PON A N D GUALERZI
Foster, J . F. (1968), Chem. Soc., Spec. Publ. No. 23. Friedman, M., Krull, L. H.. and Cavins, J. F. (1970), J . Biol. Chem. 245, 3868. Gans, H., and Tan, B. H . ( I 967), Clin. Chim. Acta 17, 1 1 I . Glasgow, J . F. T., Matthew, J. L., Hercz, A., Levison, H., and Sass-Kortsak, A. ( 1 973), A m . J . Med. 54, 18 !. Hade, E. P. K., and Tanford, C. (1967), J . A m . Chem. SOC. 89, 5034. Heimburger, N . (1972), in Pulmonary Emphysema and Proteolysis, Mittman, C., Ed., New York, N.Y., Academic Press, p 307. Heimburger, N., Haupt, H., and Schwick, H. G . (1971), Proc. I n t . Res. Conf. Proteinase Inhibitors Ist, 1970. 1 . Hirs, C . H . W . (1967), Methods Enzymol. 11, 197. Janatova, J . (1974), J . Med. (Basel) 5, 149. Johnson, D. A,, Pannell, N . R., and Travis, J. (1974), Biochem. Biophys. Res. Commun. 57, 584. Kress, L. F., and Laskowski, M. (1973), Prep. Biochem. 3, 541. Kueppers, F., and Bearn, A. G . (1966), Proc. Soc. Exp. Biol. Med. 121, 1207. Liener, I . E., Garrison, 0. R., and Pravda, Z. (l973), Biochem. Biophys. Res. Commun. 51, 436. Miller, F., and Metzger, H. (1965), J . Biol. Chem. 240, 4740. Myerowitz, R . L., Handzel, Z. T., and Robbins, J. B. (l972), Clin. Chim. Acta 39, 307. Ornstein, L. (1964), Ann. N.Y. Acad. Sci. 121, 321. Ouchterlony, 0 . (1958), Prog. Allergy 5, 1.
Pannell, R., Johnson, D., and Travis, J. (1974), Biochemistry 13, 5439. Panyim, S., and Chalkley, R. (1969), Arch. Biochem. Biop h y s . 130, 337. Rosevear, J . W., and Smith, E. L. (1961), J . Biol. Chem. 236, 425. Salaman, M. R., and Williamson, A. R. (1971), Biochem. J . 122, 93. Schultze, H . E., Heide, K., and Haupt, H . (1962), Klin. Wochschr. 40, 427. Schultze, H. E., and Heremans, J . F. (1966), in Molecular Biology of Human Proteins, Schultze, H. E., and Heremans, J . F., Ed., Amsterdam, Elsevier, p 190. Schwick, V., Heimburger, N., and Haupt, H . (1966), 2. Gesamte Inn. Med. Ihre Grenzgeb. 21, 193. Shamash, Y., and Rimon, A. (1966), Biochim. Biophys. Acta 121, 35. Sharp, H . L., Bridges, R. A., Krivit, W., and Freier, E. F. (1969), J . Lab. Clin. Med. 73, 934. Spencer, E. M., and King, T . P. (1971). J . B i d . Chem. 246, 201. Tomasi, T. B., and Hauptman. S. P. (1974). J . Immunol. 1 12, 2274. Weber, K., and Osborn, M. (1969), J . Biol. Chem. 244, 4406. Williamson, A. R., Salaman, M. R., and Kreth, H . W . ( 1 973), Ann. N.Y. Acad. Sci. 209, 21 0. Winzler, R. J. (1955), Methods Biochem. Anal. 2, 279. Yphantis, D. A. (1964), Biochemistry 3, 297.
The Role of 16s rRNA in Ribosomal Binding of IF-3+ Cynthia L. Pon* and Claudio Gualerzi
ABSTRACT: The binding of initiation factor IF-3 to Escherichia coli 3 0 s ribosomal subunits has been found to be inhibited by rRNA ligands such as ethidium bromide, polyamines, and monovalent alkali metals. The order of effectiveness of the polyamines (spermine > spermidine > putrescine) and alkali metals (Li+ > N a + > K') in inhibiting the ribosomal binding of IF-3 parallels their degree of affinity for the RNA. Furthermore, the binding of IF-3 to 3 0 s subunits chemically modified by photooxidation with rose bengal, nitration with tetranitromethane, and reaction with
kethoxal, monoperphthalic acid, and p-chloromercuribenzoic acid was studied. Results obtained after the direct treatment of the 3 0 s subunits with the above chemical reagents or upon reconstitution of 3 0 s particles having a modified rRNA or ribosomal proteins indicate that the IF-3 binding site is preferentially lost when the rRNA becomes modified. It was found that IF-3 could bind normally to 3 0 s subunits lacking protein S1 or proteins S11, S12, S19, and S21 (and perhaps S14) which had been cross-linked to IF-3 in other laboratories.
