photoinduced protein-rna cross-linking in mammalian 80-s ribosomes

Eur. J. Biochem. 106, 33-40 (1980)

Photoinduced Protein-RNA Cross-Linking in Mammalian 80-S Ribosomes

Anne-Marie REBOUD, Monique BUISSON, Simone DUBOST, and Jean-Paul REBOUD

Laboratoire de Biochimie Medicale, Universite Lyon 3

(Received November 28, 1979)

RNA-protein interactions in the 80-S rat liver ribosomes were studied by measuring cross- linking of proteins to rRNAs induced by ultraviolet radiation, as already reported for free 40-S and 60-S subunits. Our results are compatible with the model in which most of the ribosomal proteins are accessible to rRNAs in the native conformational state of the ribosomes. Subunit association in 80-S ribosomes does not seem to induce modifications in protein-RNA interactions as measured by this irradiation technique. However, two proteins, SS and S I ~ , appeared to be significantly less cross-linked with RNA in ribosomes than in free subunits.

Ribosomes which had been frozen and thawed several times were highly sensitive to ultraviolet radiation. Such treatment in the cold chiefly modified their 60-S subunit moiety.

The dissociation of 80-S ribosomes to subunits and their subsequent reassociation constitute essential steps in ribosomal function. Several authors noted striking differences in the patterns of reaction of the 40-60-S free subunits and the 40-60-S moiety of the 80-S ribosomes towards a variety of protein re- agents: salts or chemical and enzymatic reagents (for review see [l]). They suggested that these differences result from either direct shielding or conformational changes induced in subunits during their association. However, in this last case, when protein reagents are used, it is not possible to distinguish between modifi- cations of protein-protein and protein-RNA inter- actions.

In the present study the extent of protein-RNA interaction in 80-S ribosomes, as measured by ultra- violet irradiation, was compared to those already determined in free 40-S and 60-S subunits [2,3]. Utili- sation of ultraviolet irradiation has the main advantage that the probe is of zero length; that is, the two partners in the reaction are within covalent binding distance of each other when cross-linking occurs. Also, there is no perturbation of the natural complex by the introduction of an extraneous chemical moiety. It has been well established that ultraviolet irradiation, although giving rise to aspecific and non-cleavable protein-RNA cross-linking, is useful for topographic studies on ribosomes and subunits [2-91.

MATERIALS AND METHODS

Ribosomes from rat liver were isolated according to a method adapted from that of Moldave [lo]. When ribosomes labelled on their protein moiety had to be used, they were treated with formaldehyde and b~ro[~H]hydride for 20 min at 20 "C prior to irradia- tion [ll]. In the other cases they were methylated under the same conditions, but using unlabelled re- agents, in order to obtain comparable results [3]. The A260 of a 1 mg/ml solution was considered to be equal to 14.0.

Ultraviolet Irradiation Conditions

Five identical samples of ribosomes in buffer A: 50 mM triethanolamine, pH 7.3, 30 mM KC1, 1.5 mM MgCl2 containing 20 mM 2-mercaptoethanol were irradiated at 253.7 nm using a low-pressure mercury lamp under conditions already reported for 40-S and 60-S subunits: 2.2 mg/ml, 2 mm solution depth, at 4 "C or 25 0.1 "C in a thermocryostat. At given time intervals one of the samples was removed for analysis and the others shaken manually before further irradiation. As the volumes used were very small (less than 180 pl), magnetic stirring could not be used. However we observed that circulation of thermostated water by itself ensured homogenization of the samples

34 Photoinduced Protein-RNA Cross-Linking in 80-S Ribosomes

during irradiation experiments. Incident radiation doses were determined by ferrioxalate actinometry [I 21. No material precipitated during irradiation.

Electrophoresis Studies

Unreacted proteins from 5.5 AZ60 units of ribo- somes, subjected to increasing doses of radiation, were obtained using different extraction procedures : so- dium dodecyl sulfate for monodimensional gel elec- trophoresis, acetic acid [13] and LiCl/urea [14] for monodimensional and bidimensional gel electropho- resis. In some experiments, acidic hydrolysis (in 5 % trichloroacetic acid at 90°C for 7 min) was used to degrade rRNAs from either irradiated ribosomes or from the remaining insoluble protein-RNA complexes after acetic acid extraction.

