OIG'G

Molec. gen. Genet. 152, 253-257 (1977)

© by Springer-Verlag 1977

A New Nucleic Acid-Protein Cross-Linking Reagent Chr. Oste 1, R. Parfait 1, A. Bollen z, R.R. Crichton 1 i Laboratoire de Biochimie, Universit~ Catholique de Louvain, 1, Place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium 2 Laboratoire de G6n6tique, Universit6 Libre de Bruxelles, 67, rue des Chevaux, B-1640 Rhode-St-Gen6se, Belgium

Summary. A new photoactivable reagent is described, which allows the formation of RNA-protein crosslinks via disulfide bridges in combination with mercaptobutyrimidate. The reconstituted L24 protein-23S RNA complex from the large subunit of E. coli ribosomes has been used as a model system for the cross-linking. The main advantages of the reagent are the absence of U.V. generated cross-links, since photoactivation is carried out at 360 nm, on one hand and the ease of cleavage of the cross-link by mild reduction (/?mercaptoethanol) on the other.

O ,C-OC H N--N-C 2 B I ,C O" \NH CH-COOH I

~H2 SH

23 S RNA-L 2/~ complex hv 360 nm L 2z,- 23S RNA O \ ~ - C-OC2HB C O~" "NH-CH-COOH CH +H2

Introduction ®

Interactions between proteins and nucleic acids play an important role in biology. In the ribosome these interactions are involved not only in the structure and assembly of the particles, but also in their function (Pongs et al., 1974). A useful and stable model system for the study of rRNA-ribosomal protein interactions is offered by the complex of 23S RNA with the ribosomal protein L24. The protein L24 is resistant to trypsin digestion both in the 50S and 70S particles and in reconstituted complexes of 23S RNA and L24 (Crichton and Wittmann, 1973). As part of a continuing programme on this complex we have developed a new photoactivable crosslinking reagent: N-(1-carboxy,2 thiol ethyl)-diazomalonamic ethyl ester (" N2SH") (Fig. 1) to prepare stable L24-23S RNA complexes which can be subsequently dissociated by reduction. The principle was to react the 23S RNA in the RNA-protein complex with " N z S H " , the protein with methyl mercaptobutyrimidate ("C4SH"), and then to form cross-links by oxidation using H 2 O 2. We have also developed optimal conditions for the dissociation of the L2423S RNA complex.

--

~ -SH l L 2/~

~NH^ C[ SH-(CH~)~-C~ Z Z ~ OCH3

o

SH ~ ] 23 S RNA

H2021 [ SH-CH~CH~OH ~-S-S

i

L % --23 S RNA Fig. 1. " N 2 S H " : N-(1-carboxy, 2thiol ethyl)-diazomalonamic ethyl ester: mode of action

Material and Methods 1. Organic Synthesis The synthesis of " N z S H " has been carried out by addition of phosgene to ethyldiazoacetate, formation of the corresponding acti-

254

Chr. Oste et al. : A New Nucleic Acid-Protein Cross-Linking Reagent

vated hydrosuccinimide ester, and condensation of L-cysteine hydrochloride to this last adduct. The complete synthesis will be described elsewhere (Oste, Chr., Bollen, A. and Loffet, A. in preparation). All other chemicals used were of analytical grade.

Table 1. General feature of the cross-linking experiment. L24-23S RNA complex (0.1 nmole, 1.2 x 104 cpm/ml) TEA buffer pH 7.8

2. Bacteria and Ribosomes

I + N2SH stock" final concentration: 25 mM

E. coli MRE 600 were grown in rich medium according to Gold and Schweiger (1971) and ribosomes isolated by the method of Eikenberry et al. (1970). Total r-RNA was prepared by repeated phenol extraction of 70S ribosomes and the purity checked by polyacrylamide gel electrophoresis (Wrede and Erdmann, 1973). The r-RNA was not further purified since protein L 24 binds specifically to the 23S RNA. 'The protein L24 was prepared from 50S ribosomal subunits by CM-cellulose chromatography (Hindennach et al., 1971) or by phosphocellulose chromatography of total 50S proteins (Zimmermann and St6ffler, 1976).

