THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
VOl.,267, No.2, Issue of January 15, pp. 1382-1390,1992 Printed in U.S.A.
Structure Function Relationshipsin the Ribosomal Stalk Proteins of Archaebacteria" (Received for publication, February 13, 1991)
Andreas K. E. Kopke$Qll,Peter A. LeggattS, and Alastair T. Matheson$ From the $Department of Biochemistry and Microbiology, University of Victoria, P. 0. Box 3055, Victoria, British Columbia, V8W 3P6 Canada and the EiMax-Planck-Institut f u r Molekulare Genetik, Abteilung Wittmann, Ihnestrassee 73, 1000 Berlin 33,Federal Republic of Germany
The ribosomal L12 protein gene of Sulfolobus solfa- only one protein present infour copies, the ribosomal protein and overex- L12 (Wittmann-Liebold, 1986). This proteinhas been setaricus (SsoL12) has beensubcloned pressed in Escherichia coli. Five protein L12 mutants quenced (Terhorst et al., 1973), shown to exist as two dimers were designed: two NH2-terminal and two COOH-ter- bound to the L10 protein (Pettersson et al., 1976), and anaminal truncated mutants and one mutant lacking the lyzed for its function (Hamel et al., 1972; Agthoven et al., highly charged part of the COOH-terminal region. The 1975; Gudkov et al., 1980). The COOH-terminal half of the mutant protein genes were overexpressed in E. coli protein has been crystallized (Leijonmarck and Liljas, 1988), and theproducts purified. The amino acid composition analyzed by two-dimensional NMR insolution and compared was verified and the NH2terminally truncated mutants to thecrystal structure (Aquist and Tapia,1990). were subjected toEdman degradation. To summarize these findings it was shown that the50 S E. The SsoLl2 protein was selectively removed from coli ribosome contains two copies of L12 and two copies of an entire S. aolfataricus ribosomes by an ethanol wash. The remaining ribosomal core particlesshowed a sub- NHz terminally acetylated version, called L7, present as two stantial decrease in the in vitrotranslational activity. dimers. One dimer constitutes the stalk protuberance while S. solfataricus L12 protein overexpressed in E. coli the other was located at the base of the stalk in the 50 S (SsoLl2") was incorporated into these ribosomal cores particle (Olson et al., 1986). The L12 protein dimers are both bound to the L10 protein, which in turn is bound to the 23 S and restored their translational activity. Mutants lacking any part of the COOH-terminal re- rRNA close to thebinding site of the L11 protein (Beauclerk gion could beincorporated into these cores, as proven et al., 1983). By a simple salt-ethanol wash (Hamel et al., by two-dimensional polyacrylamide gels of the recon- 1972) the L12 protein can be readily extracted from the 50 S stituted particles. Mutant SsoLl2 MC2 (residue 1-70) subunit. The ribosomal 50 S cores produced by this procedure was sufficient for dimerization and incorporation into possess only about 30% of the translationalactivity, possibly ribosomes. In contrast to the COOH terminally trun- due to incomplete removal of the L12 protein. The functions cated mutants, L12 proteinslackingthe 12 highly of the L12 protein are presumed to be the interaction with conserved NH2-terminal residues or the entire NH2- the different factors, the activation of the GTPase activity terminal region (44 amino acids) are unable to bind to for the elongation factors, and possibly the active movement ribosomes, suggesting that the SsoLl2 protein binds of the tRNAs(see Moller and Maassen, 1986; Moller 1990, as with itsNH2-terminal portion to the ribosome. None of the mutants could significantly increase the reviews). The amino acid composition of this protein is chartranslational activity of the core particles suggesting acteristic; it contains many alanines and glutamic acid resithat every deleted part of the protein was needed