Protein samples were electrophoresed in one diinen- sion using sodium dodecyl sulfate [15] or in two dimensions using, for the first dimension, either a basic or an acidic buffer system: pH 8.6 or 5.5 [16]. The conditions of the second dimensions are the same in both systems (pH 4.5). The code for numbering the proteins has already been reported [ l l ] and cor- responds to that adopted recently as 'uniform nomen- clature' [16,17]. Staining intensity of each protein spot was measured as previously described using 25 '%; pyridine [18]. The recovery of the total stainable ma- terial from the electrophoregrams or irradiated prod- ucts was higher when using one-dimensional gel elec- trophoresis than with the two-dimensional technique because of RNA nicking. Initially cross-linked pro- teins might have small pieces of RNA still attached to them and would therefore be excluded from two- dimensional gel electrophoresis, whereas they could still penetrate into the gels using one-dimensional electrophoresis carried out in the presence of sodium dodecyl sulfate [3].

Melting-Out Curves

They were determined on control and irradiated ribosomes kept in buffer A as previously described [3].

RESULTS MCTIlODS OF ASSAY OF KNA-PROTEIN CROSS-LINKING REACTION

The existence of covalent protein-RNA cross- linking in ribosomes submitted to increasing doses of radiation was demonstrated by means of two methods already reported in previous papers concerning the photoinduced cross-linking of ribosomal proteins to the 18-S and 28-S RNA molecules in the compact 40-S and 60-S subunits [2,3]. The first method con- sisted in the direct measurement of the amount of

labelled ribosomal proteins which remained associated to rRNAs after addition of sodium dodecyl sulfate to the irradiated 80-S ribosomes. The second method was indirect, and only suitable for experiments in- volving sufficiently high levels of cross-linking : un- reacted proteins extracted from irradiated subunits were analyzed by one or two-dimensional gel electro- phoresis and the decrease of their staining intensity was followed as a function of radiation doses. Both methods clearly showed a dose-dependent protein- RNA cross-linking. The dependence of cross-linking reaction upon radiation doses, as analyzed by one- dimensional gel, is shown in Fig. 1 (O), for ribosomes kept in a pH 7.4 buffer. It can be seen that photo- induced disappearance of stainable material occurs for radiation dose of 10" quanta (157;). With 4.32 x 10'' quanta about twice as much cross-linking was observed.

VARIABILITY OF DATA

During our investigations we noticed variations in the extent of protein-RNA cross-linking. These variations depended on the ribosome preparations. We noticed that repeated freezing and thawing of ribosomes increased their ability to form protein- RNA cross-links. 75 %, of proteins were recovered from ribosomes frozen once and irradiated with 4.32 x lo1* quanta; see Fig. 1 (0). After two freeze- thaw steps this recovery dropped to 45';/, (0). It is important to note that we could only detect differences between these ribosomes after they had been irradiated. Repeated freezing and thawing increased simultane- ously the extent of protein-RNA cross-linking and the inactivation of the ultraviolet-irradiated material, although it did not apparently change the properties of the controls, nor their biological activity and theirs' values nor the sedimentation profile of their RNAs (results not shown). Controls and irradiated ribosomes which had been previously frozen and thawed once, twice or three times were dissociated into subunits, in order to determine the site of the structural modifi- cation. Comparison of the sedimentation profiles of these ribosomal subunits suggested that it was the large subunit which was mainly damaged during cold treatment (results not shown). This was confirmed by following inactivation of each subunit in cold-treated and irradiated ribosomes, as measured after supple- menting these ribosomes with standard 40-S or 60-S subunits in pol y(U)-dcpendent polyphenylalanine syn- thesis, under conditions in which ribosomes frozen once before irradiation remained fully active. A typical example of the results obtained is shown in Table 1. Preincubating ribosomes for 10 min at pH 8.6 or sub- jecting them to reductive methylation, which affects neither their ability to function in v i t ro nor the migra- tion characteristics of their proteins, proved to be effec-