1 ml

0.3 ml

0.5 ml

Step A + UV; l h , 4°C

0.4 ml

0.3 ml

0.3 ml

0.3 ml

3. Preparation of'23S RNA-protein L24 Complex

Step B

The complex was reconstituted using the conditions of Traub and Nomura (1968). The r-RNA, at a concentration of 5 mg/ml in TMK buffer, pH 7.4 (30 mM Tris-HC1, 20 mM Mg (OAc)2, 350 mM KC1) was precipitated overnight at - 2 0 ° C , by addition of two volumes of ethanol and pelleted at 10,000 rpm for 45 min. The pellet was dried under vacuum and resuspendcd in 1 ml of TMK buffer pH 7.4. Radioactive L24 protein was prepared by reductive methylation (Moore and Crichton, 1973). The r-RNA solution was added to a lyophilised mixture of 10 gl of unlabelled L24 (1.45 mg/ml) and 10 gl of labelled L24 (1.1 x 104 cpm/gl; 10~ cpm/~tg) and subsequently incubated for 1 h a t 43°c. The complex formed under these conditions was chromatographed on a Biogel A 0.5 m column (30 c m x 1 cm) in TEA buffer pH 7.8 (50 mM triethanolamine, 50 mM KC1, 1 mM MgClz). The fractions excluded from the gel containing the RNA-protein complex were pooled. A control experiment showed that after incubation of (14C)L24 with 16S rRNA, no L24 was found in the excluded fractions, confirming the specificity of the interaction between L24 and 23S RNA.

Biogel A 0.5 m chromatography in dissociating conditions after heating in NaC1 2.5 M, EDTA 30 raM, 45 min at 45 ° C

Step D

Excluded fractions (6-7) concentration of the samples by dialysis against pure glycerol

Step E

4. Irradiation Technique

Biogel A 0.5 m chromatography in reducing dissociatg conditions after heating in NaC1 2.5 M, EDTA raM, fl-mercaptoethanol 3%, 45 rain at 45°C

For irradiation all samples (0.3-0.5 ml) were placed in Pyrex tubes (7 cm x 0.6 cm), briefly degassed and the tubes tightly shut. The tubes were immersed in water inside a water-cooled pyrex jacket and the system thermostated at 6° C. A "Philips Biosol" U.V. lamp of 500 W was used as light source, at a distance of 20 cm. The radiation at 360.8_+ 1.5 rim received by the sample in this system was about 104 ergs/cm2 s.

Step C

i

I

I

l

1 Step F

a The reagents were prepared as stock solutions in 1 M TEA

Step C. All samples coming from steps A and B were then submitted to oxidation by addition of 1/200 volume of HaO2 (30%).

5. Cross-linking Experiments (Table 1) Step D. All the above samples plus an additional control which Step A. To a 1 ml sample of L24-23S RNA complex (about 0.1 nmol, 1.2x 104 cpm). " N z S H " was added from a 0.5 M stock solution in TEA, to a final concentration of 25 raM. A 0.7 ml fraction of this sample was then submitted to U.V. irradiation as described above and the remaining 0.3 ml was not irradiated.

Step B. 0.4 ml of the irradiated sample was kept as control and the remainder (0.3 ml) was treated with "C4 SH" at a final concentration of 25 mM, for 20 min at 0°C. The non-irradiated sample from step A and 0.3 ml of original complex were also reacted with "C4 SH" in the sam e way.

had been UV irradiated, were submitted to dissociating conditions (see below) and analysed on Biogel A 0.5 m.

Step E. For each sample the fractions from the Biogel which contained undissociated L24-23S RNA complex were pooled and concentrated by dialysis against glycerol. Step F. Finally a second analysis on Biogel A 0.5m was carried out for each sample, this time in reducing dissociating conditions (see below). For each sample the amount of undissociated complex was redetermined.

