- Email: [email protected]
Identification of the protein cross-linked to 3′-terminus of 5 S RNA in rat liver ribosomal 60 S subunits
Biochinlica et Biophvsica ,4eta, 697 { 1992) 20-- 24
20
Elsevier Biomedical Pres~ BBA 91047
IDENTIFICATION OF THE PROTEIN CROSS-LINKED TO 3'-TERMINUS OF 5 S RNA IN RAT LIVER RIBOSOMAL 60 S SUBUNITS K A Z U O TERAO, TOSHIO U C H I U M I and K I K U O O G A T A
Department of Biochemistry, Niigata University School of Medicine, Niigata, Niigata 951 (Japan) (Received October 20th 1981)
Key words: Ribosomal protein; RNA-protein complex," Cross-linking," rRNA," (Rat liver)
To cross-link the 3'-terminus of 5 S RNA to its neighbouring proteins, ribosomal 60 S subunits of rat liver were oxidized with sodium periodate and reduced with sodium borohydride. 5 S RNP was then isolated by EDTA treatment followed by sucrose density-gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis. The protein with a slower mobility than the L5 protein, which was thought to be cross-linked 5 S RNP, was labeled with 1251, treated with RNAase, and analyzed by two-dimensional polyacrylamide gel electrophoresis, followed by radioantography. A radioactive spot located anodically from L5 protein was observed, suggesting that it is the L5 protein-oligonucleotide complex. When analyzed by SDS slab polyacrylamide gel electrophoresis followed by radioautography, the peptide pattern of the a-chymotrypsin digest of this 12SI-labeled protein-oligonucleotide complex was similar to that of the digest of 12Sl-labeled L5 protein. The results indicate that L5 protein binds to the 3'-terminal region of 5 S RNA in rat liver 60 S subunits.
Introduction Previously, we [1] showed that the protein moiety of 5 S RNA-protein complex (5 S RNP) released from rat liver 60 S subunits by EDTA treatment was L5 protein according to the proposed uniform nomenclature* [2]. On the other hand, affinity chromatography through Sepharose containing the immobilized 5 S RNA showed that L6 and L19 proteins but not L5 protein were bound to 5 S RNA [4,5]. Therefore, a following possibility was pointed out that EDTA unfolds 60 S subunits and causes proteins to lose their specific locations on ribosomal RNA [5]. Recently, however, we indicated that the L5 protein was
* L3 according to our nomenclature [3]. Abbreviation: 5 S RNP, 5 S RNA-protein complex. 0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
cross-linked to 5 S RNA in 60 S subunits by ultraviolet-irradiation [6]. Furthermore, Metspalu et al. have shown that L5 protein is firmly bound to 5 S RNA by means of affinity chromatography which was not eluted by high salt buffer containing EDTA [4,5], but by 8 M urea/4 M LiC1 [7]. To extend our studies, we intended to identify 60 S protein located near the 3'-terminus of 5 S RNA by sodium periodate oxidation of 60 S subunits followed by sodium borohydride reduction [8,9]. Materials and Methods Chemicals. Na125I (17 Ci/mg) was obtained from New England Nuclear, Boston. Bovine pancreatic RNAase A (EC 2.7.7.16) was purchased from Worthington Biochemical Corporation. ctChymotrypsin (EC 3.4.21.1) from bovine pancreas was obtained from Sigma Chemical Company.
21
Preparation of ribosomal subunits and crossfinking. The cross-linking of 60 S subunits pre-
Results
pared from rat liver [10] was carried out according to the slight modification of the methods of Van Duin et al. [8] and of Srobada and McConkey [9]. In brief, 2 ml 60 S ribosomal subunits (7.6 mg RNA) in Medium I (40 mM NaC1/I mM MgCI2/10 mM Hepes/0.1 mM EDTA, pH 6.3) were oxidized by adding one-tenth volume of Medium I containing 220 mM sodium periodate. Control 60 S subunits were treated as described above without the addition of sodium periodate. The ribosomal subunits were left in the dark for 45 min at room temperature. They were dialyzed against Medium I containing 2% glycerol, pH 8.0, for 1 h at 4°C and then against Medium I for 1 h at 4°C to remove periodate. The solution was adjusted to 10 mM sodium borohydride to induce the protein-RNA linkage. After standing for 30 rain at 4°C, one-tenth volume of 250 mM EDTA was added and the solution was subjected to sucrose density-gradient centrifugation to isolate 5 S RNA-protein complex as described previously [l].