A.-M. Reboud. M. Buisson, S. Dubost, and J.-P. Reboud 3.5

\

I I I I I

1 2 3 4 5

10'' Dose (quanta)

Fig. 1. Effect q f storrige urld preincuhution c~onditiorzs on the dose- dependent disappearance qf total ribosomal proteins nieasurcd rr.sirlg one-dinzensional polyacrylumide gel. The intensity of the \Lain material was determined by integration of absorbance peaks. The resultant data are plotted as fractions of the total initial stain intensity. Ribosomes frozen once were preincubated 10 min at 4 'C, either in a pH 7.4 (0) or in a pH 8.6 (A) buffer prior irradiation at pH 7.4 and 4°C (see text). Ribosomes frozen twice were subjected to the same preincubation conditions at pH 7.4 (0) or at pH 8.6 (A) before irradiation

tive for diminishing protein-RNA cross-linking and in- activation even after repeated freezing and thawing (compare curves A and 0 in Fig. 1 and see Table 1). Hence, all the following experiments were carried out on once-frozen ribosomes methylated with unlabelled reagents for 10 min at 20°C. This treatment had the further advantage that the results could be directly compared with those obtained using 80-S ribosomes labelled in vitro on their protein moiety.

EFFECT OF IRRADIATION ON RIBOSOMAL COMPONENTS

Proteins

Photoinduced cross-linkage of ribosomal proteins to the rRNAs was followed by measurement of the disappearance of proteins from either the acetic acid or LiCliurea or dodecyl sulfate extracts prepared from ultraviolet-irradiated ribosomes. Protein analysis was first made by one-dimensional gel electrophoresis in the presence of dodecyl sulfate with identical results whatever the protein extraction method used. The electrophoretic patterns suggest that no photoinduced peptide bond cleavage occurred during ultraviolet irradiation. High-molecular-weight material never ap- peared in soluble extracts from ribosomes unless these had been heavily irradiated at 25 "C. Two conclusions can therefore be drawn: (a) ribosomal proteins cross- link to rRNAs by covalent bonds which are not labile

Table 1. Effect of freeze-thaw treatment before irradiation of riho- sorne.s on the residual activity of each subunit Ribosomes were subjected first to freeze-thaw treatments as indicated and then irradiated with 1.08 x 10" quanta. Aliquots (16 pg) were taken out and assayed for poly(U)-dependent polyphenylalanine synthesis [I91 either in conjunction with standard subunits [ I 1 pg 60-S (a) or 5 pg 40-S (b)] or alone. In this last case the results obtained were identical with those in (b). Results are expressed as a percentage of activity of unirradiated material. The values in parentheses were obtained using ribosomes either methylated [l 11 or preincubated for 10 min at pH 8.6 before irradiation at pH 7.4. In the other cases, ribosomes were maintained at p H 7.4 throughout the whole experiment

Number of freeze- thaw steps in 80-S ribosome

Activity of subunits organized

~~ ~~~ ~~~~~

40-S subunits 6 0 3 subunits (a) (b)

7" ~ ~ ~~

I 100 (100) 82 (82) 2 82 (85) 12 (45) 3 68 (73) 0 (30)

during acetic extraction procedure, otherwise different results would be expected using other methods of protein extraction ; (b) protein-protein cross-linkage is very unlikely in ribosomes irradiated at 4'.C. Data indicating that acidic hydrolysis of rRNAs from ribo- somes irradiated at 4 "C almost completely reversed the effect of irradiation on the protein pattern (either on one or two-dimensional gel electrophoresis) also confirmed the absence of peptide bond cleavage and protein-protein complexes and strongly suggest that proteins are complexed to RNA individually. Although protein-protein-RNA complexes labile in hot tri- chloroacetic acid would not be seen, such associations remain unlikely at 4'C in view of the fact that no protein-protein cross-link was detected at this tem- perature (see above).