Chr. Oste et al. : A New Nucleic Acid-Protein Cross-Linking Reagent

255

6. Complex Dissociation Assays

0

5 [

10 15 I

0

I

5 I

10 15 I

I

A

For dissociation, samples were adjusted to 2.5 M NaC1, 30 mM EDTA and heated for 45 rain at 45°C. To measure the amount of dissociation, samples were chromatographed at room temperature on Biogel A 0.5 m columns (30 cm x 1 cm) in the same buffer conditions. Dissociation in reducing conditions was carried out in the same way as above but with 3% fi-mercaptoethanol.

B

0.5 I

%

Results

i

x

T h e p r i n c i p l e o f the c r o s s - l i n k i n g e x p e r i m e n t s was the following. First, the c o m p l e x was i r r a d i a t e d in the presence o f " N z S H " . T h e result o f this step was the f o r m a t i o n o f a stable c o v a l e n t b o n d b e t w e e n the r e a g e n t a n d the R N A o f the c o m p l e x . " C 4 S H " was t h e n a d d e d a n d b o u n d c o v a l e n t l y to the p r o t e i n in the c o m p l e x . I n the final step, a n oxidising agent ( H 2 0 2 ) was i n t r o d u c e d to f o r m disulfide b r i d g e s bet w e e n the free S H g r o u p s o f the two reagents. These b r i d g e s were stable u n d e r c o n d i t i o n s in which the c o m p l e x was dissociated, b u t were easily cleaved by addition of fi-mercaptoethanol. F o r this p r o c e d u r e c o n d i t i o n s h a d to be f o u n d in w h i c h the L24-23S R N A c o m p l e x c o u l d be c o m p l e t e l y d i s s o c i a t e d w i t h o u t the use o f reducing agents. T a b l e 2 shows the results o b t a i n e d with a series o f d i s s o c i a t i n g agents. T h i o c y a n a t e , a g o o d c h a o t r o p i c ion, gave the best d i s s o c i a t i o n , b u t c o u l d n o t be u s e d b e c a u s e o f its r e d u c i n g power. DissociaTable 2. Results of the dissociation assays

Dissociation conditions

25° C

SDS 0.1%

10%

10 12%

Urea 6 M

22 25%

27-31%

5 7%

13-15%

NaC1 1.5 M, EDTA 20 mM

17-21%

25%

NaC1 1.5 M, EDTA 20 raM+ DMSO 5%

28%

31-34%

NaC1 1.5 M, EDTA 20 mM+ Urea 6 M

11%

16-19%

Guanidine C1 7 M

35%

41%

KSCN 1 M, KC1 0.15 M, Na cacodyl. 0.013 M pH 7.0 CaCI2 1 M NaC1 2.5 M, EDTA 30 mM

D

C

"1-

03

0.5

10 15

0

5

10 15 fractions

Fig. 2A-D. Profiles of Biogel A 0.5 m chromatography. Chromatography conditions were: column dimensions: length. 30 cm, inner diameter : 1 cm. Elution rate : 19 ml/hour; fraction volume: 0.91 ml. A Complex formed in standard conditions eluted in TEA buffer. L24-23S RNA peak in fractions (6-7)=excluded complex: fractions (14-15)=free L24. B Dissociation of the complex (Fig. 2A) in 2.5 M NaC1, 30 mM EDTA at 45°C for 45 rain and elution in these conditions. C Cross-linked complex (sample ff 2, Table 3) treated as in B. D Cross-linked complex (excluded peak in Fig. 2 C) after reduction with fi-mercaptoethanol. Small volumes of the fractions were analysed by scintillation counting

% complex undissociated 45° C

SDS 0.1%+urea 6 M

0 u

3%

8%

63%

75%

8%

12%

All of the dissociation conditions tested were evaluated for the amount of complex remaining undissociated, eluting in fractions 6-7 on a Biogel A 0.5 m chromatography in standard conditions, as compared to the total quantity of labelled complex applied on the column

