in Fig. 6. This protein appeared as a hyper-sharp peak in fraction 15, which, because of the large size of this protein and its consequent high radioactivity, can even be seen in the 14C- profile of the hydrolysate (Fig.5, fraction 15, arrow- ed). This peak was not coincident with either of the two fragment peaks in Fig.6, and XI has therefore been omitted for clarity. A similar peak of Sl can be seen in Fig.2 (fraction 14); again this peak was not coincident with other protein peaks, and it appears that S1 can move independently into the RNA - protein gels. (S1 has an isoelectric point of less than 7.6 [13].)

A number of different complex hydrolysis pat- terns were analysed in this way, and the compo- sitions of the fragments in the various hydrolysates were deduced from Calcomp plots like that of Fig.6. Unlike peak I1 (Fig.2) or “Band 111” [I], the results were not always entirely reproducible. By this we mean that if a particular hydrolysis gave an RNA protein €ragment containing five proteins, repetition of the experiment might give the same fragment with one protein more, or one protein less, or occasionally with one protein differ- ent altogether. However, this does not invalidate the results, since the same groupings of proteins recur in different experiments, with each set of data serving to re-inforce another. This point is illustrated by the histograms in Fig. 7 (cf. Fig. 3). Fig. 7 A shows a protein gel from a ribonuclease TI hydrolysate made in 2 M urea under condition (g) (see Methods). Bearing in mind that the radioactivity values in the histogram are not divided by molecular weight, this shows a fragment containing D,, El, F,, F, and G, in approximately equimolar amounts. (The radio- activity in F, in this case was probably a cross- contamination from FJ. This fragment is therefore similar to but not identical with the faster-moving fragment of Fig. 6. Fig. 7 B shows on the other hand a fragment which is similar to the slower-moving frag- ment of Fig. 6; it contains (after division by molecular weight) equal amounts of proteins E, or E,, G,, G,, G, and possibly G,. The protein gel in this case was from a ribonuclease T, hydrolysate made in the

300

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Fig. 7. Histograms of proteins in RNA . protein fragments (cf. Pig. 3) . Cross-hatched areas indicate stained protein bands. (A) Proteins from a fragment obtained with ribonuclease TI (condition g, see Methods). (B) Proteins from a fragment

obtained with ribonuclease TI (Condition c)

presence of 2 mM EDTA instead of 2 M urea (con- dition c, see Methods). We use this example for illustration because it suggests that EDTA particles wil l prove a useful source of fragments in the future. However, since it is only a preliminary result, this fragment is not included in the tables (see below). EDTA treatment has in fact been used by Kagawa et al. [14] in essentially similar studies an 50-S ribosomes from E . coli. It is interesting that hydro- lysis in the presence of EDTA can yield apeciiic fragments, whereas hydrolysis a t very low magnesium ion concentrations apparently does not (see above). Nevertheless it is already clear that the salt condi- tions required for formation of specific RNA - pro- tein fragments are not the same as those necessary for the specific reconstitution of single proteins with ribosomal RNA (e.g. [15]).

The results of all our experiments are summarized in Tables 1 and 2. Table 1 shows the protein compo- sitions of each fragment in both nomenclatures (cf. Fig. l), and lists the various different experimental

Vo1.29, No.3,1972 J. MORGAN and R. BRIMACOMBE 549

Table 1. 8ummary of RNA * protein fragments The specific proteins found in fragments are listed in both nomenclatures [l, 91, with the experimental conditions under which each was obtained. The hydrolysis conditions are

described in Methods

Hydrolysis conditions

nuIuber Proteins in fragment T1 RNAase Pancreatic RNAasc

Fragment.

a 514 and S19 are not distinguishable (Fig.1). Hydrolysis was with 0.03 pg/ml of enzyme. Already published, “Band 111” [l].

d S16 and 517 are not distinguishable (Fig.1).

conditions under which each particular fragment was obtained. The fragments will be discussed indi- vidually under Conclusions, and will be referred to by the numbers assigned to them in Table 1. In Table 2 the results are presented quantitatively, in terms of the molar ratios of the proteins specific to each fragment. These ratios were calculated from Calcomp plots such as Fig.4 and 6. The values of protein radioactivity divided by molecular weight in peak regions of the plot (e.g. fractions 10 to 12, or 18 to 22, in Fig.6) were summed for each of the specXc proteins in the fragment. These totals were then normalised, taking the median total as 1.00. Contributions from all other (non-specific) proteins in the same region of the Calcomp-plot were summed, averaged and normalised to this same median value, to give a measure of mean background protein

noise”. Table 2 indicates the total number of individual protein analyses used to make the deter- minations, and gives the molar composition of the 30-5 particle, averaged over all the experiments.

The data of Table 2 demonstrate that the frag- ments fulfil two of the criteria for specificity which we discussed previously [i], viz. the specific proteins are all present in equimolar amounts in each frag- ment, and the background protein “noise” level is acceptably low in every case. Further, in all thc experiments, the ratio of RNA to protein in the fragment peaks of the RNA protein gels was very

“

Table 2. Xtoichiometry of proteins in Rh’A * protein fragments The amount of each specific protein present in each fragment and the mean protein background “noise” level were determined as described in the text. Combined protein molecular weights were calculated using the values given in Fig. 1. The fragment

numbers are those given in Table 1

Amount in fragment no. 30-S controla Protein

1 2 3 4 5 0 7 8 9

0.95 0.80 - 1.10 __ 0.50 _ _ 0.90 ~- 1.25

__ 1.05 0.85 1.10 1.05 1.20

- __ __ -.. :::: ] 2.850 1.950 2.00c 1 ._

Fl -.. 1.05 0.80 1.20 - - -

F2 G,

- - 1.00 1.00 0.95 - 1.15 - 0.85 1.10

- 0.55 - 0.90 1.20 1.30

__ - c,, - -

G3 G; Ga ~- -

- - - _ _ - - - _. - - ~.

._ -

Background 0.30 0.15 0.25 0.20 0.15 0.30 0.05 0.30 0.30 - - “noise” f0 . l f0.05 AO.1 f 0 . l 1 0 . 1 h0.l f0.05 f 0 . l *O.l

Number of samples 5 15 9 7 5 4 10 3 4 23 x combined

protein M , 48.5 62.2 76.1 89.4 75.1 89.0 27.2 51.7 78.9 300.0d Mean values from controls under all conditions (a) to (g) (see Methods). E, and E3 were not separated. F1 and F. were not separated.

d See t l O , i l ] .

550 RNA * Protein Fragments from Escherichia coli 30-S Ribosomes Eur. J. Biochem.

0 0.2 0.4 0.6 Gel rnobilitv fcf. brornoohenol blue)

Fig.8. Mobility of RNA protein fragments in 5O/, composite gels. Log of total protein molecular weight in a fragment (Table 2) is plotted against mobility of that fragment relative to the mobility of bromophenol blue. Each fragment is identified by the number given to it in Table 1. 0, Un- hydrolysed 30-5 ribosomes and fragments Nos. 2 and 7 (see text); 0, all other fragments (each point represents one experiment); x , dimer positions of fragments Nos. 8

and 9; A, A, anomalgous fragment “X” (see text)

similar to that observed in the 30-5 controls (see Fig. 1 and 5) . This is quite important because a large excess of protein in an RNA-protein fragment could mean that some extra proteins were non- specifically adsorbed to the fragment, whereas a large excess of RNA would imply that some proteins had been lost, leaving (‘gaps’’ in the fragment. Small changes in RNAIprotein ratio would of course be expected, arising from different local environ- ments of the various proteins. There is however another criterion which must be observed [l], which is that the mobility of the RNA - protein fragment in the gel must be consistent with the protein composition assigned to it. Ideally, if all the fragments had the same RNAIprotein ratio, and were all of similar conformation, then their mobility in the gel should show the usual linear relationship with the logarithm of their mass. For a constant RNA/protein ratio, the mass is propor- tional to the combined molecular weights of the proteins in the fragment. This combined molecular

weight is listed for each fragment at the bottom of Table 2, and the values are plotted (as logarithms) in Fig.8 against the mobility of each fragment in the RNA - protein gel. The straight line in Fig.8 is defined by three sets of points (solid circles). These are the 30-5 particle, which contains 300000 daltons of protein [lo, 111, and fragments Nos. 2 and 7 (Tables I and 2); fragment No. 2 (“Band 111” [I]) and fragment No. 7 (peak 11, Fig.2) both run in the RNA - protein gels as sharp peaks, free from other overlapping fragments, and are com- pletely reproducible. I n addition, the RNA from both these fragments has been shown to be of a size which is consistent with the protein composi- tions assigned to the fragments (see above); they are therefore good reference points for the mobility plot. The other fragments, from complex overlapping peaks such as those of Fig.5 and 6, are represented by open circles in Fig.8. The fit with the straight line is inevitably crude, since small variations in RNA/protein ratio do occur, and the results are from several different electrophoretic systems (see Methods). However, the factor of crucial importance to the argument is that while several of the points lie to the left of the line, there are none sigdicantly to the right of it. A point lying to the left of the Iine in Fig.8 means that the fragment concerned moves too slowly in the RNA * protein gel for the composi- tion assigned to it. This could arise from any of three reasons, none of which would invalidate the speci- ficity of the assignment. Firstly, the fragment could be retarded by non-specific attachment to other contaminating fragments. This would be manifested by a high level of background “noise”. Secondly, it could merely appear to be retarded by containing an extra protein which does not run in the sarkosyl gel system, possibly 52, 511 or 512 for example (see Fig. 1). This would result in our building up a picture of the 3 0 3 particle which would be deficient in that particular protein. (However, it should be noted that if such a protein were able to penetrate the sarkosyl gel a t all, it would be detected on histograms such as those Fig.7.) Thirdly, the fragment could be retarded by running a.s a specific dimer ; this possibility has already been raised [I], and we suggest that the low mobilities of fragments Nos. 8 and 9 (Fig. 8, and see Fig. 6) are due to dimer formation. The crosses in Fig. 8 indicate the respective positions for dimers of these fragments, and these lie reasonably close to the line. The fact that both fragments have similar protein composi- tions may have some significance in respect t o a tendency to dimerise. Dimer formation could also account for the low mobilities of one experimental point for each of fragments Nos. 2 and 3 (Fig.8).

I n contrast, any point which lies significantly to the right of the line in Fig.8 cannot be regarded as a genuine fragment. This is because a fragment

Vo1.29, N0.3, 1972 J. MORGAN and R. BRIMACOMBE 551

which is moving too rapidly in the RNA protein gel for its assigned composition can only be explained in terms of a mixture of smaller fragments moving independently, but with the same mobility. This is illustrated by the points represented by triangles in Fig.8; the solid triangle indicates the position of an apparently specific four-protein fragment “X”, which satisfied all our other criteria (see above). In spite of this, its mobility indicates that it must have been a fortuitous equimolar mixture of two two-protein fragments of similar size (open triangles). With no way of telling which two proteins were together, the data were meaningless and had to be abandoned. Considerable confusion could obviously result if some such mobility criterion is not applied, and its absence makees it difficult to assess the validity of ribosomal fragments which have been reported by other workers, notably Schendel et al. [I61 and Kagawa et al. [14].

CONCLUSIONS The fragments Listed in Tables I and 2 show a

very clear pattern of specificity. Using the interna- tional nomenclature [9], fragment No. l (Table l) contains S7, S9, and 514 or ,919. Fragment No.2 contains the same three proteins together with 513, not XI0 as previously reported [l]. However, S10 appears in the next fragment, No. 3, which contains S7, 59, 1310, S13, and 514 or 819. This five-protein piece is sometimes found instead of fragment No. 2 under our original hydrolysis conditions ([I], con- dition a, see Methods). Fragment No.4 contains one more protein again, and has three proteins from the combined bands F, + F, (Table 2); this accounts for all the proteins known to migrate in this part of the sarkosyl gel, and the fragment is therefore S7, S9, SlO, 513, 514 and 519. The frequency with which this group of fragments recurs suggests that the 30-5 ribosome may have a “weak point”. This is interesting in the light of the electron micrographs published by Nonomura et al. [17], which indicate that the small subparticle of the rat liver ribosome has a groove or division near one end.

In fragments Nos. 5 and 6, a new protein, S20, appears. There is some ambiguity here as to which of the proteins from bands F, and F, are present (Table 2), but both fragments clearly contain 57 and S9 in addition to 520, together with two proteins from the trio S13, S14 and S19. This ambiguity is not serious, since S13, 514 and S19 are all close neighbours, as shown by fragment No. 4. Fragment No. 6 also contains SIO.

Fragment No. 7 is completely different from the first six, and is the two-protein fragment containing 58 and 515, which has been described in detail in the first part of this paper. Fragment No. 8 is different again, but here our protein assignments are

Fig.9. Assembly m p of the 30-5 subparticle (21, re-arranged to incorporate the data of Table 1. International nomenclature [B] is used. Different cross-hatchings identify the proteins from fragments Nos. 4, 7 and 8 (see text and Table 1)

less certain; the fragment contains S6, and S20 (cf. fragments Nos. 5 and 6) and either S16 or 517. It also contains protein G,, which we have tentatively identified as S21 (Fig. 1). Finally, fragment No. 9 is a combination of fragments Nos. 7 and 8. It contains two proteins from bands E, + E, (Table 2) which must be S6 and 58, together with S20, S15, SIB or 517, and the presumed 521. The preliminary ex- periments described above (Fig. 7B) suggest that hydrolysis in the presence of EDTA may provide more information about these particular proteins. I n this context, it is important to note that when the ribosome is in an “unfolded” conformation, some proteins which are close neighbours in the compact 30-S particle will presumably become separated ; it is however reasonable to assume that the unfolded particle will not contain any new close- neighbour relationships which were not present in the compact particle. Data on fragments obtained from unfolded particles are therefore still relevant to the structure of the compact ribosome.

In Fig.9, we have attempted to combine the fragment data of Table I with the “assembly map” of Nashimoto et al. [2]. To do this, the assembly map

552 J. MORGAN and R. BRIMACOMBE: RNA Protein Fragments from Escherichia coli 30-5 Ribosomes Eur. J. Biochem.

has been re-arranged to some extent so as t o group together those proteins which we have found in RNA - protein fragments; these groupings are indi- cated by the cross-hatching in the diagram. It is clear that most of the proteins found together in individual fragments are closely related in the assembly process. Proteins from fragments Nos. 1 to 4 form one group, proteins from fragment No. 7 a second and proteins from fragment No. 8 a third. These last two groups are close together in the map, accounting for fragment No. 9, and protein S20 is close to S7, S9 and S13, to account for fragments Nos. 5 and 6. None of Nomura’s interactions have been omitted from this map, and it is interesting to note that Green (personal communication) has found, by reconstitution experiments, tha,t proteins 58 and S15 interact during assembly and that S7 influences the binding of S13. Fig.9 also takes into account the data recently published by Bickle et al. [la], from protein cross-linking experiments with bis-imido esters. These authors conclude that proteins S6, S7 and S9 are adjacent in the ribosome, and that S9 is adjacent to 55. Proteins S19 and S l l or 512 constitute a third group, and S12 has been included in Fig.9 for this reason. Finally, Schendel et al. [16] have reported a manuscript in preparation by C. T. Shih and G. R. Craven, indicating that proteins Si8, Sl1 and S21 are cross-linked by treat- ment with tetranitromethane.

Fig. 9 is not intended to be structural map of the 30-5 particle; it is obviously impossible to put forward any detailed model a t this stage. We sug- gest however that this map can be continually revised as new data become available, eventually incorporating the RNA as a backbone, and if necessa- ry going into three dimensions. The map should then gradually evolve into a representation of the actual topography of the 30-5 ribosome.

We are very grateful to Mr M. R. B. Clarke of the Institute of Computer Science, University of London, for writing the Calcomp programmes and plotting the data.

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J. Morgan and R. Brimacombe M.R.C. National Institute for Medical Research Mill Hill, London, NW7 lAA, Great Britain