The sensitivity to ultraviolet irradiation of each individual protein was assessed by following the dose- dependent reduction of its initial stain intensity on two-dimensional gel electrophoresis. Different pat- terns were obtained according to the proteins con- sidered when irradiation was performed at 4 -C (Fig. 2). Cross-linking of individual proteins was higher and less specific for the same radiation dose at 25 'C than at 4"C, as observed previously in the case of free ribosomal subunits [2,3]. Furthermore, comparison of the curves with those concerning the same proteins but in irradiated free ribosomal subunits was made. Such comparison is valid, since the absorbed dose of radiation was identical in both cases. Indeed solutions of the same absorbance were irradiated under identical conditions. Results suggest that the state of the par- ticles (free or complexed into monosomes) has little influence on the reactivity of proteins (including the

36 Photoinduced Protein-RNA Cross-Linking in 80-S Ribosomes

1.0

0.5

1.0

0.5

c c 0 1 .0 .- m LL L

0.5

I

I I I I I I I I I I I I I A 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4

. ”(

=. c vl c 0

C

.- c ._ - m

C

+-

.- .- ._ c

I

27a

I

1.0

0.5

23

0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4

1 0 ” Dose (quanta)

Fig. 2. Dose-dependent disappearance ofproteinsfrorn two-dinimsional poljacrylarnide gels. The intensity of the stained spots was measured as described in Materials and Methods. The fractions of initial stain intensities remaining at various dose levels of ultraviolet radiations were expressed using a semilog plot. Only well-resolved proteins have been considered in this experiment. All of these were determined using the pH 8.6 system (for the first dimension) except Plb and Pz, which were determined using the pH 5.5 system. Each point represents the average value obtained from three separate irradiation experiments. The maximal deviation of the data between the experiments was & 10%. Proteins were recovered from either 80-S ribosomes (A) or free subunits in previous experiments ([2,3]; ----)

A.-M. Reboud, M. Buisson, S. Dubost, and J.-P. Reboud

100

50

u F e F 0

01 C

0, .- c e a

37

I I I I

I I I , \ " 0 1 2 3 4 0 1 2 3 4

Id'' Dose (quanta)

Fig. 3 . Variation in the amounts of proteins extractable from ribosomes and subunits irradiated with increasing doses at 4 "C and 25 "C. The total stain recovered from well-resolved 80-S ribosome protein spots from irradiated ribosomes was measured and expressed as a fraction of the stain intensity of the same protein fraction recovered from control non-irradiated ribosomes (A; see also Fig.2). This protein fraction has been recovered from either complexed subunits (-), or free subunits in previous experiments ([2,3]; ---). (0, 0) 40-S protein fraction; (0,o) 6 0 4 protein fraction. (A) 4°C; (B) 25°C

phosphoacidic, P l b and P2) up to 2 x 10" quanta. A few proteins appear to be a little more easily cross- linked in the complete ribosome than in isolated sub- units, but the differences remain very small and are probably within the limits of experimental error. On the other hand, proteins S9 and S13 seem to be significantly more resistant in 80-S ribosomes. It is interesting to note that these two proteins were re- cently reported to be located at the interface between the two subunits [20]. For higher radiation doses, proteins were much more resistant to ultraviolet irradiation in monosomes than in free subunits. Therefore, the classification of 80-S ribosomal pro- teins into low-dose-reactive and high-dose-reactive proteins is very similar to that obtained using isolated subunits, since only data concerning low-dose radia- tion at 4 "C signify specific intermolecular protein- RNA cross-linking (see above and also [2,3] for the basis of this procedure). All these results are, in a sense, summarized in Fig. 3A and B, which shows the dose-dependent decrease at 4 "C and 25 'C of total staining of the proteins which are well resolved in 8 0 4 ribosome patterns (see Fig. 2). Patterns concern- ing ribosomes (solid lines) and free subunits (inter-

rupted lines) have been reported here. As was expected, the 80-S ribosome protein fraction was more reactive at 25 "C than at 4 "C and exactly the same results were obtained using the whole 80-S ribosome proteins (results not shown). Isolated or complexed subunits have almost the same behavior when irradiated with low-doses of radiation (lo1' quanta). Thereafter, at higher radiation doses both subunits in the complexed state showed a lower sensitivity than when in the isolated form. At 4 "C complexed or free 40-S subunits were less reactive than their 60-S analogs, whereas both subunits showed the same reactivity at 25 "C.