t i o n was also a c h i e v e d with p r o t e i n d e n a t u r i n g agents such as g u a n i d i n e h y d r o c h l o r i d e (TM) a n d u r e a (6M). W e rejected these c o n d i t i o n s in o r d e r to a v o i d d e n a t u r a t i o n o f the p r o t e i n . Since salt l i n k a g e s are believed to p l a y a n i m p o r t a n t role in the s t a b i l i s a t i o n o f nucleic a c i d - p r o t e i n i n t e r a c t i o n s , one c o u l d expect high salt c o n c e n t r a t i o n s to dissociate the c o m p l e x . T h u s we finally chose the c o n d i t i o n s ( T a b l e 2) 2.5 M NaC1, 30 m M E D T A with h e a t i n g for 45 m i n at 45°C. F i g u r e 2 (a, b) shows the e l u t i o n o f the c o m p l e x f r o m Biogel A 0.5 m (a) a n d its d i s s o c i a t i o n by the c o n d i t i o n s d e s c r i b e d a b o v e (b). T h e different crossl i n k i n g e x p e r i m e n t s a n d c o n t r o l s c a r r i e d o u t are summ a r i z e d in T a b l e 3. F i g u r e 2(c) shows h o w crossl i n k e d c o m p l e x resists dissociation. T h e results are q u i t e c o n c l u s i v e : to o b t a i n a significant a m o u n t of c o m p l e x resistant to d i s s o c i a t i o n , the s a m p l e must h a v e u n d e r g o n e all t h r e e steps o f the cross-linking p r o c e d u r e . I n the first step the " N z S H " was p h o t o a c t i v a t e d by U.V. i r r a d i a t i o n a n d r e a c t e d with the R N A . I n the s e c o n d step, m e r c a p t o b u t y r i m i d a t e

Chr. Oste et al. : A New Nucleic Acid-Protein Cross-Linking Reagent

256 Table 3. Results of the cross-linking experiments

Sample n°

Treatment of the sample +N2SH

+UV

+C4SH

% of cross-linked complex (yield)

1. 2. 3. 4. 5.

+ + + --

-+ + + -

+ + --+

-(average) 68% about 3% --

6 .

-

-

-

-

The yield of cross-linked complex is defined as the % of L24-23S R N A complex which resists non-reduCing dissociation, as determined by Biogel 0.5 m gel filtration (Fig. 2). The data given were the mean of a series of separate experiments with several preparations of the complex

reacted with free e-amino groups of the protein. Finally in the third step, disulfide bridges were formed in situ between free thiol groups of the two components, creating covalent links between the protein and the RNA. The cross-linked complex can be dissociated by reduction with fl-mercaptoethanol (Fig. 2 d).

Discussion

The cross-link exPeriments reported here indicate that " N z S H " used together with mercaptobutyrimidate (Traut et al., 1973) is indeed an effective means of cross-linking protein-RNA complexes. We observed (Table 3) a very low yield of covalent linkages between the 23S R N A and the protein L24 on UV irradiation alone. This was expected since neither component of the complex absorbs at the wavelength used (360 nm). On the other hand, primary amino groups of the nucleic acid do not seem to be accessible or reactive towards the "C4SH", since no cross-linking occurred by addition of this reagent alone. Moreover, no covalent bonds are formed in the complex between the 23S RNA and the protein by irradiation in the presence of "N2SH". This suggests some specificity of the UV-generated carbene towards the nucleic acid in the experimental conditions used here. Finally no cross-links were observed if the complex, treated with both reagents, " N 2 S H " and "C4SH" was not irradiated. We assume that the reagent " N 2 S H " reacts via carbene formation upon irradiation (Fig. 1) but the precise chemistry of substitution reactions by carbenes remains unclear. Whilst a number of side reactions with solvent and other side reactions such as Wolff rearrangement (Knowles, 1972) can be expected, a significant amount of the UV-generated carbene undergoes reaction, presumably with exposed bases in the R N A by cycloaddition