Periodate- and borohydride-treated 60S subunits (treated 60 S subunits) as well as the control 60 S subunits were treated with EDTA and analyzed by polyacrylamide gel electrophoresis at pH 7.8. 5 S RNP from the treated 60 S subunits showed only one band with the same mobility as that from the control 60 S subunits, suggesting that a greater part of 5 S RNP was not cross-linked to other components of 60 S subunits to form heavier products (data not shown). To examine whether periodate- and borohydride-treatment cross-linked 60 S proteins to Yterminus of 5 S RNA, the treated 60 S subunits were subjected to sucrose density-gradient centrifugation after EDTA treatment. 5 S RNP thus obtained was analyzed by polyacrylamide gel electrophoresis at pH 7.8. As shown in Fig. 1, 5 S RNP prepared from the treated 60 S subunits showed one band containing both protein and RNA at the same position as that from the control 60 S subunits in addition to 5 S RNA. However, when 5 S RNP from the treated 60 S subunits was subjected to SDS-polyacrylamide gel electrophoresis, one protein band with a slower mobility than L5 protein band was observed in addition to the L5 protein band. On the other hand, only the L5 protein band appeared in the case of 5 S RNP from the control 60 S subunits (Fig. 2). The results indicate that the band having a lower mobility is a cross-linked 5 S RNP induced by periodate- and borohydride-treatment, because it is not dissociated by SDS. To identify the protein moiety of cross-linked 5 S RNP, two stained gel discs containing two protein bands on SDS-polyacrylamide gel electrophoresis of 5 S RNP from the treated 60 S subunits were cut out. These discs were then radioiodinated with ~25I, and extracted proteins were mixed with carrier 60 S ribosomal proteins, treated with RNAase, and then subjected to two-dimensional urea-polyacrylamide gel electrophoresis. The gel slab was analyzed by radioautography as described previously [6,11]. As shown in Fig. 3, the protein having the same mobility as L5 protein on the SDS-polyacrylamide gel has one radioactive spot at the position of L5 protein on the two-dimensional gel, whereas the
Electrophoretic analysis and radioiodination of proteins in the gel discs. Polyacrylamide gel electrophoresis at pH 7.8 and SDS-polyacrylamide gel electrophoresis of 5 S RNP were carried out as described previously [6]. Proteins of 5 S RNP in the SDS-polyacrylamide gel discs were radioiodinated and, after RNAase digestion, analyzed by two-dimensional acrylamide gel electrophoresis followed by radioautography as described previously [6,11]. In the case of peptide analysis of L5 protein, the same methods were used except that RNAase treatment was not carried out. Peptide analysis. After the identification of the position of the 125I-labeled protein by radioautography on two-dimensional acrylamide gel electrophoresis, the gel piece containing the 125I-labeled protein was loaded directly onto the SDSpolyacrylamide gel well and was treated with achymotrypsin as described by Cleveland et al. [12]. After SDS slab polyacrylamide gel electrophoresis [13], the gel was stained with Coomassie brilliant blue and then radioautographed as described previously [ 11].
22
!~i~i~i~~!~I !~f
L5
B
5SRNP
,u
ii ~ ii
5SRNA
i¸¸'¸~¸¸i !i~ii ~ i ~
1
!~
2
3
4
1
2
3
Fig. 1. Polyacrylamide gel electrophoresis of 5 S RNP. 5 S RNPs from the treated 60 S subunits and the control 60 S subunits were subjected to polyacrylamide gel electrophoresis at pH 7.8 [6] and stained with Coomassie brilliant blue (lane 1 and 2) and Azur B (lane 3 and 4). Lane I and 3; 5 S RNP from the control 60 S subunits, lane 2 and 4 : 5 S RNP from periodate- and borohydride-treated 60 S subunits.
Fig. 2. SDS-polyacrylamide gel electrophoresis of 5 S RNP. 5 S RNP prepared from the treated 60 S subunits and the control 60 S subunits were applied to SDS-polyacrylamide gel electrophoresis [6] and stained with Coomassie brilliant blue. Lane I; 60 S ribosomal proteins. Lane 2 : 5 S RNP from the control 6 0 S subunits. Lane 3 : 5 S RNP from the periodate- and borohydride-treated 60 S subunits.