rRNAs

As could be expected, the ultraviolet-induced cross-linkage reaction simultaneously modifies the properties of proteins and rRNAs in 80-S ribosomes. Photoreactivity results in a dose-dependent decrease in the separability of the rRNAs and proteins in 4 M urea/3 M LiCl (results not shown) as observed pre- viously in the case of isolated subunits [2,3].

If one considers the sedimentation patterns of rRNAs extracted from control and irradiated ribo-

38 Photoinduced Protein-RNA Cross-Linking in 80-S Ribosomes

100

- U E

E

$ 50

> 0 "

Q

In

2 u m In ln

0 0 1 2 3 4 5

10.'' Dose (quanta)

Fig. 4. Varitrtiori it1 the miounts of S - S ( i t id i .H- .T RIYA extractable jvonz rihosonies subjectid to increasiiig dosrs of' radiation. 5-S RNA (A) and 5.8-S RNA (0) were extracted from ribosomes irradiated at 4 C ( A . 0 ) or at 2 5 ' C (A,.). The conditions of incubation were 3 min at 25 C for 5-S RNA extraction and 3 min at 60°C followed by a rapid cooling at 4 ° C for 5.8-S RNA release, in 40 mM Tris-HCI p€I 7.3, 20 mM sodium acetate, 1 mM EDTA (buffer B) and 1 (wlv) sodium dodecyl sulfate. The solutions were then loaded onto a 12% polyacrylamide gel in buffer B. The tanks contained buffer B together with 0.2 7; sodium dodecyl sulfate. Electrophoresis was run for 4.75 h at 90 V. The amounts of both rRNAs were deduced from the peak areas recorded at 260 nm and expressed as a percentage of the 5-S and 5.8-S RNA contents of control ribosomes. (---) The variation in the amounts of these rRNAs extractable from irradiated 60-S subunits [3]

somes much the same results were obtained as with isolated 40-S and 60-S subunits a t 4 "C or 25 "C. In the case of irradiated samples, rRNA peaks were pro- gressively reduced. They became broader, although neither nucleotides nor fragments were seen (results not shown). Irradiation at 25°C caused at least one chain scission of the large rRNAs, probably of the 1 8 3 RNA, as seen repeatedly from samples irradiated with (0.5-4.32) x 10" quanta and then treated with sodium dodecyl sulfate under conditions which do not denature the native secondary structure of the rRNA molecule. A sharp peak corresponding to a RNA fragment of about 60000 molecular weight appeared in 12'),:, polyacrylamide gel, which was never observed using heavily irradiated 60-S subunit samples. In one experiment out of four performed at 25 T , we have observed a faster RNA sedimenting at 37.3 S (molec- ular weight of 2.5 x lo6) ' which would be consistent with cross-linkage between 18-S RNA and 2 8 3 RNA.

Fig. 4 illustrates the dose-dependent decrease of the 5-S and 5 . 8 3 RNA amounts recovered from ribo- somes irradiated at 4 "C and at 25 "C. In both cases

I That value is calculated using the equation developed by Kur- land [21]: s = 0.98 x MP.56 for E . coli rRNA.

the same progressive disappearance of material was observed (solid line). In ribosomes 5-S and 5 . 8 3 RNA were slightly more reactive than when they formed part of the free 60-S subunits (see [ 3 ] and in Fig.4 interrupted line).