or by insertion. From previous studies with a similar type of reagent (Bispink and Matthaei, 1973) it has been reported that 85% of the available reagent not involved in side reactions reacted with RNA. The results found with " N z S H " (Table 3) compare well with the above figure, especially since the yield found here includes the formation of disulfide bridges in the complex. With " N 2 S H " we do not have the problems of spontaneous generation of covalent cross-links during UV irradiation (Salomon and Elad, 1974; Schoemaker and Schimmel, 1974; M611er and Brimacombe, 1975), since we are working at a wavelength where neither rRNA nor ribosomal proteins absorb. This is confirmed by our control experiments (Table 3). In conclusion, it should be pointed out that photoactivable reactive groups such as carbenes and nitrenes have advantages but also disadvantages. They are highly active, but their chemistry is not well characterised, and they are rather non-specific as indicated by the importance of their side reactions (Knowles, 1972). However, they can be produced in rather mild conditions, especially when the wavelength used for irradiation is sufficiently high to avoid non-specific, radiation-induced reactions. We think that the main advantage of " N 2 S H " for RNA-protein cross-linking it its cleavability under mildly reducing conditions, thus facilitating the task of identifying which part of the reagent has reacted with which component of the systeml Acknowledgements. This work has been supported by the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture, in the case of C.O. We are grateful to A. Loffet and J. Gobert for helpful assistance in the organic synthesis. We thank R.R. Traut for critical reading of the manuscript.

References Bi-spink, L., Matthaei, M. : Photoaffinity labelling of 23S R N A in E. coli ribosomes with poly-U coded ethyl-2-diazomalonyl PhetRNA. FEBS Lett. 37, 291-294 (1973) Crichton, R.R., Wittmann, H.G. : A native ribonucleoprotein complex from E. eoli ribosomes. Proc. nat. Acad. Sci. (Wash.) 70, 665-668 (1973) Eikenberry, E.F., Bickle, T.A., Traut, R.R., Price, C.A. : Separation of large quantities of ribosomal subunits by zonal ultracentrifugation. Europ. J. Biochem. 12, 113 116 (1970) Gold, L., Schweiger, M.: Synthesis of bacteriophage specific enzymes directed by D N A in vitro. Methods in Enzymology XX, 537-542 (1971) Hindennach, I., Kaltschmidt, E., Wittmann, H.G.: Ribosomal proteins: isolation of proteins from 50S ribosomal subunit of E. coli. Europ. J. Biochem. 23, 1~16 (1971) Knowles, J.R. : Photogenerated reagents for biological receptor-site labeling. Accts. Chem. Res. 5, 155 160 (1972) M611er, K., Brimacombe, R.: Specific cross-linking of proteins $7 and L4 to ribosomal RNA, by UV irradiation of Escherichia coli ribosomal subunits. Molec. gen. Genet. 141, 343 355 (1975)

Chr. Oste et al. : A New Nucleic Acid-Protein Cross-Linking Reagent Moore, G., Crichton, R.R. : Reductive methylation: a method for preparing functionally active radioactive ribosomes. FEBS Lett. 37, 74 78 (1973) Pongs, O., Nierhaus, K.H., Erdmann, V.A., Wittmann, H.G. : Active sites in Escheriehia coli ribosomes. FEBS Lett. 40, 528-537 (1974) Salomon, I., Elad, D.: Ultraviolet and °/-ray induced reactions of nucleic acid constituents: reactions of purines with amines. Photochem. Photobiol. 19, 21-27 (1974) Schoemaker, H.J., Schimmel, P.R.: Photo-induced joining of a t-RNA with its cognate aminoacyl-RNA synthetase. J. molec. Biol. 84, 503 513 (1974) Traub, P., Nomura, M.: Structure and function of E. coli ribosomes: V. Reconstitution of functionally active 30S ribosomal particles from RNA and proteins. Proc. nat. Acad. Sci. (Wash.) 59, 777-784 (1968)

257 Traut, R.R., Bollen, A., Tung-Tien Sun, Hershey, J.W.B., Sindberg, J., Pierce, L.R.: Methyl 4-mercaptobutyrimidate as a cleavable cross-linking reagent and its application to the E. coil ribosome. Biochemistry 12, 17, 3266-3273 (1973) Wrede, R., Erdmann, V.A.: Activities of B. stearothermophilus 50S ribosomes reconstituted with prokaryotic and eukaryotic 5S RNA. FEBS Lett. 33, 315-319 (1973) Zimmermann, R.A., St6ffler, G.: Purification of proteins from the 50S ribosomal subunit of E. coli by ion exchange chromatography. Biochemistry 9, 2007-2017 (1976)

Communicated by H.G. Wittmann Received December 4, 1976 / February 17, 1977