Fig. 3. Autoradiography of two-dimensional gel electrophoresis of radioiodinated proteins in the SDS-polyacrylamide gel discs. Proteins of 5 S RNP in the SDS-polyacrylamide gel discs after electrophoresis were radioiodinated and subjected to twodimensional urea-polyacrylamide gel electrophoresis followed by radioautography [6]. A; Protein showing the same mobility as L5 protein on the SDS-polyacrylamide gel electrophoresis. B; Protein showing a lower mobility than L5 protein on the SDS-polyaerylamide gel.
protein having a lower mobility than the L5 protein has the diffuse radioactive spot distributed anodically from the L5 protein spot. Since oligonucleotide cross-linked to L5 protein may remain even after RNAase treatment of 5 S RNP, these results are explained by assuming the presence of oligonucleotides attached to the L5 protein as observed in the cases of ribosomal proteins cross-linked to rRNA by periodate oxidation [9] and by ultraviolet-irradiation [6,14,15]. To confirm that the protein moiety of this protein-oligonucleotide complex is L5 protein, we compared the peptide pattern of the protein of this complex with that of L5 protein according to the technique of Cleveland et al. [12], by using mzsIlabeled proteins. The results are shown in Fig. 4. The finding that the radioactive patterns of peptides on SDS-polyacrylamide gel, which were
23
1
2
3
4
5
Fig. 4. Comparison of partial proteolytic products from the ~25I-labeled cross-linked protein and the L5 protein. Twodimensional gel pieces containing ~25I-labeled protein were digested by a-chymotrypsin according to the method of Cleveland et al. [12], using the following enzyme concentrations; 10 /~g (lanes 2 and 3), 20 /~g (lane 4) and 30 /~g (lane 5). The radioautographs of the SDS-polyacrylamide gels after electrophoresis are shown. Lane 2; cross-linked protein. Lanes 1, 3, 4 and 5; L5 protein. The arrow indicates the undigested L5 protein.
produced from these two kinds of proteins by a-chymotrypsin, are similar with respect to the molecular weight and the relative density on the radioautograph, may provide conclusive evidence that the cross-linked protein is L5 protein. Discussion
It is well known that 5 S RNA exists in large ribosomal subunits from procaryotes to eukaryotes as 5 S RNA-protein complexes. Recently, much evidence has been accumulated showing that the structure and function of 5 S RNP are conserved during evolution. 5 S RNP of both procaryotes and eukaryotes has GTP and ATP hydrolyzing activity [16-19]. 5S RNA of procaryotes and eukaryotes interacts not only with tRNA [18,19] but also with the 3'-terminal of rRNA of corre-
sponding small subunits [20,21]. Concerning the protein moiety of 5 S RNP, Nazar et al. [22] suggested that the protein moiety of yeast 5 S RNP corresponding to rat liver L5 protein has the amino acid sequence expecting the fusion of L 18 and L5 protein of E. coli 50 S subunits which are known to interact with 5 S RNA. Furthermore, from the experiment using limited ribonuclease digestion of the 5 S RNP and free 5 S RNA, Nazar [23] has suggested that the protein moiety of yeast 5 S RNP interacts mainly with the Y-half of the 5 S RNA molecule. Similar results were reported for 5 S RNP from E. coli [24] and H. cutirubrum [25]. Taking these observations together with the present results, it appears that the primary protein binding site of 5 S RNA has also been well conserved from procaryotes to mammals. The findings described above suggest the importance of 5 S RNP on protein synthesis although its detailed role has not yet been clarified. References 1 Terao, K., Takahashi, Y. and Ogata, K. (1975) Biochim. Biophys. Acta 402, 230-237 2 McConkey, E.H., Bielka, H., Gordon, J., Lastik, S.H., Lin, A., Ogata, K., Reboud, J.-P., Traugh, J.A., Traut, R.R., Warner, J.R., Welfle, H. and Wool, I.G. (1979) Mol. Gen. Genet. 169, 1-6 3 Terao, K. and Ogata, K. (1975) Biochim. Biophys. Acta 402, 214-229 4 Metspalu, A., Saarma, M., Villems, R., Ustav, M. and Lind, A. (1978) Eur. J. Biochem. 91, 73-81 5 Ulbrich, N. and Wool, I.G. (1978) J. Biol. Chem. 253, 9049-9052 6 Terao, K., Uchiumi, T. and Ogata, K. (1980) Biochim. Biophys. Acta 609, 306-312 7 Metspalu, A., Toots, I., Saarma, M. and Villems, R. (1980) FEBS Lett. 119, 81-84 8 Van Duin, J., Kurland, C.G., Dondon, J. and GrebergManago, M. (1975) FEBS Lett. 59, 287-290 9 Sroboda, A.J. and McConkey, E.H. (1978) Biochem. Biophys. Res. Commun. 81, I 145- I 152 10 Ogata, K. and Terao, K. (1979) Methods Enzymol. 59, 502-515 I 1 Terao, K., Uchiurni, T., Kabayashi, Y. and Ogata, K. (1980) Biochim. Biophys. Acta 621, 72-82 12 Cleveland, D.W., Fischer, M.W. and Laemmli, U.K. (1977) J. Biol. Chem. 252, 1102-1106 13 Laemmli, U.K. (1970) Nature 227, 680-685 14 Mrller, K. and Brimacombe, R. (1975) Mot. Gen. Genet. 141,343-355 15 Terao, K. and Ogata, K. (1979) J. Biochem. 86, 605-617 16 Grummt, F., Grummt, I. and Erdmann, V.A. (1974) Eur. J. Biochem. 43,343-348
24 17 Benson, S., Olsnes, S., Pihl, A., Skorve, J. and Abraham, A.K. (1975) Eur. J. Biochem. 59, 573-580 18 Ogata, K., Terao, K. and Uchiumi, T. (1980) J. Biochem. 87, 517-524 19 Erdmann, V.A. (1976) in Progress in Nucleic Acid Research and Molecular Biology (Cohn, W.E.. ed.), Vol. 18, pp. 45-90, Academic Press, New York 20 Azad, A.A. (1979) Nucl. Acids Res. 7, 1913-1929 21 Azad, A.A. and Deacon, N.J. (1980) Nucl. Acids Res. 8, 4365-4376
22 Nazar, R.N., Yaguchi, M., Willick, (i.E., Rollin, C.F. and Roy, C. (1979) Eur. J. Biochem. 102, 573-582 23 Nazar, R.N. (1979) J. Biol. Chem. 254, 7724-7729 24 (}ray, P.N., Bellemarke, G. and Monier, R. (1973) J. Mol. Biol. 77, 133-152 25 Nazar, R.N., Willick, G.E. and Natheson, A.T. (1979) J. Biol. Chem. 254, 1506-1512
20
Elsevier Biomedical Pres~ BBA 91047
IDENTIFICATION OF THE PROTEIN CROSS-LINKED TO 3'-TERMINUS OF 5 S RNA IN RAT LIVER RIBOSOMAL 60 S SUBUNITS K A Z U O TERAO, TOSHIO U C H I U M I and K I K U O O G A T A
Department of Biochemistry, Niigata University School of Medicine, Niigata, Niigata 951 (Japan) (Received October 20th 1981)
Key words: Ribosomal protein; RNA-protein complex," Cross-linking," rRNA," (Rat liver)
To cross-link the 3'-terminus of 5 S RNA to its neighbouring proteins, ribosomal 60 S subunits of rat liver were oxidized with sodium periodate and reduced with sodium borohydride. 5 S RNP was then isolated by EDTA treatment followed by sucrose density-gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis. The protein with a slower mobility than the L5 protein, which was thought to be cross-linked 5 S RNP, was labeled with 1251, treated with RNAase, and analyzed by two-dimensional polyacrylamide gel electrophoresis, followed by radioantography. A radioactive spot located anodically from L5 protein was observed, suggesting that it is the L5 protein-oligonucleotide complex. When analyzed by SDS slab polyacrylamide gel electrophoresis followed by radioautography, the peptide pattern of the a-chymotrypsin digest of this 12SI-labeled protein-oligonucleotide complex was similar to that of the digest of 12Sl-labeled L5 protein. The results indicate that L5 protein binds to the 3'-terminal region of 5 S RNA in rat liver 60 S subunits.