Activity und Sedimentation of'Irmdiuted Ribosomes

In Fig. 5A, ribosomes irradiated with increasing doses of radiation at 4 "C or at 25 "C were assayed for poly(U)-directed polyphenylalanine synthesis. Non-irradiated ribosomes kept at 4 "C and 25 "C during the whole experiment retained all their activity, whereas ultraviolet irradiation progressively inacti- vated them. The damage incurred was slightly higher at 25°C than at 4"C, and at these temperatures for 4.32 x 10" quanta, the activity of ribosomes was respectively reduced to 33 and 50 "/, of its normal level. Identical results were obtained for the endoge- neous activity measured in the absence of poly(U). The inactivation of 40-S and 60-S subunits in the 80-S ribosomes, followed by hybridization of irradi- ated ribosomes with standard 60-S or 40-S subunits, appeared to be identical to that of whole ribosomes when irradiation was performed at 25 'T. At 4 ° C the 40-S subunits were slightly less damaged than the 60-S. We observed that irradiated samples were more easily inactivated than non-irradiated controls, when kept in the cold room. The inactivation increased as a function of the radiation doses used. In Fig.5B, ribosomal activity is plotted against the percentage of either total protein or 5 - 5.8-S RNA recoveries. This dependence became critical in a relatively low range of protein decrease (about 40 x) as compared with the plots obtained for isolated 60-S and 4 0 4 subunits (70- 90 :4, [2,3]). Inactivation occurred for a smaller decrease of 5 - 5.8-S RNA recovery.

It was observed that control ribosomes contained only monomers and small amounts of subunits. The absence from the sedimentation patterns of ribosomal dimers 90 - 1 10 S derived from mechanical breakdown of polysomes, must be related to the reaction of methylation at pH 8.6, which dissociated them into subunits [I 11. The rapid return to pH 7.4 reassociated- back ribosomal subunits into monosomes. At 4 "C and at 25 "C, the diagrams (not shown) indicate the same gradual decrease of the monomer peak area for in- creasing doses of radiation, without any change in their sedimentation coefficient value nor any evidence of digestion products, in contrast to what was ob- served with free subunits [3].

Melting- Out Tenzperu tures

The temperature t,, at which 50% of the increase in absorbance is observed, has been determined for ribosomes irradiated with increasing doses of ultra-

A.-M. Reboud, M. Buisson, S. Dubost, and J.-P. Reboud 39

100

- ." > c 5 0 .- c u 4

0 0 1 2 3 4 5

1 0 ' ~ Dose (quanta)

0 5 0 100

Proteins or 5 S 5.8 S RNA

recovered (%)

Fig. 5. Inuctivution ofphotoirradiated ribosomes. Aliquots of ribosomes irradiated with increasing doses were assayed for poly(U)-directed polyphenylalanine synthesis. The results are expressed as a percentage of the activity of non-irradiated controls kept under the same conditions (see text). (A) Given as a function of radiation doses a t 4 "C (A) or at 25 "C (A). (B) Given as function of the amounts of 5 4 , 5.8-S RNAs (0, see Fig.4) or of whole 80-S proteins (A) recovered on two-dimcnsional gel electrophoresis from ribosomes irradiated at 4 'c (0, A) or at 25 'c (W, A)

violet radiation, at 4 "C and 25 "C. The same results were obtained at both temperatures. t , remained at its initial value (48°C) for 10" quanta but fell pro- gressively, for samples irradiated with higher radiation doses, down to 39 "C for 4.32 x 10" quanta. Irradiation progressively destabilized the rRNAs secondary struc- ture, which is in agreement with previous results con- cerning rat liver 60-S subunits [ 3 ] .

DISCUSSION

The data presented in this report support the con- clusion that ultraviolet irradiation of 80-S ribosomes results in the covalent cross-linkage of the ribosomal proteins to the rRNAs. The basis of the arguments developed in the text has been discussed elsewhere for irradiated 40-S and 60-S subunits [2,3].