Previous studies directed toward identifying the nature of the ribosomal binding site for initiation factor IF-3 have suggested that this factor binds to a segment of the 1 6 s rRNA and that the ribosomal proteins (r-proteins)' confer specificity to the binding (Gualerzi and Pon, 1973). Since IF-3 has recently been cross-linked to some r-proteins, the most prominent being S12 (Hawley et al., 1974) or S12 and
SI 1 (Traut et al., 1974; R . R. Traut, personal communication), the question has been raised as to whether these rproteins are directly involved in the binding o f IF-3 or whether the cross-linking simply reflects the proximity of these proteins to the ribosome-bound IF-3. This problem is of particular relevance, since the involvement of protein SI 2 in the initiation of R17 coat protein translation has been suggested (Held et al., 1974), while the role o f IF-3 in directing the binding of m R N A to 3 0 s subunits (Haselkorn and Rothman-Denes, 1973), and the importance of both S12 and S11 in controlling the codon-anticodon interaction on the ribosomes have long been recognized (Nomura et al.. 1969).
+ From the Max-Planck-Institut fur Molekulare Genetik, Abt. Wittmann, Berlin-Dahlem, Germany. Receiued August I , 1975. Abbreviations used are: r-protein, ribosomal protein; C(N02)4, tetranitromethane; p-CIHgBzO, p-chloromercuribenzoic acid.
'
804
BIOCHEMISTRY,
VOL.
15, NO. 4, 1916
RIBOSOMAL BINDING OF
IF-3
In the present study we have further investigated the properties and the nature of the IF-3 ribosomal binding site and the role of some r-proteins, including S12 and S11, in the binding of initiation factor IF-3. Materials and Methods Materials. Escherichia coli M R E 600 frozen cells were obtained from the Microbiological Research Establishment, Porton Down, Salisbury, U.K. Ethidium bromide was kindly supplied by Dr. A. Favre, Paris. Polyamines were purchased from Sigma Chemical Co. Rose bengal was a kind gift of Dr. G. Jaureguiberry, Paris, and before use was recrystallized as described by Brand et al. (1967). Tetranitromethane was obtained from Serva, Heidelberg, and kethoxa1 from Nutritional Biochemical Co. Monoperphthalic acid was generously provided by Dr. V. Erdmann, Berlin. Actinomycin D, poly(U), ATP, GTP, phosphoenolpyruvate, pyruvate kinase, and E . coli t R N A were purchased from Boehringer-Mannheim. [l4C]Phenylalanine (522 mCi/ mmol) was obtained from Amersham-Buchler and [I4C]formaldehyde (57 mCi/mmol) from New England Nuclear Corp. General Preparations. The preparation of E. coli M R E 600 ribosomes and ribosomal subunits, supernatant enzymes, and purified initiation factor IF-3, and the in vitro labeling of IF-3 by reductive alkylation have been described (Gualerzi et al., 1971, 1973). Ribosomal Binding of Radioactive IF-3. For the standard assay of the ribosomal binding of IF-3, 3 0 s ribosomal subunits (1 ,4260 unit unless otherwise specified) were incubated for 15 min a t 37 OC in 0.5 ml of buffer (10 m M TrisH C l ( p H 7.7), 8 m M Mg(OAc)z, 100 m M NH4C1, and 5 m M 2-mercaptoethanol) with approximately 2 pg of [methyl-14C]IF-3 (2-3 X l o 4 cpm per pug a t 80% counting efficiency). After centrifugation on a 10-30% sucrose gradient in the above buffer a t 42 000 rpm for 3 h in a S W 60-Ti rotor, the radioactivity associated with the 3 0 s subunit was determined as previously described (Gualerzi and Pon, 1973). Chemical Modifications. Photooxidation of the 3 0 s subunits with rose bengal was performed essentially as described by Noller et al. (1971) for the times indicated in Figure 5 with the exception that the amount of rose bengal was reduced to 1.2 pg/ml. Nitration with tetranitromethane was carried out under the conditions described by Shih and Craven (1973) with the exception that the 3 0 s particles were suspended in buffer containing 10 m M Mg(0Ac)z (instead of 20 m M ) and that tetranitromethane was given in a 4000-fold molar excess (instead of 600-fold) over 3 0 s subunits. The incubation was a t 28 O C for the times indicated in Figure 5. Treatment of 3 0 s particles with kethoxal was performed as described by Noller and Chaires (1972) with the exception that the cacodylate buffer system was replaced by 10 m M maleate buffer (pH 6.8) containing 60 m M NH4Cl and 15 m M Mg(0Ac)z. The binding of IF-3 to kethoxal treated 3 0 s subunits as well a s the poly(U) dependent polyphenylalanine synthesis with these ribosomes were also carried out in maleate buffer. Monoperphthalic acid treatment of the 3 0 s subunits was performed as described by Erdmann et al. (1973) except that the concentration of monoperphthalic acid was varied as indicated in Figure 5. Reconstitution of Total and Protein Deficient 30s Particles. For the reconstitution of 3 0 s subunits containing chemically modified r R N A or r-proteins, the 3 0 s ribosomal