Introduction Previously, we [1] showed that the protein moiety of 5 S RNA-protein complex (5 S RNP) released from rat liver 60 S subunits by EDTA treatment was L5 protein according to the proposed uniform nomenclature* [2]. On the other hand, affinity chromatography through Sepharose containing the immobilized 5 S RNA showed that L6 and L19 proteins but not L5 protein were bound to 5 S RNA [4,5]. Therefore, a following possibility was pointed out that EDTA unfolds 60 S subunits and causes proteins to lose their specific locations on ribosomal RNA [5]. Recently, however, we indicated that the L5 protein was
* L3 according to our nomenclature [3]. Abbreviation: 5 S RNP, 5 S RNA-protein complex. 0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
cross-linked to 5 S RNA in 60 S subunits by ultraviolet-irradiation [6]. Furthermore, Metspalu et al. have shown that L5 protein is firmly bound to 5 S RNA by means of affinity chromatography which was not eluted by high salt buffer containing EDTA [4,5], but by 8 M urea/4 M LiC1 [7]. To extend our studies, we intended to identify 60 S protein located near the 3'-terminus of 5 S RNA by sodium periodate oxidation of 60 S subunits followed by sodium borohydride reduction [8,9]. Materials and Methods Chemicals. Na125I (17 Ci/mg) was obtained from New England Nuclear, Boston. Bovine pancreatic RNAase A (EC 2.7.7.16) was purchased from Worthington Biochemical Corporation. ctChymotrypsin (EC 3.4.21.1) from bovine pancreas was obtained from Sigma Chemical Company.
21
Preparation of ribosomal subunits and crossfinking. The cross-linking of 60 S subunits pre-
Results
pared from rat liver [10] was carried out according to the slight modification of the methods of Van Duin et al. [8] and of Srobada and McConkey [9]. In brief, 2 ml 60 S ribosomal subunits (7.6 mg RNA) in Medium I (40 mM NaC1/I mM MgCI2/10 mM Hepes/0.1 mM EDTA, pH 6.3) were oxidized by adding one-tenth volume of Medium I containing 220 mM sodium periodate. Control 60 S subunits were treated as described above without the addition of sodium periodate. The ribosomal subunits were left in the dark for 45 min at room temperature. They were dialyzed against Medium I containing 2% glycerol, pH 8.0, for 1 h at 4°C and then against Medium I for 1 h at 4°C to remove periodate. The solution was adjusted to 10 mM sodium borohydride to induce the protein-RNA linkage. After standing for 30 rain at 4°C, one-tenth volume of 250 mM EDTA was added and the solution was subjected to sucrose density-gradient centrifugation to isolate 5 S RNA-protein complex as described previously [l].
Periodate- and borohydride-treated 60S subunits (treated 60 S subunits) as well as the control 60 S subunits were treated with EDTA and analyzed by polyacrylamide gel electrophoresis at pH 7.8. 5 S RNP from the treated 60 S subunits showed only one band with the same mobility as that from the control 60 S subunits, suggesting that a greater part of 5 S RNP was not cross-linked to other components of 60 S subunits to form heavier products (data not shown). To examine whether periodate- and borohydride-treatment cross-linked 60 S proteins to Yterminus of 5 S RNA, the treated 60 S subunits were subjected to sucrose density-gradient centrifugation after EDTA treatment. 5 S RNP thus obtained was analyzed by polyacrylamide gel electrophoresis at pH 7.8. As shown in Fig. 1, 5 S RNP prepared from the treated 60 S subunits showed one band containing both protein and RNA at the same position as that from the control 60 S subunits in addition to 5 S RNA. However, when 5 S RNP from the treated 60 S subunits was subjected to SDS-polyacrylamide gel electrophoresis, one protein band with a slower mobility than L5 protein band was observed in addition to the L5 protein band. On the other hand, only the L5 protein band appeared in the case of 5 S RNP from the control 60 S subunits (Fig. 2). The results indicate that the band having a lower mobility is a cross-linked 5 S RNP induced by periodate- and borohydride-treatment, because it is not dissociated by SDS. To identify the protein moiety of cross-linked 5 S RNP, two stained gel discs containing two protein bands on SDS-polyacrylamide gel electrophoresis of 5 S RNP from the treated 60 S subunits were cut out. These discs were then radioiodinated with ~25I, and extracted proteins were mixed with carrier 60 S ribosomal proteins, treated with RNAase, and then subjected to two-dimensional urea-polyacrylamide gel electrophoresis. The gel slab was analyzed by radioautography as described previously [6,11]. As shown in Fig. 3, the protein having the same mobility as L5 protein on the SDS-polyacrylamide gel has one radioactive spot at the position of L5 protein on the two-dimensional gel, whereas the
Electrophoretic analysis and radioiodination of proteins in the gel discs. Polyacrylamide gel electrophoresis at pH 7.8 and SDS-polyacrylamide gel electrophoresis of 5 S RNP were carried out as described previously [6]. Proteins of 5 S RNP in the SDS-polyacrylamide gel discs were radioiodinated and, after RNAase digestion, analyzed by two-dimensional acrylamide gel electrophoresis followed by radioautography as described previously [6,11]. In the case of peptide analysis of L5 protein, the same methods were used except that RNAase treatment was not carried out. Peptide analysis. After the identification of the position of the 125I-labeled protein by radioautography on two-dimensional acrylamide gel electrophoresis, the gel piece containing the 125I-labeled protein was loaded directly onto the SDSpolyacrylamide gel well and was treated with achymotrypsin as described by Cleveland et al. [12]. After SDS slab polyacrylamide gel electrophoresis [13], the gel was stained with Coomassie brilliant blue and then radioautographed as described previously [ 11].