Scveral data in the literature demonstrate that ribosomes exist in different conformations. It was not surprising, therefore, to find that photoinduced pro- tein-RNA cross-linkage depends on ribosome prepa- ration and on preincubation conditions. Thus, as shown in Fig. 1 and in Table 1, repeated freeze-thaw operations render 80-S ribosomes very sensitive to ultraviolet irradiation, although no modification in ribosome properties has apparently occurred. Disso- ciation experiments and biological activity measure- ments of irradiated ribosomes supplemented with in- tact subunits provided evidence that cold treatment modifies mainly 60-S subunits. RNA secondary struc- ture appeared to be altered in these subunits when

frozen several times, since the subunit melting-out temperature progressively decreased as a function of the number of freeze-thaw steps (M. J. Marion, un- published results). On the other hand, tertiary struc- ture of the subunits seems to be preserved, as can be deduced from their unchanged sedimentation pattern and biological activity. Denaturation of the native secondary structure of rRNA would be expected to increase the number of base-protein interactions, since pyrimidines in single-stranded polynucleotides are far more reactive in photoaddition reactions with small molecules than pyrimidines in helical polynucleotides [6]. It is interesting to note that spontaneous conforma- tional modification of eukaryotic 5-S RNA also oc- curred at - 15 "C [22].

Here it is shown that the sensitivity of a great number of 40-S and 60-S ribosomal proteins to low radiation doses is about the same when the subunits are free or form part of the 80-S ribosome. Therefore, one may suppose that subunit association into 80-S ribosomes does not cause a real change of conforma- tion in their structure, at least at the protein-RNA interaction level. It must be pointed out that such hypothesis which could be sustained only after a detailed analysis of the number and localisation of the different crosslinks2 would not be in contradiction with results obtained using protein reagents, and

' Such studies are hampered, in mammalian ribosomes, by the difficulty in obtaining rRNAs uniformly labelled with a high specific radioactivity.

40 A,-M. Reboud, M. Buisson, S. Dubost, and J.-P. Reboud: Photoinduced Protein-RNA Cross-Linking in 80-S Ribosomes

which suggested that association and dissociation processes modify subunit surface areas [l]. The ob- servation that for high radiation doses fewer protein groups are reactive in 80-S ribosomes than in free subunits, is compatible with smaller photoinduced change in the 80-S ribosome topography, as shown by sedimentation analysis and photoinactivation. This stabilizing effect should result from the interaction between subunits. The fact that some proteins (L25, L29, L31, S22 - 23, S28) totally disappeared indicates that ultraviolet radiation has acted on each subunit.

The main disadvantage of the preparations used (80-S not pretreated with puromycin) is that they con- tained numerous factors, e.g. aminoacyl-tRNA, pep- tidyl-tRNA, messenger RNA, which might affect interaction between 40-S and 60-S subunits. How reductive methylation at pH 8.6, which reversibly dissociated about half of the polysomes and disomes into subunits [ll], modified the tightness of binding of these macromolecular factors is not known. On the other hand, combination of equimolecular amounts of 40-S and 60-S subunits would have introduced another ambiguity, since we know that several different types of couples between 40-S and 60-S can be formed, including 40-S dimers and 60-S dimers. These studies of crude 80-S ribosomes were also hampered by the presence of a latent ribonuclease. However, no attempts were made to remove it because the washing of ribo- somes by high concentrations of salt has a particularly dramatic effect on protein reactivity [23] and because this enzyme remains even in subunits prepared with 0.88 M KC1 [24]. That the photoinduced decomposi- tion of the ribosomal structure produces an activation of ribonuclease is suggested by the variability of results concerning 18 - 28-S RNA cross-linkage and the pro- gressive inactivation of irradiated material kept at 4 "C as compared with the controls. However, although chain breakage of the RNA components due to nu- clease action or photoinduced breakage was observed, it did not alter the tertiary structure of ribosomes, since these remained active. Consequently our photo- chemical analysis of protein-RNA interactions re- mains valid and at 4 ° C most of the 80-S ribosomal proteins are accessible to rRNAs in the native con- formational state of 80-S ribosomes.

This work has been supported by grants from te Centre National de la Recherche Scientifique (E.R.A. no. 399), the hstitul

National de la SantP et de la Recherche Medicale (78.1.159.3) and the Delegation GPnerale a la Recherche Scientifique et Technique (79.7.0160).

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