subunits were allowed to react as described above and then divided into three portions: the first was used directly to determine IF-3 binding activity and poly(U) dependent polyphenylalanine synthesis; the other two portions were used for the extraction of the r R N A and r-proteins, respectively. The extractions of the r R N A and r-proteins as well as the subsequent reconstitutions were carried out essentially as described by Traub et al. (1971). The activity of the reconstituted particles was determined and compared to that of particles reconstituted from r-proteins and r R N A derived from unreacted 3 0 s particles. The latter were always found to be no less than 90% as active as unfractionated 3 0 s subunits. For the preparation of protein deficient particles, 1.5 M LiCl cores of 3 0 s ribosomal subunits were incubated as described by Traub et al. (1971) with mixtures of r-proteins lacking the specified proteins. The 1.5 M LiCl split proteins were separated by chromatography on phosphocellulose in the PC-1 system as described by Held et al. (1973). The purity of the proteins was determined by two-dimensional polyacrylamide gel electrophoresis (Kaltschmidt and Wittmann, 1970). The size of the plates, however, was reduced to 10 cm X 10 cm X 0.3 cm and the electrophoretic runs were performed a t 2-4 OC. Poly(U) Dependent Polyphenylalanine Synthesis. To determine the activity of the 3 0 s ribosomal subunits in poly(U) dependent polyphenylalanine synthesis, their concentrations in the reaction mixture were varied to obtain a linear relationship between the amount of 3 0 s subunits in the assay and the amount of polyphenylalanine synthesized. Each reaction mixture contained 10 m M Tris-HC1 (pH 7.7), 15 m M Mg(OAc)2, 110 m M NH4C1, 1 m M dithiothreitol, 2 m M ATP, 0.4 m M GTP, 10 m M phosphoenolpyruvate, 1.2 pg of pyruvate kinase, 10 pg of poly(U), 0.05 pCi of [14C]phenylalanine (522 mCi/mmol), 100-150 pg of E. coli MRE600 SlOO protein, 0.25 ,4260 unit of 5 0 s ribosomal subunits, and variable amounts of 3 0 s subunits in a total volume of 0.1 ml. Results ( a ) Effect of R N A Ligands Ethidium Bromide. Ethidium bromide is an N-heterocyclic amine which intercalates within the double helical structures of D N A and R N A (Waring, 1965, 1966)1 and binds to ribosomes through the r R N A thereby inhibiting poly(U) dependent polyphenylalanine synthesis (Wolfe, 1971). Although it is generally assumed that the binding of ethidium bromide to ribosomes occurs mainly through the ionic interactions between this cationic dye and the anionic phosphate groups of the 1 6 s r R N A , we have found that the addition of 3 0 s subunits into a solution of ethidium bromide causes an increased fluorescence of the dye as well as a “red shift” of its absorption maximum (not shown). Therefore, it seems that some ethidium bromide can also be intercalated within the r R N A bases of the 3 0 s subunit since only intercalation results in enhancement of fluorescence (Lepecq and Paoletti, 1967) and in a metachromatic effect (Waring, 1965). The experiment of Figure 1 shows the effect of ethidium bromide on the ribosomal binding of IF-3. As seen from the figure, the binding of IF-3 is strongly inhibited by low concentrations of ethidium bromide and virtually abolished a t a n ethidium bromide concentration of 2 X M. In a control experiment, the binding of IF-3 to 3 0 s ribosomal BIOCHEMISTRY,
VOL.
1 5 , NO. 4, 1976
805
PON A N D GUALERZI
IA
150--
U C 3
1 0 3
c?
I
0
Efh a i
I 1 50 n mol. a d d e d
I
I
\
!
100
5c
Effect of ethidium bromide and actinomycin D on the ribosomal binding of IF-3. The binding of IF-3 was determined essentially as described under Materials and Methods. I n addition, each tube contained, in a volume of 0.5 ml, the indicated amounts of either ethidium bromide or actinomycin D. One hundred percent binding of IF-3 is equivalent to 16 090 cpm per A260 unit of 3 0 s ribosomes.
-1
F I G U R E 1.
2
0
10