22
!~i~i~i~~!~I !~f
L5
B
5SRNP
,u
ii ~ ii
5SRNA
i¸¸'¸~¸¸i !i~ii ~ i ~
1
!~
2
3
4
1
2
3
Fig. 1. Polyacrylamide gel electrophoresis of 5 S RNP. 5 S RNPs from the treated 60 S subunits and the control 60 S subunits were subjected to polyacrylamide gel electrophoresis at pH 7.8 [6] and stained with Coomassie brilliant blue (lane 1 and 2) and Azur B (lane 3 and 4). Lane I and 3; 5 S RNP from the control 60 S subunits, lane 2 and 4 : 5 S RNP from periodate- and borohydride-treated 60 S subunits.
Fig. 2. SDS-polyacrylamide gel electrophoresis of 5 S RNP. 5 S RNP prepared from the treated 60 S subunits and the control 60 S subunits were applied to SDS-polyacrylamide gel electrophoresis [6] and stained with Coomassie brilliant blue. Lane I; 60 S ribosomal proteins. Lane 2 : 5 S RNP from the control 6 0 S subunits. Lane 3 : 5 S RNP from the periodate- and borohydride-treated 60 S subunits.
Fig. 3. Autoradiography of two-dimensional gel electrophoresis of radioiodinated proteins in the SDS-polyacrylamide gel discs. Proteins of 5 S RNP in the SDS-polyacrylamide gel discs after electrophoresis were radioiodinated and subjected to twodimensional urea-polyacrylamide gel electrophoresis followed by radioautography [6]. A; Protein showing the same mobility as L5 protein on the SDS-polyacrylamide gel electrophoresis. B; Protein showing a lower mobility than L5 protein on the SDS-polyaerylamide gel.
protein having a lower mobility than the L5 protein has the diffuse radioactive spot distributed anodically from the L5 protein spot. Since oligonucleotide cross-linked to L5 protein may remain even after RNAase treatment of 5 S RNP, these results are explained by assuming the presence of oligonucleotides attached to the L5 protein as observed in the cases of ribosomal proteins cross-linked to rRNA by periodate oxidation [9] and by ultraviolet-irradiation [6,14,15]. To confirm that the protein moiety of this protein-oligonucleotide complex is L5 protein, we compared the peptide pattern of the protein of this complex with that of L5 protein according to the technique of Cleveland et al. [12], by using mzsIlabeled proteins. The results are shown in Fig. 4. The finding that the radioactive patterns of peptides on SDS-polyacrylamide gel, which were
23
1
2
3
4
5
Fig. 4. Comparison of partial proteolytic products from the ~25I-labeled cross-linked protein and the L5 protein. Twodimensional gel pieces containing ~25I-labeled protein were digested by a-chymotrypsin according to the method of Cleveland et al. [12], using the following enzyme concentrations; 10 /~g (lanes 2 and 3), 20 /~g (lane 4) and 30 /~g (lane 5). The radioautographs of the SDS-polyacrylamide gels after electrophoresis are shown. Lane 2; cross-linked protein. Lanes 1, 3, 4 and 5; L5 protein. The arrow indicates the undigested L5 protein.
produced from these two kinds of proteins by a-chymotrypsin, are similar with respect to the molecular weight and the relative density on the radioautograph, may provide conclusive evidence that the cross-linked protein is L5 protein. Discussion
It is well known that 5 S RNA exists in large ribosomal subunits from procaryotes to eukaryotes as 5 S RNA-protein complexes. Recently, much evidence has been accumulated showing that the structure and function of 5 S RNP are conserved during evolution. 5 S RNP of both procaryotes and eukaryotes has GTP and ATP hydrolyzing activity [16-19]. 5S RNA of procaryotes and eukaryotes interacts not only with tRNA [18,19] but also with the 3'-terminal of rRNA of corre-
sponding small subunits [20,21]. Concerning the protein moiety of 5 S RNP, Nazar et al. [22] suggested that the protein moiety of yeast 5 S RNP corresponding to rat liver L5 protein has the amino acid sequence expecting the fusion of L 18 and L5 protein of E. coli 50 S subunits which are known to interact with 5 S RNA. Furthermore, from the experiment using limited ribonuclease digestion of the 5 S RNP and free 5 S RNA, Nazar [23] has suggested that the protein moiety of yeast 5 S RNP interacts mainly with the Y-half of the 5 S RNA molecule. Similar results were reported for 5 S RNP from E. coli [24] and H. cutirubrum [25]. Taking these observations together with the present results, it appears that the primary protein binding site of 5 S RNA has also been well conserved from procaryotes to mammals. The findings described above suggest the importance of 5 S RNP on protein synthesis although its detailed role has not yet been clarified. References 1 Terao, K., Takahashi, Y. and Ogata, K. (1975) Biochim. Biophys. Acta 402, 230-237 2 McConkey, E.H., Bielka, H., Gordon, J., Lastik, S.H., Lin, A., Ogata, K., Reboud, J.-P., Traugh, J.A., Traut, R.R., Warner, J.R., Welfle, H. and Wool, I.G. (1979) Mol. Gen. Genet. 169, 1-6 3 Terao, K. and Ogata, K. (1975) Biochim. Biophys. Acta 402, 214-229 4 Metspalu, A., Saarma, M., Villems, R., Ustav, M. and Lind, A. (1978) Eur. J. Biochem. 91, 73-81 5 Ulbrich, N. and Wool, I.G. (1978) J. Biol. Chem. 253, 9049-9052 6 Terao, K., Uchiumi, T. and Ogata, K. (1980) Biochim. Biophys. Acta 609, 306-312 7 Metspalu, A., Toots, I., Saarma, M. and Villems, R. (1980) FEBS Lett. 119, 81-84 8 Van Duin, J., Kurland, C.G., Dondon, J. and GrebergManago, M. (1975) FEBS Lett. 59, 287-290 9 Sroboda, A.J. and McConkey, E.H. (1978) Biochem. Biophys. Res. Commun. 81, I 145- I 152 10 Ogata, K. and Terao, K. (1979) Methods Enzymol. 59, 502-515 I 1 Terao, K., Uchiurni, T., Kabayashi, Y. and Ogata, K. (1980) Biochim. Biophys. Acta 621, 72-82 12 Cleveland, D.W., Fischer, M.W. and Laemmli, U.K. (1977) J. Biol. Chem. 252, 1102-1106 13 Laemmli, U.K. (1970) Nature 227, 680-685 14 Mrller, K. and Brimacombe, R. (1975) Mot. Gen. Genet. 141,343-355 15 Terao, K. and Ogata, K. (1979) J. Biochem. 86, 605-617 16 Grummt, F., Grummt, I. and Erdmann, V.A. (1974) Eur. J. Biochem. 43,343-348
24 17 Benson, S., Olsnes, S., Pihl, A., Skorve, J. and Abraham, A.K. (1975) Eur. J. Biochem. 59, 573-580 18 Ogata, K., Terao, K. and Uchiumi, T. (1980) J. Biochem. 87, 517-524 19 Erdmann, V.A. (1976) in Progress in Nucleic Acid Research and Molecular Biology (Cohn, W.E.. ed.), Vol. 18, pp. 45-90, Academic Press, New York 20 Azad, A.A. (1979) Nucl. Acids Res. 7, 1913-1929 21 Azad, A.A. and Deacon, N.J. (1980) Nucl. Acids Res. 8, 4365-4376
22 Nazar, R.N., Yaguchi, M., Willick, (i.E., Rollin, C.F. and Roy, C. (1979) Eur. J. Biochem. 102, 573-582 23 Nazar, R.N. (1979) J. Biol. Chem. 254, 7724-7729 24 (}ray, P.N., Bellemarke, G. and Monier, R. (1973) J. Mol. Biol. 77, 133-152 25 Nazar, R.N., Willick, G.E. and Natheson, A.T. (1979) J. Biol. Chem. 254, 1506-1512