The primary structure of an acidic ribosomal protein from streptomyces griseus

164

Biochimica et Biophysica A cta, 701 (1982) 164-172 Elsevier Biomedical Press

BBA 31044

THE PRIMARY STRUCTURE OF AN ACIDIC RIBOSOMAL PROTEIN FROM STREPTOMYCES GRISEUS TAKUZI ITOH a, MASANORI SUGIYAMA b and KEN-ICHI I-IIGO a a Department of Biochemistry and Biophysics, Research Institute/or Nuclear Medicine and Biologv, tliroshima Umversity, Kasumi 1-2-3 and/' Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Sendamachi 3, Hiroshima 730 (Japan) (Received September 7th, 1981)

Key words: Ribosomal protein: Amino acid sequence," (Strep. griseus)

The complete primary structure of a L7/L12-type acidic ribosomal protein (SA1) from actinomycetes or mycelial bacterium Streptomyces griseus has been determined. SAI is composed of 125 amino acid residues and has the composition: AspT, Thrs, Ser s, Giuls, Gin4, Pro4, Glys, AlaT.4, Vail3 , Met 1, Ile4, Leul2, Phes, Lyst4 and Arg I. The molecular weight of SA1 is 13069. The amino acid sequence was determined by a combination of automated Edman degradation of the intact protein in a Beckman sequenator, and 4-N,Ndimethylaminoazobenzene 4'-isothiocyanate/phenylisothiocyanate double-coupling degradation of the peptides obtained by digestions with trypsin, chymotrypsin, pepsin and Staphylococcus aureus protease of the intact protein. When the amino-terminal sequence of the SA1 (the first 39 residues) was compared to those of equivalent proteins from other eubacteria, the highest degree of similarity was found with that from Arthrobacter glaciMis. The acidic ribosomal proteins from these two Gram-positive bacteria (S. gr/seus and A. glaciMis) are somewhat distinct, in amino acid sequence and in amino acid compositions, from those of other Gram-negative and Gram-positive bacteria so far studied. However, they still appear to retain the characteristic prokaryotic-type structure. Introduction During protein synthesis, the ribosomes perform a number of discrete functions which are Supplementary data to this article are deposited with and can be obtained from Elsevier/North-Holland Biomedical Press B.V., BBA Data Deposition, P.O. Box 1345 1000 BH Amsterdam, The Netherlands. Reference should be made to No. BBA/DD/209/31044/701 (1982) 164. The supplementary information includes: thin-layer peptide maps of the trypsin, chymotrypsin, pepsin, Staphylococcus aureus protease peptides, amino acid composition of these peptides, other peptides derived from further digestion with Staphylococcus aureus protease a n d / o r pepsin of tryptic peptide T4 and T2, and comparison of the N-terminal amino acid sequence of acidic protein from various prokaryotic sources (38-39 residues) (Fig. 7). Abbreviations: DABITC, 4-N, N-dimethylaminoazobenzene 4'isothiocyanate; TPCK, L-(tosylamido-2-phenyl)ethyl chloromethylketone. 0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

modulated by supernatant protein factors; the initiation, elongation and termination factors. In both prokaryotic and eukaryotic organisms, L7/L12type acidic proteins from the ribosomal large subunit are involved in the functioning of many of these factors [1]. The acidic proteins L 7 / L I 2 from Escherichia coli ribosomes are at present the most thoroughly studied ribosomal proteins. Involvement of this protein in the functioning of ribosome has been reviewed [21. Recently, the L7/L12 from E. coli has been crystallized [3] and a model has been proposed for its structure in solution [4-6]. Ribosomes from other bacterial species have proteins homologous to L7/LI2. The ubiquity of this protein as a ribosomal constituent is indicated by some degree of conservation in their aminoterminal amino acid sequences in p.rokaryotic or in