di- dues and is rare in arginines, histidines, tyrosines, cysteines, rectly or indirectly for translational activity. Our re- andtryptophans (Wittmann-Liebold, 1986). The physical sults suggest that theCOOH terminally truncated mu- data and the x-ray structure show that the L12 protein can tants were bound to ribosomes but not functional for be divided intothree domains: the rod-like NHz-terminal domain, the very flexible hinge region, andthe globular translation. Cores preincubated with these COOH terminally COOH-terminal domain (Liljas et al., 1986a). While the NH2truncated mutants regained activity when a second terminal domain binds the L12 protein to the 50 S subunit, incubation with the entireoverexpressed SsoLl2" pro- the COOH-terminal region interacts with the different factors tein followed. This finding suggests that archaebacter- (Nag et al., 1987). ial L12 proteins are freely exchanged on the ribosome. The large ribosomal subunits of eukaryotes and archaebacteria also contain four copies of an acidic protein (SaenzRobles et al., 1988; Casiano et al., 1990), and like its eubacterial counterpart it is located in the stalk protuberance. These The 50 S ribosomal subunit of Escherichia coli contains proteins have been sequenced and compared to those of the * This work wassupported in part by BCHRF Research Grant 125 eubacteria (see Wittmann-Liebold et al., 1990; Matheson et (91-1) (to A. T. M.). The costs of publication of this article were al., 1990, as reviews). It has been found that the equivalent defrayed in part by the payment of page charges. This article must L12 and L10 proteins of the different organisms fall into two therefore be hereby marked "advertisement" in accordance with 18 distinct groups: (i) the eubacterial type and (ii) the archaeU.S.C. Section 1734 solelyto indicate this fact. bacterial and eucaryotic type (Ramirezet al., 1989; Kopke and This paper is dedicated to Dr. B. Wittmann-Liebold. Wittmann-Liebold, 1989; Kopke et al., 1989). While the sePartially supported by the Deutsche Forschungsgemeinschaft. To whom correspondence should be addressed Max-Planck-Institute fur quence similarity within these groups is significant, no unExperimentelle Medizin, Herman Rein Str. 3, 3400 Gottingen, F. R. ambiguous alignment could be found between these groups. In contrast, other ribosomal proteins, e.g. L2, S8, and S11 G.
1382
Ribosomal Proteins Stalk
of Archaebacteria
1383
show extensive sequence similaritiesthroughout all kingdoms a 2% agarose gel and eluted using the "Geneclean" kit (Bio 101 Inc., La Jolla, CA). The 224-bp band was digested with DdeI (59- and 165(Wittmann-Liebold et al., 1990). Many different models for bp bands), separated on a 2% agarose gel and the 165-bp band was the alignment orrearrangement of protein L12during the eluted as described. This 165-bp band and the161-bp band from the evolution of these molecules have been proposed, but none of first digest were ligated together with the SsoL12MN1 linker and the these proposals is based on significant functional or statistical pUC 19 vector (Fig. 2). The recombinant clones were checked by evidence (Amons et al., 1979; Yaguchi et al., 1980; Lin et al., restriction enzyme digests (Fig. 3) and DNA sequencing (using the 1982; Matheson, 1985; Liljas et al., 1986b; Otaka et al., 1989; Sequenase Kit, United States Biochemical Corporation, Cleveland, Ohio). Ramirez et al., 1989). SsoL12* was digested with DraI (153- and 263The archaebacterial and eukaryotic L12 proteinscontain a bpSsoLl2MN2"The bands) and the fragments were separated on an "Gene Pak Fax" highly charged COOH-terminal region which is not found in column (Waters, Milford, MA). The 263-bp fragment was precipitated the eubacteria. The whole COOH-terminal region of the ar- from the collected HPLC fractions by the addition of 1 volume of 2chaebacterial and eukaryotic L12 proteins was found to be propanol, redissolved in TE-buffer (10 mM Tris-C1, pH 8, 1 mM most similar to the COOH-terminal region of the L10 proteins EDTA) and ligated with the SsoL12MN2 linker in pUC 19 (Fig. 2). The recombinant clones were verified as described above (Fig. 3). of the sameorganism,ratherthanbeinghighlyconserved SsoLI2MCl"The pUC 19SsoL12* plasmid was digested with throughout all organisms. It was proposed that this highly EcoRI and AvaII (222-, 287-, 1024-, and 1512-bp fragments), the conserved sequencein the COOH-terminal regions of L12 andmixture was separated on the ion-exchange HPLC column (''Gene L10 is due to a specificselective pressure, absent in eubacteria, Pak Fax"), and the 287-bp band was ligated with the SsoL12MC2 linker into thepUC19 vector (Fig. 2). enforcing the coevolution of these tworibosomalproteins SsoL12MC2"The pUC19 SsoL12MCl plasmid was digested with (Kopke et al., 1989). Since the L10 and L12equivalent proteinsfrom archaebac- PstI (2928- and 72-bp fragment) and separated on a 0.8% agarose gel. 2928-bp band was religated to form the SsoL12MC2 plasmid teriaandeukaryoteshavecharacteristics that are not ob- The (Fig. 2). served in the eubacterialsequencesareevaluationof the SsoL12MC3"The pUC 19 SsoL12* plasmid was digested with structure/function relations for these proteins is necessary. EcoRI and Sun and the 397-bp gene fragment was separated from In addition, attempts to incorporate archaebacterial L12 pro- the pUC 19 vector on the ion-exchange HPLC column. The gene teins into E. coli ribosomes (Boublik et al., 1979), as well as fragment wascleaved by PstI and AvaII (224-, 65-, and 108-bp attempts to incorporate eubacterial L12 proteins in Halobac- fragments) and again separated on the anion-exchange HPLC colThe 224- and the 108-bp fragment together with the Ssoterium marismortui ribosomes (Kopke et al., 1990) failed. In umn. L12MC3 linker were ligated into the pUC 19 vector (Fig. 2). The contrast, the L12 protein of Bacillus stearothermophilus (eu- recombinant clones were checked as described above (Fig. 3). bacterium) was successfully incorporated into E. coli (eubacTests for Overexpression of the Cloned Genes in Small Culturesterium) ribosomes (Boubliket al., 1979), and the L12 protein The overexpression of the different mutant proteins in E. coli was of Methanococcus vannielii (archaebacterium)was incorpo- monitored in 10 ml cultures, whichwere induced with 0.01 mM after reaching an optical denrated into ribosomes of Halobacterium marismortui (archae- isopropyl-l-thio-P-D-galactopyranoside sity of Asso = 1.0. Total cell proteins were obtained from induced E. bacterium) (Kopke et al., 1990). coli cells by adding SDS loading dye to a constant amount of cells, To investigate the structure/function relationship of the and analyzed on15% SDS-high-Tris-polyarcylamidegels (Fig. 4) L12proteins in archaebacteria,mutantL12proteingenes (Fling and Gregerson, 1986) . Purificution of Overexpressed Mutant Proteins-Overexpression from Sulfolobus solfataricus havebeenproduced,overexpressed,purified,andincorporated into the S. solfataricus was carried out with the various overexpression plasmids in the host ribosome.Poly(U) translational assays were performedto test E. coli BL21-DE3, grown in 2 liters of LB medium under the optimal conditions previously described for SsoL12' (Kopke et al., 1991).The incorporated proteins fortheir function. culture was incubated for an additional 4h, following induction with 0.01 mM isopropyl-l-thio-P-D-galactopyranoside at an optical density of AeSa = 1.0. The cells were harvested by low speed centrifugation (5000 X g for 10 min), resuspended in 20 ml of TMA I (10 mM TrisDNA handling and cloning procedures were performed according HCl, pH 7.8, 10 mM MgC12, 30mM NH4C1,6 mM P-mercaptoethanol) to Sambrook et al. (1989). Separation of DNA fragments was partly and opened by sonication. The cell debris was removedby high speed done on HPLC' as previously described (Kopke et al., 1989). The centrifugation in a SS34 rotor (27,000 X g for 10 rnin), and the synthesis of the oligonucleotide linkers and theoverexpression in test supernatant was centrifuged overnight in an ultracentrifuge (Ti-50 cultures under optimized conditions were done as previously described rotor, 40 krpm) to precipitate the E. coli ribosomes. This supernatant for the SsoL12' protein (Kopke et al., 1991). was analyzed on SDS-polyacrylamidegels (Fig. 4),titrated with acetic E. coli Strains Used-E. coli XL1 blue has the following chromo- acid to pH 3.0, and incubated a t 0 "C for 30 min. The precipitated somal mutations: recAl, Alac, endAl, gyrA96, thi, hsdRl7 (rk-,mk+), proteins were removedby centrifugation (27,000 X g for 10 rnin). The supE44, thi-1, lambda-, (lac-), relA1, and an F' with the mutations total supernatant of a preparation from 2 liters of mediumwere proAB, lacPAZM15, TnlO (tet") (Bullok et al., 1987). concentrated to 5 ml and separated on a size exclusion column E. coli BL21 DE3 has the mutations F ,ompT, rB-mB- for the (Sephacryl S-100 HR, inner diameter, 25 mm; bed height, 850 mm; BL21 strain and the DE3 lysogen contains the minimum region of flow rate, 2 ml/min; running buffer: 40 mM sodium trifluoroacetate, phage 21, a fragment of the lac1 gene, the lacUV5 promotor, beginning pH 3, 200 mM NaC1, 1 mM dithiothreitol). All fractions (fraction size of lac2 gene and the gene for T7 RNA polymerase (Studier and 4 ml) were screened for the overexpressed proteins on mini SDSMoffatt, 1986). polyacrylamide gels. The fractions containing the desired protein Cloning of SsoLl2 and the Mutant Protein Genes-The SsoLl2 were combined, dialyzed against water, and lyophilized. If necessary, gene was subcloned, modified, and overexpressed as described in the proteins were further purified by HPLC reversed-phase chromaKopke et al. (1991). The cloned gene with a ShineDalgarno sequence tography. identical to thatof the MvaLl2gene (Kopke et al., 1990) was excised HPLC Reuersed-phase Separations-For a fast and reliable purifiby EcoRI and Hind111 as a gene cartridge (SsoL12') from the pUC 19 cation of these proteins,the enriched protein mixtures were separated cloning vector and used for all subsequent modifications (Fig. 1).The on a reversed-phase HPLC column (8 X 250 mm, Nucleosil C4,5-pm gene cartridges were than subcloned in pT7-5 (Tabor and Richardson, particles, 30-nm pore size, from Macharey & Nagel, Germany; or 1985) for overexpression. Brownlee RP-8, 10-pm particle size, 100-nm pore size, from Applied SsoL12MNl"The SsoL12*was digested by BumHI and PstI. The Biosystems, CA). The solvents were 0.1% trifluoroacetic acid in water 5', 224-bp fragment, and the 3', 161-bp fragment were separated on as buffer A and acetonitril or 2-propanol as buffer B. Linear gradients from 0 to 50% buffer B in about 60 min (flow rate 1 ml/min) were ' The abbreviations used are: HPLC, high performance liquid chro- employed and optimized for each application. The fractions were matography; bp, base pair; SDS, sodium dodecyl sulfate; HEPES, 4- screened by SDS-polyacrylamide gels for the overexpressed protein. (2-hydroxyethyl)-l-piperazineethanesulfonic acid. The combined fractions containing the overexpressed protein were MATERIALS ANDMETHODS
Ribosomal Stalk Proteinsof Archaebacteria
1384 R1
SJOL12
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R3
R2
b*
-4
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-
R5
U
10 20 30 40 50 60 70 80 90 100 MEYIYASLLLHAAKKEISEENIKNVLSAAGINDEVRLKAV~LKEVNIDEILKTATAMPV~VAAPAGQTQQ~EKKEEKKEEEKKGPSEEEIGGGLSSLFG
SsOL12Q
IMNI IMN2 IMCl
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9
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FIG. 1. The amino acid sequence ofSsoLl2 has been divided intofive regions (RI-R5indicated on top of thesequence).The SsoLI2, SsoLlZMNI, SsoLlZMN2, SsoLIZMCI, SsoL12MC2,and SsoL12MC3protein genes have been produced, subcloned into the expression vector pT7-5, and overexpressed in E, coli. The hatched bar represents the coded sequence identical to the genuine SsoLl2 sequence. Extra amino acids coded are due to the cloning procedure and shown for these sequences. NHz-terminal methionines in brackets were removed during post-translational modification by E. coli. dimensional polyacrylamide gels (Geyl et al., 1981) for their protein constituents. Poly(U) Translational Assays-The activity of the reconstituted ribosomes was tested utilizing a poly(U) assay similar to that deSsoL12MN1 scribed by Londei et al. (1986). To prepare the s. solfataricus S150 Ode I H i n D III protein fraction the supernatant of the ribosomal sedimentation in the ultracentrifuge was treated as follows. The protein mixture was SsoLI2MNZ precipitated by addition of solid ammonium sulfate to 70% saturation E o RI ....._... resuspended in one-fifth of the original volume of S150-storagebuffer (10 mM Tris-HC1, pH 7, 1 mM MgCl,, 5 mM P-mercaptoethanol, and 10%glycerol in water) and dialyzed overnight against the same buffer SsoLIZMCI to remove residual ammonium sulfate. The solution was stored in Avo 11 HinD /I1 aliquots at -80 "C. To obtain a S. solfataricus tRNA mixture, 40 ml of ribosomal supernatant was extracted twice with phenol, once with chloroform/isoamylalcohol(99:1)and precipitated by the addition of SsoL12MCZ Prr I HmD 111 Eco RI 3 volumes of ethanol. After incubation a t -20 "C for 20 min, the precipitated RNA fraction was collected by centrifugation and redissolved in 10 mM Tris-HC1 at pH 7. Aliquots of the tRNA mixture . .. . were stored at -80 "C. Eco RI PSI I Ava I1 HinD Ill The poly(U) translational assay contained 0.2-0.4 A,,, of S. solfataricus ribosomes or core particles in a reaction volume of 50 pl. The mixture contained 25 pl of two times poly(U) buffer (40 mM HEPESFIG. 2. The appropriate gene fragments were first ligated KOH, pH 7.0,20 mM KCl, 36 mM magnesium acetate, 6 mM sperminewith their oligonucleotidelinkers in the pUC 19 vector. The HCl, 10 mM P-mercaptoethanol), 1p1 of 100 mM GTP, pH 7.0, 1.5 p1 recombinant plasmids were cut with EcoRI and Hind111 and the resulting gene cartridge was subcloned into pT7-5 for expression, of200 mM ATP, pH 7.0, 1 pl of 10 mg/ml poly(U), 1 pl of tRNA mixture (180Az60/ml),5 pl of S150-fraction, 5 p1 of [14C]phenylalanine resulting in the constructs shown above. (18 GBq/mmol, Du Pont-New England Nuclear), 0.5 pl of water, and 10 pl of reconstitution mixture. Final concentrations inthe assay due rechromatographed where necessary. to the addition of 10 pl of reconstitution buffer and 5 pl of S150 Depleting Sulfolobussolfataricus Ribosomes of SsoLl2-Ribosomes fraction in the mixture: 20 mM HEPES-KOH, pH 7.0, 10 mM KC1, of S. solfataricus have been isolated and purified as described by 20mM magnesium acetate, 3 mM spermine-HCl, 6 mM P-mercaptoLondei et al. (1986). The ribosomes were resuspended in R-buffer (40 ethanol, 2 mM GTP, 6 mM ATP, 0.2 mg/ml poly(U), 8 mM NH,C1,5 mM NH4C1, 20mM Tris-HC1,pH 7.4,lO mM magnesium acetate, and mM Tris-HC1, and 1% glycerol. The poly(U) assay mixture was 5 mM P-mercaptoethanol), extracted with 0.5 volumes of ice-cold incubated at 80 "C for 30 min. 1 mlof 5% trichloroacetic acid was ethanol a t 0 "C for 15 min with constant stirring and subsequently added and the mixture was incubated for 10 min a t 90"C. The precipitated by addition of a second 0.5 volume of ice-cold ethanol precipitated material was collected on a glass fiber filter, washed under constant stirring at 0 "C for 5 min, similar to Hamel et al. subsequently with 5% trichloroactetic acid and ethanol/ether (1:l). (1972) but without high salt concentration. The extraction was re- The radioactivity on the filters was measured utilizing Aquasol scinpeated once more under identical conditions. The extracted ribosomes tillation liquid (Du Pont-New England Nuclear) and a scintillation were collectedby centrifugation at 14 krpm in a table top centrifuge. counter (Beckman Instruments LS8100, Fullerton, CA). The supernatant and theribosome cores were analyzed by SDS- and The 10 pl of reconstitution mixture for the translational assay were two-dimensional-polyacrylamidegel electrophoresis (Fling and Gre- prepared as follows: 5 pl of R-Buffer containing 0.2 or 0.4 A260 gerson, 1986;Geyl et al., 1981). S. solfataricus ribosomes and core ribosomal cores were ( a ) incubated together with additional 5 pl of particles were stored in R-buffer containing 20% glycerolat -20 "C. R-buffer containing 1 pgof purified overexpressed mutant protein Reconstitution of the Overexpressed Proteins into S. solfataricus for 30 min at 60 "C; or( b ) incubated together with 2.5 pl of R-buffer Ribosome Cores-40 A260 units of Sso ribosomal cores were incubated containing 1 pgof mutant protein for 15 min a t 60 "C and then with about 100 pg of overexpressed and purified protein SsoLlT or additional 2.5 pl of R-buffer containing 1-2 pg of SsoL12"for further protein mutant, in300 pl of R-buffer for 30 min at 60 "C.The reaction 15 min. For the control experiments 10 pl of R-buffer contained either mixture was incubated on ice for 5 min, loaded on 7 ml of an ice-cold ( a ) the same amount of entire ribosomes, ( b ) the same amount of sucrose cushion (18%(w/v) sucrose in 500 mM NH4Cl, 20 mM Tris- ribosomal cores, or (c) no ribosomal particles. HC1, pH 7.4, 10 mM magnesium acetate, and 5 mM P-mercaptoethanol), and ultracentrifuged in a Ti-50 rotor at 50 krpm for 3 h as also RESULTS ANDDISCUSSION used for ribosomepurifications. The ribosomes sediment through the The stalk protein domain of the archaebacterium S. solfuhigh salt cushion buffer, while unbound proteins dissociate from the ribosomes and stay in solution. The precipitated ribosomes were turicus has been chosen as a model for the group of similar analyzed by protein extraction (Nierhaus and Dohme, 1979) and two- archaebacterial and eukaryotic LlO/L12 proteins. S. solfuturSSOLlZ
E m RI
Rsa I
HlnD 111
Ribosomal Stalk Proteins of Archaebacteria
1385
EmR I
PSI
SSOL12. SsoLlZMNl sSOL12MN2
HinD
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I
I
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-
em^,
EcoR I
I
I
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I I
I
SsoLIZMC3
I 1
I
0
I I
I
SsoL12MCZ
SsoLI2MCl
I
I
I
I HinD III
HinD n/
IHinD III
I
100
200
300
400
FIG. 3. Left, restriction patterns of all mutants separated ona 2% agarose gel to verify gene sizeand orientation. * in thislegend. Right, restriction Some bands are due to partial digestion of the plasmid and therefore marked with map of the designed mutant genes. Only the enzymes usedfor the restriction analysis are shown.A, “1-kb ladder” band sizes (from bottom to top):75,134,154,201,220,298,344,396,506/517, 1018 bp, and others.R, SsoL12MNl digested with EcoRI and HindIII (385 bp, pUC 19). C, SsoL12MN1 digested with EcoRI and PstI(179 and 192 bp, pUC 19). D, SsoL12MN2 digested with EcoRI and HindIII (290 bp, pUC 19). E, SsoL12MN2 digested with EcoRI and PstI (179 and97bp,pUC 19). F, SsoL12MCl digested withEcoRIandHindIII (309bp, pUC19). G, SsoL12MCl digested with EcoRI and PstI (224, 71, and 295 bp*, pUC 19). H, SsoLl2 MC2 digested with EcoRI and HindIII (241 bp, pUC 19). I , SsoL12 MC2 digested with EcoRI and PstI (224 bp, pUC 19). J , SsoL12MC3 digested with EcoRI and HindIII (384 bp, pUC 19). K,SsoL12MC3 digested with EcoRI and PstI (224, 146, and 370 bp*, pUC19).
icus appears to be of ancient origin (Zillig et al., 1989) and possesses the highest sequence similarity between the highly charged COOH-terminal regions of its L10 and L12 proteins, a property unique to theeukaryotic and archaebacterial stalk protein group (Matheson etal., 1990; Kopke and WittmannLiebold, 1989). By a comparison of the ribosomal stalk structure/function relationships in S. solfataricus to theknown E. coli structure/ function relationshipsit should be revealed if the two different kinds of amino acid sequences have similar or different structure/function relationships. It is possible that the unique properties of the eukaryotic and archaebacterial sequences are reflected in different mechanismsor additional functions. Division of the SsoLlP Protein into Distinct Regions-To investigate the function of different parts of the SsoL12 protein structure, it was divided into five regions (Fig. 1): R1, the highly conserved NH, terminus; R2, a less conserved region in the NHZ-terminal third of the protein; R3, the highly flexible hinge region, comprising many alanine and proline residues; R4, the highly charged region in theCOOH-terminal third of the sequence; and R5, the COOH-terminal region, conserved in all organisms. Regions four and five are very similar between the L10 and L12 proteins of the same organism. This partitioning has lead to thedesign of five different mutant proteins, lacking different parts of the protein sequence (Fig. 1). Production of the Mutant L12 Proteins-The original SsoLl2 protein gene andthemutantprotein genes were cloned into pUC 19 and their DNA sequence verified by restriction patterns (Fig. 3) and nucleotide sequencing. All these genes had the Shine-Dalgarno sequence of the methanococcal L12 protein gene ligated to their 5’ end, since this Shine-Dalgarno sequence had been shown to be highly efficient for overexpression (Kopke et al., 1990, 1991). The EcoRI-Hind111 cut gene cartridges were subcloned (orientation directed) into the expression vector pT7-5 and checked for their restriction patterns. Finally, these plasmids were transformed into E. coli BL21-DE3 and testedfor overexpression as described under “Materials andMethods.”
FIG. 4. Analysis of total E. coli cell protein before induction (a),a f t e r 4 h of induction( b ) ,a f t e r cell l y s i s a n d u l t r a c e n t r i f ugation (c), for t h e m u t a n tgenes indicated on top of the lanes.
The analysis of the totalprotein mixture onSDS-polyacrylamide gels, following 4 h of overexpression, showed a significant production of the SsoLl2 mutant proteins, when compared to the totalcell protein mixture before induction (Fig. 4). Overexpressed proteins were purified from the cytosolic cell fraction as described under “Materials and Methods.’’ The proteins were purified by reversed-phase HPLC separation when used for amino acid analysis or protein sequencing in order to get a salt-free preparation. Proteins intended for reconstitution analysisand activity tests were sometimes used directly following the size exclusion separation (purity >go%), dialysis, and concentration and notalways purified by HPLC. All mutant protein preparations were checked on SDS-acrylamide gels (Fig, 5) and two-dimensional gels (not shown). The overexpressed and purified mutant proteins were confirmed by amino acid analysis (Table I). