A Preliminary Three-Dimensional Arrangement of the Proteins in the Escherichia coli 30-S Ribosomal Sub-Particle

Eur. J. Biochem. 37,472-480 (1973)

A Preliminary Three-Dimensional Arrangement of the Proteins in the Escherichia coZi 3 0 3 Ribosomal Sub-particle

Joan MORGAX and Richard BR-COMBE National Institute for Medical Research, Mill Hill, London

(Received April 19,1973)

1. 30-S ribosomal subparticles from Escherichia mli were hydrolysed with ribonuclease TI, pancreatic ribonuclease or micrococcal nuclease in the presence of 2 M urea, and various concentra- tions of magnesium and ethanol. The RNA - protein fragments produced were separated on 50l0 polyacrylamidelagarose composite gels, and fractions from these gels were subjected to protein analysis on i7.5O/, periodate-soluble polyacrylamide gels run in the detergent sarkosyl, using the technique already published.

2. A wide range of RNA * protein fragments was obtained by this procedure, each containing a few specific ribosomal proteins. The strict criteria already published for determining the specificity of the proteins in each fragment were applied. The RNA - protein fragments divide into two distinct groups, those containing some or all of proteins 57, S9, SiO, 513, Si4 and Si9, and those containing some or all of proteins S4, 55, S6, S8, Sii, Sl5, S16(i7), 518 and S20. Proteins S1, S2, 53, Si2 and 521 were not found in specific fragments.

3. The individual proteins found together in specific RNA - protein fragments are interpreted as being close neighbours in the 30-S particle. The range of fragments observed is sufficient to enable the data to be combined with Nomura’s “assembly map)’ and data from protein cross- linking experiments, into a preliminary three-dimensional arrangement of the proteins.

In previous papers we have described a method for the analysis of ribonucleoprotein (RNA - protein) fragments from Escherichia coli ribosomes [i], and have used this method to characterize a series of specific fragments from the 30-5 particle [2]. In this paper we present a further series of fragments, obtained by mild nuclease digestion of the 30-S ribosome with ribonuclease TI, micrococcal nuclease or pancreatic ribonuclease. These two series of fragments account for 16 out of the 21 ribosomal proteins, and the data have been combined with the assembly map of Nashimoto et d. [3], into a preliminary three- dimensional model. The protein arrangement takes account of results obtained by cross-linking of ribosomal proteins with bi-functional reagents [4,5,6], and a possible linear sequence of some of the proteins along the ribosomal RNA is also discussed.

Abbreviations. RXA * protein, ribonucleoprotein; sarkosyl, N-lauryl sarcosine.

Enzymee. Ribonuclease TI (.EC 2.7.7.26) ; pancreatic ribonuclease (EC 2.7.7.16) ; micrococcal nuclease (EC 3.1.4.7).

Definition. Azao unit is the quantity of material contained in I ml of a solution which has an absorbance of I at 260 nxn, when measured in a 1-cm path-length cell.

MATERIALS AND METHODS Preparation of Ribosomes

Radioactive and non-radioactive 30-5 ribosomal sub-particles from E . coli MRE 600 (obtained from MRE, Porton, U.K.) were prepared exactly as described previously [2], except that the isolated subparticles were kept stored at -20 “C in 10 mM Tris- HC1 pH 7.8, 0.3 mM magnesium acetate, Containing 10-20°/0 ethanol. The ethanol was only dialysed away immediately before use in hydrolysis reactions.

Separation and Analysis of RNA .Protein Fragments Radioactive 30-S ribosomes were hydrolysed

with ribonuclease TI, pancreatic ribonuclease, or micrococcal nuclease (all from Sigma) for 4.5 h at room temperature. Reaction mixtures contained 8-10 ABso units of ribosomes in 0.2-0.4 ml. The hydrolysates were separated and analysed by electrophoresis on 501, polyacrylamide/0.5 agarose composite gel slabs [7], exactly as before [2]. The hydrolysis buffers contained i0 mM Tris-HC1 pH 7.8, 20 mM KCI, and 2 M urea [2], with various amounts of added magnesium acetate, ethanol and enzyme, as

Vol.37, No.3, 1973 J. MORGAK and R. BRIMACOMBE 473

Table 1. Experhental codi t ions Hydrolysis and gel electrophoresis buffers contained the components listed in Materials and Methods, together with these

additions

Magnesium and ethanol

Hydrolysis Gel Type of nnclease Quantity of nuckase per A2so unit ribosomes in hydrolysis

Reference letter

MLg EtOH m EtOH

A B C D E P G H I J K

mnf I" 5.0 -

1 .o 0.3 - 0.3 - 0.3 -

0.3 10 0.3 10 0.3b 10 0.3 10 0.3 10 0.3 10

-

rnM

8.0 1 .o 0.3 0.3 0.3 0.3 0.3 0.3 0 . 3 c 0.3 0.3

" 0

- R.KAase T, 58 U RNAase T, and 68 U pancreatic RNAase 0.7 ng RNAase T, 13 U

- Pancreatic BPI'Aase 0.2 ng -~ Xcrococcal nuclease a

- RNAase T, 50 U - Pancreatic RNAase 0.7 ng - Micrococcal nuclease a

- RNAase T, 13 U 10 RNAase T, 40 U 10 Pancreatic RNAase 0.7 ng

- - 1 0.003 U

0.005 U

a Hydrolysis buffer contained 2 mil1 CaCl, for this calcium-dependent enzyme. Hydrolysis buffer contained 25 mM Tris-citrate pH 8.8. Gel reservoir contained 25 mM Tris-chloride pH 8.8, instcad of Tris-citrate.

listed in Table 1. For electrophoresis, the gel contained 25 mM Tris-HC1 pH 7.8 and 20 mM KC1 [2], and the reservoir buffers 25 mM Tris-citrate pH 8.8 and 20 mM KC1. Both gel and reservoir buffers also contained added amounts of magnesium acetate and ethanol as indicated in Table 1.

Proteins in the radioactive RNA protein fragments were analysed on 17.5 o / o polyacrylamide gels containing 0.5O/, sarkosyl (Geigy, U.K.), and 1.5O/, N,N'-diallyl tartardiamide [S], exactly as before [2 ] .

RESULTS AND DISCUSSION In our previous paper, we showed that the 30-S

ribosomal particle could be split with nuclease into a series of specific RNA . protein fragments, by including 2 M urea in the hydrolysis buffer [ 2 ] . Here we have extended this series of fragments, by varying the concentration of magnesium in the hydrolysis and electrophoresis buffers, and also by adding loo/, ethanol to the system. The addition of ethanol was prompted by the simple observation that ribosomes stored in 10-2Oo/, ethanol showed less tendency to aggregate (see Methods). Details of the nucleases used, and of the hydrolytic and electrophoretic buffers, are listed in Table 1.

The ribosomal hydrolysates were analysed by the method which we have already described [l,Z]. To recapitulate very briefly the principle of the technique, 30-S ribosomes labelled with [3H]uridine and a mixture of I4C-labelled amino acids are used in the hydrolysis. The radioactive RNA protein fragments are then separated on a 5 polyacrylamide/agarose composite gel, and individual fractions of this gel are

analysed for protein by loading the gel slices directly onto a second 17.5O/, polyacrylamide gel. The second gel contains the detergent sarkosyl, and the periodate- sensitive gel cross-linker N,N'-diallyl tartardiamide [8 ] . The detergent disocciates protein from RNA, and separates the proteins on a molecular weight basis. An aliquot of unlabelled 30-S ribosomes is added to each sample to give a shainable pattern of total 30-S protein, and ribonuclease is also added to di- gest away the liberated RNA. After running, the gel is analysed by cutting out each stained protein band, dissolving it in periodic acid, and assaying for lac- radioactivity.

Before describing the results, we must first discuss the resolution of some ambiguities in the identification of the ribosomal proteins in the sarkosyl gel system. A typical protein separation is shown in Fig. 1 with each protein band identified in the international nomenclature [Q]. The identifications were made both by running unlabelled purified 30-S proteins on a gel in adjacent slots to total 3 0 3 protein, and also by running radioactive proteins isolated by chromatog- raphy on CM-cellulose [lo] in the same slot as total 30-5 protein. The former method identifies the individual protein bands in the 30-S pattern, while the latter, by simulating the experimental conditions of our RNA * protein fragment analysis, shows which of the less well-resolved bands can be positively distinguished by a radioactive measurement. The most important ambiguity has arisen through the strange behaviour of S15, of which Fjg.1 shows a particularly capricious example ; 515 can run with S20, S11 or S21, or across all three as in Fig.1. Purificd isolated S15 exhibits the same behaviour, for which we can offer no explanation. Recognition

474 $rrangement of Escherickia coli 30-S Ribosomal Proteins Eur. J. Biochem.

s1-

s2-

53- S 1-

s7- s5-

S12?-

s9- S 8-

S6- S13-

S I O t l L ~ s20- s21--

S 18- S 16.1 7 ~

515-

Fig.1. Separation of 30-S ribosomal proteins on a 17.5a/a polyac yhmide-sarkoyl gel (20-cm long). The three slots are from a standard protein analysis (see Methods), and show the variable mobility of protein S15. Bands are identified in the

international nomenclature [9]

of this property enables 515 to be identified (in a series of samples) in the presence of any or all of proteins S11,520 or S21, since the radioactivity and stain are precisely coincident in the gel. The two-protein fragment previously reported to contain S15 and S8 [ 2 ] was correctly quoted, but, as a result of the variable mobility of S15, the tentative identification of S21 in two other fragments [ 2 ] was incorrect. The tests on other poorly-resolved pairs of proteins showed that S8 and 56 are distinguishable in some but not all cases, and that S18 can almost always be distin- guishedfrom S16(17) (Fig. I). SIB and S17 themselves

are not resolved. Protein Sl9, which we previously identified [2] as running with S14, is in fact distinguishable from S10, S13 and S14, and it is proteins 810 and S14 which run together. However, purified SIO sometimes gave two bands in the gel, the fainter of which ran with S13, and therefore S10 can be con- fused with S13. Since S10, S13 and S14 have all been found with Sl9 in a single RNA . protein fragment [ 2 ] , the lack of resolution in this area of the sarkosyl gel is not too serious. In the ensuing discussion, a notation such as SlO(13) implies that we are referring to S10 or S13, but not both together. The advantage of the one-dimensional detergent gel for analysing RNA . protein fragments remains, in that i t has the capability of rapid handling of large numbers ofsam- ples, but i t is clear that in a few key cases, ambiguity in protein identification will need to be resolved by two-dimensional electrophoresis [l I]. To this end, a procedure for non-detergent extraction of the proteins from an RNA * protein gel slice is currently being worked out. Protein XI2 is now the only protein whose position in the sarkosyl gel is uncertain.

We will describe in detail three examples of RNA .protein fragment analyses. The first of thesc i s a ribonuclease TI hydrolysate in 2 M urea, with 5 mM magnesium (condition A, Table I), and the electrophoretic profile of this hydrolysate is shown in Fig.2A. This pattern of hydrolysis (cf. [I]) shows a large amount of aggregated RNA -protein a t the origin, a peak of apparently unhydrolysed 30-S material, and two clear RXA * protein fragment peaks. As before [1,2] the proteins from each RNA * protein gel slice were analysed, and the radioactivity in each protein band was divided by the corresponding protein molecular weight, to give a measurc of the relative molarities of the proteins. The molerular weight values of (21 were used in this procedure. The results are plotted on the same graph for each protein, which is shown in this case in Fig. 3. The main peak on this graph (fraction 20) is the well-established fragment [2] containing 57, S9, S19 and two out of the three proteins S10, 513 and S14, all in equimolar amounts. Roth and Nierhaus [I21 have described a ribosomal fragment which contains the same proteins, including Sl0 and S13 but not S14. For simplicity we therefore assumc for the present that the main peak in Pig.3 likewise contains SIO and S13, without Sl4 . The ambiguity remains, but, as noted above, is not serious. I n addition to this pcak, Fig.3 shows two important features. The first of these is the peak on the left (fraction 14), which contains proteins S4, S5, 56, S8, Sll , S15, S16(17), S18 and S20 in equimolar amounts with a low level of contaminating protein “noise”. Previously, we were not able to obtain good specificity in this region of the gel, and were also unable to show the presence of S4 or 85. In this particular set of protein gels, S16(17) and S18 were not cleanly resolved, although S18 and either 516 or

475 Vol.37, Ko.3, 1973 J. MORGAN and R. BRIMACOMBE

25000

20 000

15000

10 000

5000 Z' E - . r n -

0 5

8000 mz I

Q z n

6000

4000

2000

3

Fraction number

Fig. 2. Electrophoretic profile of RNA . protein fragments from two ribonuclease TI hydrolysates. (A) Condition A (Table l), (R) condition C . The fractions are 0.l7-em sliees from a 5°/o composite gel, and the direction of electrophoresis is from left to right. The bromophenol blue dye marker ran to about fraction 60 in both cases. (----) Radioactivity in 14C-labelled protein. (-) Radioactivity in [3H]RNA. The arrows indicate the peak positions of unhydrolysed 30-5 control samples, which had radioactivity ratios ([aH]RPI'A/

[14C]protein) of 8.0 (A), and 7.0 (B)

S17 were obviously present. These proteins are therefore plotted as the sum of S16(17) and S18, dividcd by two. Similarly, the variable position of Sl5 indicated clearly that S20, X i 1 and S15 were all three present (cf . Fig. 1 ), and these were most simply plotted as the sum of S15, S11 and 520, divided by three. Protein S1 also ran with this peak, but since we have already shown that S1 can move indepen- dently into the RNA . protein gel [2], this protein has not been included. The two peaks of Fig.3 show a very clean break in the ribosome, accounting for all the proteins except S2, S3, 512, S21, and (probably) S14.

The other important feature of Fig.3 is that in fractions 23 to 25 there is a slight but unmistakable

A

(S;6(17j - Sl8) 1 2

9 13 17 21 25 Fract ion number

Fig. 3. Protein analysis from the rihonuclease T, hydrolyaate of Fig.2A. Radioactivity of each protein in every RNA .protein gel slice is shown, after division by protein molecular weight (see text and [2]). Fraction numbers correspond to

those in Fig. 2 A

shoulder of a fragment which contains only S7 and 519. The RNA/protein ratio of the hydrolysate in this region (Fig.2A) is not significantly different from that of the main peak (fraction 20), and i t follows that in fractions 23 to 25, some RN-4 has been lost as well as proteins S10, S13 and S9. An analogous but much more pronounced shoulder appears in the hydrolysate which we take as our second example. In this case the hydrolysis was made with ribonuclease T, in the presence of 2 M urea, 0.3 mM magnesium and loo/, ethanol (condition J, Table I). The ethanol to some extent counteracts the effect of urea (see e.g. [13]), but, gives very much better results than if both are omitted. The effect of 100/, ethanol in the gel is to increase the pore size slightly, but nevertheless thc hydrolysis profile was similar to that of Fig. 2A and is not illustrated. The protein analysis in the region of the main peak (corresponding to the peak in fraction 20 of Fig.3) is shown in Fig.4. Little explanation is needed. Bearing in mind the possible presence of S14 (see above), the main peak (around fraction 40) contains the same five proteins as before,

476 Eur. ,I. Riochem. Arrangement of Escherichia coli 3 0 3 Ribosomal Proteins

519

510 (13 ) 5 9 513 (10) s7

n i ~

33 37 41 45 49 Fraction number

Fig. 4. Protein analysis from a ribonuclease T, hydrolysate under condition J (ipahle 1) . Radioactivity of each protein is divided by molecular weight, as in Fig. 3. The peak is analogous to the faster-moving peak of Fig. 2A; fraction numbers

refer to the appropriate hydrolyaate (not illustrated)

but the shoulder (fraction 44) has quite clearly lost S9 and S10 or 513, leaving S7, S19 and S13 or SIO. Again, the RNA/protein ratio of the hydrolysate was similar in the regions of both main peak and shoulder, indicating that some RNA must have been removed as well as the two proteins. If only the proteins had been lost, then the RNAlprotein ratio of the shoulder would have been about 1.6 times greater than that of' the main peak. This particular hydrolysis is very reproducible, and the importance of shoulders such as that in fraction 44 will be mentioned later.

Our third example shows fragments which are derived from the large fragment of Fig.3 (pcak in fraction 14). In this case the hydrolysis was again with ribonuclease TI, in 2 M urea and 0.3 mM magnesium, but without ethanol (condition C , Table 1). The lower magnesium concentration, in the absence of ethanol, seems to allow the largcr RNA . protein fragments to disintegrate. This process in undoubtedly helped by the chelating action of the citratc ions in the gel reservoir buffer. The electrophoretic profile of this hydrolysate is shown in Fig.2B. The profile is rather like that obtained in a similar hydrolysis a t 1 mM rnagncsium 121, in t.hat there is little or no re-

21 2 5 29 33 37 41 45 22 26 30 34 38 42 46

Fraction number

Big.5. Protein analysis from the ribonuclease TI hydrolysate of Fi9.2B. RNA - protein gel slices were analysed two a t a time (see text), and radioactivity of each protein is divided by protein molecular weight, as in Fig.3. Fraction numbers

correspond to those in Fig. 2 B

maining 30-S material, there is a large peak of fast- moving RNA fragments, and there are peaks of RNA . protein a t fractions 20 and 40. However, in Fig.2B therc is also a significant spread of RNA - protein between these two peaks, in the region of fraction 30. The protein analysis of this hydrolysate is shown in Fig. 5 ; in this case the RNA * protein gel slices were analysed two a t a time in order to increase the rather low levels of radioactivity. In fractions 21 and 22, the usual peak of S7, S19 etc. (c f . Fig. 3 and 4) can be seen disappearing on the left of the diagram. On the right (peak a t fractions 41 and 42) is a strong peak containing S8 and S15, the fragment which has already been reported [ Z ] . In the middle of the diagram, two more fragments can be seen, appearing as slow-moving shoulders on the peak of S8 and S15.

Vol.37, No.3, 1973 J. MORGAN and R. BRIMACOMBE 477

Table 2. Composition of RNA protein fragments Specific proteins found in fragments are listed, together with the experimental conditions under which each was observed

I 10-8 x M, of Braanent Experimental conditions Number Proteins in fragment

(see Table 1) comhined nrotpina

1 2 3 4 5 6

s7, s19 57, s19, SlO(13)b

s9 , s7 , s19 S9. S7. S19. S10113)b

S13, S9; S7; S19; Slob ’ S13, S9, S7, S19, SIO, S14

A, B, F B, Cc. J. I

iee 12 j E, I?, and see [2]

see r21 A, J, K, and see [2,12]

33.0 46.7 48.5 62.2 76.1 89.8

7 8 9

10 11 12 13 14

S8,S15 S5, S8, S15

S8, S15, SlG(17) S5, 58, S15, S16(17)

58,515, S16(17), 520 85, S8, S15, S16(17), S2U

84, 85, S8, 515, S16(17), 520 S4, 85, S8, S15, S16( 17), S20 with S6, S11, S18e

B, C, D, E, G, H, I, K, and see [2] E, H, G c,cc

r! ., 0, E, and see 121

E, Ed, K B, J, and cf. [I21

A, F, J

27.4 45.5 39.0 57.1 51.9 70.0 92.1

131.1

see Big.6. Ambiguity in the identification of S 10, S 13 and S 14 is discussed in the text. Hydrolysis contained less enzyme (4.5 U ribonuclease TI). Hydrolysis at 5 “C overnight, instead of 4.6 h at room tcmperature. Different hydrolyses varied in respect of these three proteins.

Although the radioactivity is rather low, these two fragments show quite distinctly above the very low level of contaminating protein noise. In fractions 25 to 30, four proteins are present in equimolar amounts, namcly S5 and S16(17) in addition to 58 and S15. Moving towards the right of the diagram, 55 disappears first, leaving S8, S15 and S16(17) in equimolar amounts in fractions 31 to 34, and then S16(17) also diappears. In this case it is difficult to infer very much from the RNA/protein ratio of the hydrolysate (Fig. 2B) in the region of these fragments, since the fast-moving RNA peak clearly trails back into the RNA . protein region (about fraction 40).

The various hydrolysis conditions listed in Table 1 make use of the varied effects of combinations of ethanol and magnesium on the hydrolysis products. In addition, some experiments were made with the Tris-citrate gel reservoir buffer added to the hydrolysis mixture (see Methods, condition H, Table l) , or conversely with Tris-chloride buffer instead of Tris-citrate in the gel reservoir (condition I ) . It may well be that the conflicting effects of the reaction components (ethanol versus urea, and magnesium versus citrate) in some way mimic the subtle balance of forces within the ribosome, and allow the RNA * protein fragments to disocciate cleanly once the RNA has been cut. The complete list of fragments which we have obtained is shown in Table 2, together with the conditions under which each has been observed. The fragments are divided into two groups, numbers 1 to 6 which derive from the faster-moving fragment of Fig.3, and numbers 7 to 14 which derive from the slower. Some of the fragments reported previously [2J are also included in this table. The

fragments account for all the 30-S proteins except for S1, S2, 53, 512 and 521. The two fragments previously reported to contain 521 [2] probably correspond to fragment number 11 and a fragment similar to number 14. As has already been noted, these fragments were wrongly identified as a result of the variable mobility of protein 515 in the sarkosyl gels. For the same reason, the fragment previously reported [2] as containing 520 together with the proteins of fragment number 5 (Table 2) could in fact have contained S15; while we feel that the latter possibil- ity is unlikely in this case, we have not yet been able to resolve this particular ambiguity to our satisfac- tion, and therefore we have omitted this fragment from the table. The fragment is clearly a important one as it provides the only link between the two halves of Table 2 , with the exception of the fragment reported by Both and Xierhaus [I21 which corresponds to fragment 13 together with protein S13.

It is clear from Table 2 that proteins 57 and Sl9 (fragment I ) , and S8 and 515 (fragment 7 ) form a common “core” to their respective halves of the table. The most stable and reproducible fragments under the hydrolysis conditions we have used are numbers 2, 4, 5, 7, 13 and 14, although fragment 14 varies in its content of proteins S6, S11 and S18. The other fragments, in particular numbers 8 to 12, are difficult to reproduce in the sense (mentioned before [2]) that repetition of the same experiment often leads to isolation of fragments containing one more or one less protein. Sometimes only fragment number 7 was observed in experiments expected to yield fragments 8 to 12; further work on these particular fragments is in progress.

478 Arrangement of Escherichia coli 30-S Ribosomal Proteins Eur. 3. Biochcni.

5.50

5.25

- $ S ‘Z 5.00 -? +

R

-cl a, S

R .- E 2 4.75 v

m 0 -

4.5c

4 .2 : 0 0 .2 0.4 0.6 0.8

Re la t i ve m o b i l i t y

Fig.6. Mobility of RNA . protein fragments i n 501, composite gels. Log of’ total protein molecular weight in a fragment (Table 2) is plotted against the observed mobility range of that fragment relative to the mobility of bromophenol blue (see

text and [ S ] ) . Each fragment is identified by the number given to it in Table 2

We have already discussed in detail [2] the criteria for specificity which we apply to the RNA * protein fragments. These are [a) that the proteins in a fragment must be present in equimolar amounts, (b) t,hat the level of contaminating protein “noise” must be low [ef. Fig.3-5), (c) that the RNBlprotein ratio of the fragment must be similar to that of the 30-S particle (cf . Fig.2), and (d) that the mobility of the fragment in the polyacrylamide/agarose gel must be consistent with the protein composition assigned to it. Mobilities of all the fragments listed in Table 2 are shown in Fig. 6. In this graph, the mobility of each fragment with respect to bromophenol blue is plotted against the log of the combined molecular weights of the proteins in each fragment (listed in Table 2, see also [Z]). The straight lines are defined as before [ 2 ] by the mobilities of unhydrolysed 30-5 ribosomes, and the two most well-defined and frequently-observed fragments (numbers 5 and 7). While a plot of this nature is inevitably crude, due to the variety of electrophoretic conditions used, it can be seen that the mobilities of most of the fragments lie close to or between

the two straight lines. Notable exceptions are fragments number 1, 11 and 13. Fragment I1 appears twice on the graph, the slower mobility arising from early experiments made with ribosomes not stored in loo/, ethanol (see Methods). It is likely that this low mobility is either due to dimer formation, or to a failure in these early experiments to detect S4 and 85; these two proteins appear to be rather susceptiblc to an aggregation which is not reversible in sarkosyl gels, and failure to detect them could result in fragment number 13 being assigned as number 1 1 . Later experiments have yielded fragment 11 with the faster mobility. The low mobility of fragment 13 suggests that some proteins (probably S6, Sl1 or Sl8) may have been lost during the course of the first electrophoresis, i.e. that the initial hydrolysis product was fragment 14. Similarly the low mobility of fragment 1 (which contains S7 and S19, and which always appeared as a shoulder on fragment 4 and 5 ) is probably due to a release of this fragment during the course of the polyaerylamide/agarose gel electrophoresis. However, the important feature of Pig. 6 is

Vol. 37, No. 3, 1973 J. MORGAX and R. BRIMACOMBE 479

that none of the fragments has a mobility which is significantly too fast for the protein composition assigned to it. A mobility which is too fast would imply that the fragment concerned was in fact a fortuitous mixture of smaller pieces (see [Z]). Frag- ments obtained under conditions J and K (Table 1) are not included in Fig. 6 ; these particular hydrolysis conditions in\Tolved the inclusion of 10°/, ethanol in the RNA * protein gel, with consequent alteration in pore size (see above). Mobilities of these fragments were nevertheless self-consistent, on a plot similar to that of Fig. 6.

Interpretation in Terms of the Protein Arrangement in the 30-5’ Particle

We have already noted [ 2 ] that proteins which are found together in RNA * protein fragments are closely related during the process of ribosome assembly in vitro [3], and the fragments listed in Table 2 reinforce this conclusion. The logi- cal inference is that the “assembly map” data [3] are a direct reflection of the topographical relation- ships between the ribosomal proteins. Making this assumption, we have attempted in Fig. 7 to combine our fragment data with the assembly map into a preliminary three-dimensional model for the arrangement of the 30-5 proteins. The purpose of this figure is to demonstrate that these two independent sets of data are consistent, and also that there are very stringent restrictions on the number of arrangements which could be drawn to accomodate all the data in this manner. The model should also help us to see in which areas of the 30-S particle the data are weak, and hence which groups of proteins to try and concen- trate on in our search for new specific RNA protein fragments.

If Fig.7 is compared with the data of Table 2, it can be seen that all the proteins found together in RNA - protein fragments are close together in the figure, and the detailed arrangement has been made in such a way that all the assembly interactions [3] are between adjacent proteins. The dotted line separates the two groups of proteins of Table 2, indicating where we have found the particle to be most sensitive to breakage. Proteins S1. S2, and S12 are not included in the figure, and XI6 and XI7 are not distinguished. The protein arrangement has been arrived a t empiri- cally, and is in the form of two planar layers of proteins one centred around 58, and the other around S9. We emphasize that this symmetry is a t this stage purely for convenience in drawing the diagram, and, since nothing is known about the detailed shape of individual RNA . protein complexes within the ribosome, it is not possible to relate a schematic structure of this nature to the known physical dimensions of the 30-S particle.

Fig. 7. PrelimCnary m,ap of the three-dimensional arrangement of the 30-8 ribasomalproteins. The arrows are from the “a.ssembly map” [3], and also include the interactions reported by &!I. Green (personal communication) between 58 and S15 and 57 and S13. The dashed line divides the two groups of proteins in Table 2. The figure is slightly “exploded” for clarity

Fig. 7 takes account of results obtained by the use of bi-functional protein cross-linking reagents [4-61. Thus S7, S9 and 86 form a triad of cross-linkable proteins [4], and S8 and S5 we adjacent [S ] , as are S18 and S21 [5,6]. These groups of proteins are already related by “assembly map” interactions [3], and the fact that they can be cross-linked is added evidence for the assumption that the assembly map is a direct reflection of the protein arrangement. In addition however, Bickle et al. [4] report that S9 is adjacent to S5, and that 819 is adjacent to either Sl1 or S12. The first pair of these (S9 and S5) are already close together in Fig.7, and it can be seen that XIS, S l l and S21 could if necessary be carried “behind” the diagram to bring Sl I into contact with S19, without greatly disturbing the rest of the arrangement. The structure will undoubtedly need a number of such modifications as new data become available, and the final arrangement itself will probably require a ccrtain amount of functional flexibility. Of the proteins in Fig.7, only S3 and S21 have so far not been found in our specific fragments. The failure to find S3 is interesting, in view of its strong interaction with S5 and S10 [3]. It is possible that our hydrolysis conditions have selected for ribosomes deficient in S3, which is of a doubtful stoichiometric class in the ribosome [14]. It should be noted that in Fig.7 53 is adjacent to S14, in agreement with data on the protcins which are involved in the binding of tRNA to the ribosomal “A” site 1151. It should also be noted that the ambiguity (already discussed) in our identification of S10, S13 and 514 does not affect the protein arrangement ; all three of these proteins are adjacent to S7 and S19 (fragment I, Table 2), and frag-

480 J. MORGAN and R. BRIMACOMBE: Arrangement of Escherichia coli 30-S Ribosomal Proteins 1 ~ ” ~ . J. Bioehem.

ments number 2, 4 and 5 are accounted for in Fig. 7 , whichever of SIO, S13 and 514 are present.

In addition to the arrangement of the proteins, it is obviously important to know how the ribosomal RNA is threaded through the structure. Schaup et al. [I61 and Zimmermann et al. [I71 have shown that 54 binds in the 5’-proximal half o f the 16-S RNA. The latter authors have also shown [17] that protein S15 binds to “loop C” near the middle of the RNA, and that 87 binds in the 3‘-proximal half. Other authors [IS, 191 have suggested that RNA - protcin fragment data can be interpreted in terms of a linear sequence o f proteins along an RNA “backbone”. This idea must be treated with caution, since some proteins may bind to more than one distinct region of thc RNA, and others may have no contact with the RNA a t all, or may only be stable in large fragments. Nevertheless, the idea is a tempting one, and if it proves correct the fragments listed in Table 2 would imply that S9 is adjacent in sequence along the RNA to S7 and S19, with S10, 513 or S14 adjacent on the other side (fragments 1 to 4). Similarly, S 5 would be adjacent to 58 and Si5, with S16(17) and then S20 on the other side (fragments 7 to 11). The data of Table 2 have been presented in such a way as to help visualise this, with protein S13 (see above and [12]) forming the link between the two groups of prot,eins. These predictions can of course only really be tested by actual analysis of the RNA sequences involved with the individual proteins. Unfortunately the approach mentioned above, used by Schaup et al. [16] and Zimmermann et al. [17], is only applicable to the study of the binding sites of those few proteins (e.y. [ Z O ] ) which can be singly specifically bound to 16-S RNA; for the re- maining proteins, another approach is necessary. We have shown [21] that the RNA from a specific RNA protein fragment can be isolated and “fingerprinted”,

and have described the region of the known 16-S RNA sequence [22,23] which is involved in the binding of proteins 197, S9, Sl9 and S10 or S13. If such experiments can be extended to an analysis of the RNA sequences present in the shoulder as well as the main peak of reproducible hydrolyses such as that shown in Fig.4 (fragments number 2 and 5, Table Z) , then the regions of RNA corresponding t o proteins such as S9 and SiO(13) could be directly inferred.

We are very grateful to Dr H. G. Wittrnann for his generous gift of purified 3 0 3 proteins, and to Mr Stuart Swinburne for his untiring assistance.

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A Preliminary Three-Dimensional Arrangement of the Proteins in the Escherichia coli 30-S Ribosomal Sub-Particle