165 eukaryotic organisms including Archaebacteria (or 'metabacteria' according to Ref. 7) so far examined [8-12]. Comparative structural analysis of these proteins would give some insights into the evolutionary changes of this protein as well as the nature of active sites within the molecule. The complete primary structure of the L7/L12-type proteins from Bacillus subtilis (referred to as BL9) [13,14] and from Micrococcus lysodeikticus (referred to as MA1/2) [15] have been determined and the comparisons with L7/L12 from E. coli [16] showed 50 and 60% sequence similarity, respectively. The region from position 78 to postion 84 (Thr- Gly- Leu- Gly- Leu- Lys- Glu- Ala- Lys), which is a unique region having predictable fl-turn (or bend) structure, is especially highly conserved among these three molecules. The complete primary structure of an acidic ribosomal protein YPA1 from yeast indicated that this protein showed a high sequence similarity to the equivalent protein from a eukaryote, Artemia salina, and also showed a considerable similarity to the amino -terminal amino acid sequences of those from Archaebacteria, Halobacterium cutirubrum and Methanobacterium thermoautotrophicum. On the other hand, there is very little apparent sequence similarity between YPA1 and eubacterial acidic protein [ 17-19]. Streptomyces is Gram-positive, and one of the major genus in actinomyetes: organisms that grow in the form of a largely coenocytic, much-branched mycelium and form specialized reproductive cells; conidiospores or sporangiospores, late in the development of the mycelium. The actinomycetes are-strikingly analogous to certain eukaryotic fungi in structure and modes of reproduction. We now report the complete primary structure of an acidic ribosomal protein from this mycelial bacterium Streptomyces griseus. This protein migrates in the two-dimensional gel to approximately the same position as that of L7/L12 from E. coli or BL9 from B. subtilis. The amino acid sequence of this protein was compared with previously published complete or partial amino acid sequences of L7/Ll2-type acidic proteins from other eubacteria. //

Materials and Methods

Growth of bacteria and isolation of ribosomes. S. griseus HUT 6037 was grown in 1% glucose, 0.2% meat extract and 0.4% peptone medium [20] at 28°C. The cells, harvested in late-logarithmic growth phase, were washed with 10 mM tris-HCl (pH 7.6)/10 mM Mg-acetate/1 M KC1/5 mM Mg -Titriplex/6 mM fl-mercaptoethanol as described by Sugiyama et al. [21]. The washed cells (100g) were mixed with 800g of glass beads (0.5 mm diameter) and broken in a Vibrogen cell-mill. The crude extract obtained by several washings of the vibrated mixture with Tris-Mg buffer (50 mM Tris-HC1 (pH 7.6)/10 mM Mg-acetate/6mM flmercaptoethanol/30 mM NH4C1), containing 3 mM phenylmethylsulfonylfloride, 5 mM MgTitriplex and 0.2 mM diisoprophylfluorophosphate as protease inhibitors, was centrifuged at 8000 rev./min for 5 min in a Sorvall superspeed centrifuge to remove glass beads and cell debris. The supernatant was recentrifuged in a Spinco 30 rotor at 27000 rev./min for 45 min. The ribosomes were sedimented from this $30 fraction by centrifugation at 24000 rev./min for 15h, resuspended in Tris-Mg buffer and then washed through sucrose-salt buffer (Tris-Mg buffer containing 25% sucrose and 1 M NH4CI instead of 30 mM) by centrifugation for 17 h at 28000 rev./min. Purification of acidic ribosomal proteins. The acidic proteins were extracted, by the method of Hamel et al. [22], with an NH4C1/ethanol splitting technique modified as follows. The purified ribosomes (150 A260 units/ml in Tris-Mg buffer) were diluted with an equivalent volume of Tris-Mg buffer containing 2 M NH4C1, instead of 30 mM NH4C1, at 0°C. The ribosomal suspension was stirred for 10 min with 0.5 vol. ethanol, then a further 0.5 vol. ethanol was added (to give a final ethanol concentration of 50%) and the mixture stirred for 5 min at 0°C. After a low-speed centrifugation the supernatant was mixed with 2 vol. acetone and the proteins which precipitated after being left overnight at - 2 0 ° C were collected by the low-speed centrifugation. The protein sample were dissolved in 0.01 M ammonium acetate buffer (pH 5.7) containing 6 M urea and dialyzed against the same buffer for 20 h. The sample was applied to a DEAE-cellulose column (1.5 X 30 cm) equi-

166

librated with the same buffer. The protein was eluted by using a 2-liter linear gradient of 0.01 M ammonium acetate (pH 5.7) to 0.2 M ammonium acetate (pH 5.0) buffer containing 6 M urea with a flow rate of 20 ml/h. All steps were carried out at 4°C. The protein elution was monitored by the method of Lowry et al. and also analyzed by polyacrylamide gel electrophoresis at pH 8.6 and pH 4.5 [23,24]. Pure protein fractions indicated by the bracket in Fig. 1 (as judged by acrylamide gel electrophoresis) were pooled, desalted by gelfiltration through Bio-Gel P10 column in 15% acetic acid, concentrated with an evaporator and stored at - 70°C. Enzymatic digestion and separation of peptides. Enzymatic digestions of the protein were performed with TPCK-trypsin for 3 h in 0.1 M methylmorpholine-acetate buffer (pH 8.0) at an enzyme/substrate ratio of 1:50. Thermolysin digestion was done at pH 8.0 for 1.5h at 52°C and Staphylococcus aureus protease digestion at 37°C (pH 8.0) for 20 h, at an enzyme/substrate ratio of l : 100 and 1 : 30, respectively. Digestion with pepsin was carried out in 0.05 M HCI at 37°C for 2 h at an enzyme/substrate ratio of 1 : 50, and with carboxypeptidase Y from yeast in 0.1 M pyridineacetate buffer (pH 5.5) at 37°C. Peptides were isolated by the fingerprint technique on thin-layer plates as previously described [14,19]. Some peptides were isolated from the digest by gel filtration through Sephadex G-50 with 50% HCOOH as previously described [14]. Cleavage at aspartic acid residues. Protein

SA1 i

O.8

0.e

(3 mg) was hydrolysed with 25[ acetic acid (400/~1) in an evacuated sealed tube for 16 h at 110°C. Amino acid analysis. The amino acid compositions of proteins and peptides were determined with a Beckman model 121 M amino acid analyzer as previously described [ 14,19]. Sequence determination. Automated Edman degradation of the intact protein was done with a Beckman model 890 protein sequenator, using 0.5 M Quadrol buffer and a single-cleavage program, a modification of the method described by Hunkapiller and Hood [25]. Identification of phenylthiohydantoin amino acids was done by thin-layer chromatography on silica gel 60Fz54 (Merck) with two different solvent systems. The solvent mixtures were; first, chloroform/lpropanol/2-propanol (98 : 1 : 1, v / v / v ) ; second, chloroform/methanol (90"10, v/v) [26]. Some phenylthiohydantoin amino acids were also identified as free amino acids after hydrolysis with HI for 20h at 130°C [27]. Determination of amino acid sequences of peptides or the amino-terminal region of intact protein were performed by the double-coupling method using 4-N,N-dimethylaminoazobenzene 4'- isothiocyanate/phenylisothiocyanate [28]. The 4-N,N-dimethylaminoazobenze 4'-thiohydantoin of leucine and isoleucine were separated on polyamide thin-layer sheets in 10% formic acid/ethanol (10:9, v/v) [29]. Nomenclature. T, CH, SP and PE refer to tryptic peptides, chymotryptic peptides, S. aureus protease peptides and pepsin peptides of protein SA1, respectively. T-PE and T-SP refer to the peptides derived from pepsin and S. aureus protease digestion of tryptic peptides, respectively. T-SP-TH refers to the peptides derived from thermolysin digestion of T-SP peptide. The peptides are numbered according to their position in the sequence, starting at the N-terminal end.

0.4

R e s u l t s and D i s c u s s i o n

0.2

Purification of protein SA1. The NH4CI / ethanol extract of the 70 S ribosomes of S. griseus was fractionated on DEAE-cellulose as shown in Fig. 1. The protein SA1 was eluted as a single peak and showed a single band when analyzed on a polyacrylamide gel at either pH 4.5 or pH 8.6

| IO0

SO

Fraction

15O

Number

Fig. 1. Separation of the NH4Cl/ethanol extract from 70 S ribosome of S. griseuson DEAE-cellulose.

167

urea-gels. The position of this protein on a twodimensional gel electrophoretogram is shown in Fig. 2. The isolated protein SAI migrates in the two-dimensional gel to a position roughly the same as those of the acidic proteins L7/L12 from E. coli, BL9 from B. subtilis and MA1/2 from M. lysodeikticus. Terminal sequence of SA1.

The amino (N)terminal sequence was determined by automated Edman degradations in a Beckman sequenator up to the 35th residue except for positions 32 and 34. The positions 4 and 21 were determined to be serine by the conversion to dehydroalanine and unidentified products specific to serine after hydrolysis with HI. The N-terminal sequence up to the 14th residue was confirmed by DABITC/PITC double-coupling degradation of the intact protein (see Fig. 3). CarboxypeptidaseY released from intact protein lysine and valine after l-h incubation. Amino acid sequence of SA1. The details of sequence studies of SA1 are given in the supplementary data. The entire amino acid sequence of SA1 was determined mainly by the analyses of tryptic and S. aureus protease peptides. Alignment of the tryptic or S. aureus protease peptides was deduced from the results of automated Edman degradations of the intact protein and the sequences of the various peptides derived from cleavage of the protein with chymotrypsin, pepsin and dilute acid. A summary of sequence studies of these peptides is shown in Fig. 3 and should be

Fig. 2. Two-dimensional polyacrylamide gel electrophoresis of 70 S ribosomal protein from S. griseus [23].

consulted for the following experimental details. Peptide T2(3-24) was isolated from the digested protein by gel filtration through Sephadex G-50 and puified by cellulose thin-layer chromatography (the same condition as the second dimension of the fingerprint). The first six residues of the peptide were determined by the DABITC method, and found to be identical with the sequence of position 3 to 8 of the intact protein determined by automated Edman degradation. This tryptic peptide was further digested with pepsin, giving seven peptides. Alignment of the peptic peptides (PE-1-2-3-4-5-7) in the original peptide(T2) was determined by the results of automated Edman degradation of the intact protein or the amino acid sequence of SP peptides purified by the fingerprint method from the digested protein (SPI, 2, 3, and 4). Peptide T4(31-70) was isolated from the digested protein by gel-filtration through Sephadex G-50. The first 25 residues of peptide T4 were determined by the DABITC metod. This peptide was further digested with pepsin or S. aureus protease, giving seven and five peptides, respectively. Peptides T4-PE1 and T4-SP1 were both located at the N-terminal end of the original peptide, judging from their N-terminal sequences. The amino acid composition of five thermolysin peptides (TH1 to 5), derived from T4-SP1 that was first degraded up to six steps by the DABITC method, gave some indirect information for the C-terminal portion of T4-SP1. Peptides T4-SP5 and T4-PE6 should be C-terminal peptides because of the enzyme specificity of trypsin. The sequence of T4-PE4 established the alignment of T4-SP2 to T4-SP5. Thus, the alignment of SP peptides in the original peptide T4 must be SP1SP2-SP5. Other tryptic peptides T1, T3, T5, T6, T7, T8, T9, T10, T1 l.and T13 were isolated by gel filtration through Sephadex G-50 followed by the fingerprinting technique on cellulose thin-layer plates, and completely sequenced by the DABITC/PITC double-coupling method. A small amount of peptide T12 was isolated only by the fingerprinting technique from the digested protein. All the S. aureus protease peptides (SP) were isolated by the thin-layer fingerprint method and completely sequenced by the DABITC method. Sequences of some of these peptides determined the alignment of the tryptic peptides, and those of

168

i0 er - G l n - A s p - A s p - L e u - L e u - A l a - G l n - P h e - G l u - G l u - M e

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20 t-Thr-Leu- lle-Glu-Leu-Ser +

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60 70 A•a-G•u-G•u-A•a-G•u-G•n-Asp-G•u-Phe-Asp-Va•-ne-Leu-Thr-G•y-A•a-G•y-G•u-Lys-Lys-••e-G•n-Va•-••e-Lys T

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169 80 90 Val-Val-Ar g-Glu-Leu-Thr-Ser-Leu-Gly-Leu-Lys-Glu-Ala-Lys-Asp-Leu-Val-Asp-Gly-Thr T

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125

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T

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Fig. 3. The primary structure of an acidic ribosomalprotein (SAI) from Streptomvcesgriseus. Abbreviation: T, tryptic digestion: SP, digestion with Staph.vlococcus aureus protease; CH, digestion with chymotrypsin;PE, digestion with pepsin; T-PE, digestion with pepsin of tryptic peptide; T-SP, digestionwith Staphylococcus aureus protease of tryptic peptide; HAC, cleavagewith 2% acetic acid. Amino acids in the region indicated by (~) were identified by using the double-coupling method with DABITC/PITC; (+) identifiedby automaticliquid phase sequenator(LPSQ). the other peptides confirmed the internal sequence of large tryptic peptides. The amino acid sequence of SPI 1 was determined to be Lys-Lys-Ile-Gln-Val -Ile-Lys-Val-Val-Arg-Glu, thus confirming the alignment of T4-T5-T6. The amino acid sequence of SP17 was determined to be Ser-Leu-Lys-AlaAla-Gly-Ala-Ser-Val-Glu, and could give informa: tion on the unknown region of peptide T12. The C-terminal amino acids of all the S. aureus proteage peptides, except for SP18, were either aspartic acid or glutamic acid. These findings suggested that SPI8, determined to be Val-Lys, was the C-terminal peptide of the intact protein. Alignment of the tryptic peptides was also deduced from the sequences of various peptides derived from cleavage of the intact protein with chymotrypsin, pepsin and dilute acid. The amino acid sequence of peptide PE6 was determined to be Phe-Glu-Glu-Lys-Phe, thus confirming the alignment of T3-T4. Alignment of peptide T5-T6T7 was confirmed by the sequence of peptide CH7; Val-Ile-Lys-Val-Val-Arg-Glu-Leu. Alignment of peptide T7-T8-T9 was confirmed by the N-terminal sequence of peptide CH10. The amino

acid sequence of peptide PEI2 confirmed the alignment of T10-T11-TI2 and also gave information on the unknown region of peptide T12. Peptides derived from hydrolysis of the intact protein with 2% acetic acid were first separated with a Sephadex G-50 column, and a peptide corresponding to the positions 94 to 125 was isolated by an additional thin-layer chromatography. The first 13 residues of this peptide were determined by the DABITC method, thus confirming the alignment of T9-T10. A combination of the results described so far makes it possible to align all the peptides, and therefore to construct the entire amino acid sequence (Fig. 3). This acidic protein SA1 is composed of 125 amino acid residues and has the composition; Asp7 , Thr~, Sers, Glut8 , Gln4, Pro4, Glys, Ala24, Vail3, Met I, Ile4, Leul2, Ph%, Lysl4 and Argo. The molecular weight of this protein is 13 069. Comparison of acidic eubacterial organisms

proteins

from

various

Fig. 7 (supplementary data) shows the amino-

170

terminal amino acid sequences of L7/L12-type acidic ribosomal proteins from nine bacteria, of which three (E. coli, Vibrio costicola and an unidentified halophile NRCC 11227) are Gramnegative and the other six (Clostridium pasteurianum, Bacillus stearothermophilus, Bacillus subtilis, Micrococcus lysodeikticus, Arthrobacter glacialis and S. griseus) are Gram-positive. The complete amino acid sequences of L7/L12 protein from E. coli and the N-terminal sequences of acidic proteins from V. costicola, NRCC 11227, C. pasteurianum, B. stearothermophilus, A. glacialis have been determined by other workers [9,10,16] and the complete amino acid sequences of BL9 from B. subtilis and MA1/2 from M. lysodeikticus were reported previously [ 13-15]. Table I indicates the extent of amino acid sequence similarity in the N-terminal region (the first 39 residues) calculated from these sequence data (Fig. 7, supplementary data). A sequence from a Gram-positive species, for example, is more similar to those from within the same group, than to any from the gram-negative bacteria. Furthermore, even within the Grampositive bacteria group, the proteins from two bacteria (A. glacialis and S. griseus) show a higher degree of sequence homology to each other (74%) than to the equivalent proteins from others (46% identity on the average). They show a low degree of sequence homology to the equivalent protein

from the Gram-negative bacteria (36% identity on the average). The amino acid composition of acidic proteins from S. griseus and other bacteria so far studied are summarized in Table II and compared with each other. All the acidic proteins are alanine -rich and lack cysteine, tryptophan, histidine and tyrosine. Some characteristic features of the acidic proteins from the S. griseus group (including A. glacialis) are as follows; the acidic proteins have a significantly higher content of phenylalanine (five phenylalanine residues in S. griseus) than those from all the other bacteria (two phenylalanine residues) [9]. Three of these phenylalanine residues, specific for this group, are located in the N-terminal regions (positions 12, 23 and 27) and the rest are located in the same positions as in other bacteria (positions 31 and 59). It would thus appear that there are three types of acidic protein in the nine eubacteria investigated so far, although all of them still retain the common prokaryotictype structure; the first type is acidic protein from the Gram-negative bacteria ( E. coli, V. costicola and NRCC 11227), the second is that from Grampositive C. pasteurianum, B. stearothermophilus, B. subtilis (BL9) and M. lysodeikticus (ML1/2) and the third is that from the Gram-positive S. griseus and A. glacialis. It is also now known that acidic proteins from M. lysodeikticus give three peaks when separated by DEAE-cellulose column chro-

TABLE I E X T E N T O F A M I N O A C I D S E Q U E N C E SIMILARITY IN A M I N O - T E R M I N A L R E G I O N OF P R O K A R Y O T I C A C I D I C PROTEINS Values are calculated from the sequence data and the alignments shown in Fig. 7 (presented in supplementary_ data) and expressed as percentage. NRCC., N R C C 11227 [9]; E. coli, Escherichia coli [ 16]; V. cost., Vibrio costicola [9]: C. past.. Clostridiunt pasteurianum [9]: B. stea., Bacillus stearothermophilus [9]; M. Ivso., Micrdcoccus Ivsodeikticus M A I / 2 [15]; B. sub., Bacillus suhtilis [13]: A. glac., Arthrobacter glacialis [9]; S. grise., Streptom.vces griseus.

NRCC E. coli V. cost. C. past. B. stea. M. lyso. B. sub. ,4. glac. S. grise.

NRCC

E. coil

V. cost.

C. past.

B. stea.

M. lyso.

B. sub.

A. g(ac.

S. grise.

100 74 68 53 58 47 55 38 49

74 I00 79 50 55 50 39 28 36

68 79 I00 50 50 50 37 31 36

53 50 50 100 84 78 63 51 41

58 55 50 84 100 82 74 49 44

47 50 50 78 82 100 74 49 44

55 39 37 63 74 74 100 46 46

38 28 31 51 49 49 46 100 74

49 36 36 41 44 44 46 74 100

171 T A B L E II C O M P A R I S O N OF T H E A M I N O A C I D C O M P O S I T I O N (MOL%) OF A C I D I C P R O T E I N S F R O M V A R I O U S PROKARYOTIC SOURCES Abbreviations and references are in Table I.

Asx Thr Ser Glx Pro Gly Ala Val Met Ile Leu Phe Lys Arg

NRCC

E. coil

V. cost.

C. past.

B. stea.

M. lyso.

B. sub.

A. glac.

S. grise.

6.0 4.8 4.9 19.4 1.4 10.3 17.5 10.2 3.3 2.2 7.5 1.6 9.4 1.0

5.8 2.5 5.0 14.2 1.7 6.7 23.3 13.3 2.5 5.0 6.7 1.7 10.8 0.8

6.2 3.4 4.1 20.1 3.0 9.4 17.8 9.6 2.5 4.1 8.0 1.7 7.0 1.9

6.0 4.0 3.5 17.7 1.6 9.5 17.1 9.8 1.4 6.8 6.1 1.8 13.2 1.4

5.8 4.8 0.5 19.2 2.2 7.3 18.2 9.9 1.4 8.1 6.6 1.4 14.0 0.7

5.9 3.4 2.5 17.8 1.7 6.8 17.8 13.6 1.7 5.9 7.6 1.7 12.7 0.8

4.9 3.3 2.5 18.9 2.5 9.0 17.2 11.5 0 7.4 9.8 1.6 10.7 0.8

5.1 6.4 3.1 16.6 2.7 7.5 21.6 9.3 0 2.6 10.3 4.3 9.6 1.0

5.6 4.0 4.0 17.6 3.2 6.4 19.2 10.4 0.8 3.2 9.6 4.0 11.2 0.8

i0 E.coli NRCC M. lyso. B. sub. S. grise.

20

ALT Q E D I I N AVAEMS MNKE Q I LEA I KAMT ALN I E E I I AS VKE AT AKL S Q DDL LAQ FE EMTL

50 E. coli NRCC M . lyso. B. sub. S. grise.

A W V V

G A A -

P G G A

G A A P

G E A E A A A A A A G G A - A A A G G A P A

90 E. coli NRCC M. lyso. B. sub. S. grise.

E. coli NRCC M.lyso. B. sub. S. grise.

IITIGIL G m K E A K A IITIG[L G L K E A K E V

A ~ - ~ E ~--~ V ~ AIG AISIV EILIK I V]G A]SIV EIVIK ] VIG AISlV E[VIK l AIG AIS V~.~VIKI

30

VME VAE L V S VL E LN DL V K VL EL N DL V K I EL S E FVK

M I I F

K E E K

60 A E E E

E E Q T E~F - - K'TIE F .... IS F A E Q DiE F

40 G G G D

S T T T

A P P A

V A A

70 B:L V DIV V DILI DIV I

S S g G

i00 N - G A P A T L KIEIG M S D N A P K - - A L KIEIG V S

E A S E

K E Q K

80 V I I I

BIV KIV KIV QIV

I I I I

KIVlV KIVIV KIVIV KIVIV

RIE RIE RIE RIE

Ii0 E D G D E A K T D E A E E I K A

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E

K

EE

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120 122 118 122 125

Fig. 4. Comparison of the complete amino acid sequence of acidic ribosomal proteins from various prokary0tic sources. N R C C sequences are taken from Ref. 10 and all others are in Table I. The amino acids within the blocks indicate i~omologies among four of the acidic ribosomal protein.

172

matography (denoted MA1, 2 and 3). The amino acid sequence of protein MA3 revealed a high degree of homology to that of the third type (Itoh, T., unpublished data), while the sequence of the other MA1/2 (MA2 is the acetylated form of the protein MA1) showed a considerable homology to that of the second type as previously described [15]. Therefore, in "M. lysodeikticus, there exist two different types of acidic ribosomal protein. A comparison of the complete amino acid sequences of acidic ribosomal proteins from E. coli, NRCC 11227, M. lysodeikticus (MA1/2), B. subtilis (BL9) and S. griseus (SA1)~ as given Fig. 4, shows that the middle part, especially positions 84 to 90 (Leu-Gly-Leu-Lys-Glu-Ala-Lys), is highly conserved (a constant region) and anion binding sites of E. coli (positions 67, 68 and 71 in Fig.4), which show resemblance to the known nucleotide binding sites in many proteins [3], are also conserved. On the other hand, the N-terminal region, positions 1 to 25, are not conserved (a variable region). These different degrees of conservation within the molecule of the acidic protein might reflect some functional differences; the N-terminal part of the protein L7/L12 from E. coli is responsible for its binding to the 50 S subunit via protein L10, while the C-terminal part is needed for functional activities such as factor-dependent GTPase reaction [30,31]. Acknowledgements We wish to thank Professor S. Osawa (Nagoya University, Japan) for valuable discussions during the work. This research was supported by a Grantin-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 56580113). References 1 Matheson, A.T., MSller, W., Amons, R. and Yaguchi, M. (1979) in Ribosomes (Chambliss, G., Craven, G.R., Davies, J., Davis, K., Kahan, Land Nomura, M., eds.), pp. 297-332, University Park Press, Baltimore 2 MSller, W. (1974) in Ribosomes (Nomura, M., Tissi~res, A. and Lengyel, P., eds.), pp. 711-731, Cold Spring Harbor Lab. Press, New York 3 Leijonmarck, M., Eriksson, S. and Liljas, A. (1980) Nature 286, 824-826 4 Gudkov, A.T., Behlke, J., Utiurin, N.N. and Lim, V.1. (1977) FEBS Lett. 82, 125-129

5 Luer, C.A. and Wong, K.P. (1979) Biochemistry 18, 20192027 6 Maassen, J.A., Schop, E.N. and MOiler, W. (1981) Biochemistry 20, 1020-1025 7 0 s a w a , S. and Hori, H. (1979) in Ribosomes (Chambliss, G., Craven, G.R., Davies, J., Davis, K., Kahan, k and Nomura, M., eds.), pp. 333-355, University Park press, Baltimore 8 Higo, K., Itoh, T. and Osawa, S. (1978) in Evolution of protein molecules (Masubara, H. and Yamanaka, T., eds.), pp. 197-207, Japan Sci. Soc. Press, Tokyo 9 Visentin, L.P., Yaguchi, M. and Matheson, A.T. (1979) Can. J. Biochem. 57, 719-726 10 Yaguchi, M., Matheson, A.T., Visentin, LP. and Zuker, M. (1980) in Genetics and evolution of RNA polymerase, tRNA and ribosomes (Osawa, S., Ozeki, H., Uchida, H. and Yura, T., eds.), pp. 585-599, University of Tokyo Press, Tokyo I I Amons, R., Agthoven, A.V., Phiijms, W., Mt~ller. W., Higo, K., Itoh, T. and Osawa, S. (1977) FEBS Lett. 81,308-310 12 Matheson, A.T., Yaguchi, M., Balch, W.E. and Wolfe, R.S. (1980) Biochim. Biophys. Acta 626, 162-169 13 Itoh, T. and Wittmann-Liebold, B. (1978) FEBS Lett. 96, 392-394 14 Itoh, T. and Wittmann-Liebold, B. (1980) J. Biochem. 87, 1185-1201 15 Itoh, T. (1981) FEBS Lett. 127, 67-70 16 Terhorst, C., M611er, W., Laursen, R. and WittmannLiebold, B. (1973) Eur. J. Biochem. 34, 138-152 17 Itoh, T. (1980) FEBS Lett. 114, 119-123 18 Itoh, T., Higo, K., Otaka, E. and Osawa, S. (1980) in Genetics and evolution of RNA polymerase, tRNA and ribosomes (Osawa, S., Ozeki, H., Uchida, H. and Yura, T., eds.), pp. 609-624, University of Tokyo Press, Tokyo 19 Itoh, T. (1981) Biochim. Biophys. Acta 671, 16-24 20 Nimi, O., Kokan, A., Manabe, K.. Maehara, K. and Nomi, R. (1976) J. Ferment. Technol. 54, 587-595 21 Sugiyama, M., Kobayashi, H., Nimi, O. and Nomi, R. (1980) FEBS Letc I10, 250-252 22 Hamel, E., Koka, M. and Nakamoto, T. (1972) J. Biol. Chem. 247, 805-814 23 Kaltschmidt, E. and Wittmann, H.G. (1970) Anal. Biochem. 36, 401-412 24 Leboy, P.S., Cox, E.C. and Flaks, J.G. (1964) Proc. Natl. Acad. Sci. U.S.A. 52, 1367-1374 25 Hunkapiller, M.W. and Hood, L.E. (1978) Biochemistry 17, 2124-2133 26 Wittmann-Liebold, B., Geissler, A,W. and Mar-zining, E. (1975) J. Supramol. Struct. 3, 426-447 27 Smithies, O., Gibson, D., Fanning, E.M., Goodfliesh, R.M., Gilman, J.G. and Ballantyne, D.L. (1971) Biochemistry. I0, 4912-4921 28 Chang, J.Y., Brauer, D. and Wittmann-Liebold, B. (1978) FEBS Lett. 93, 205-214 29 Yang, C. (1979) Hoppe-Seylers Z. Physiol. Chem. 360, 1673-1675 30 Van Agthoven, A.J., Maassen, J.A., Schrier, P.I. and MiSller, W. (1975) Biochem. Biophys. Res. Commun. 64, 1184-1191 31 Gudkov, A.T. and Behlke, J. (1978) Eur. J. Biochem. 90, 309-312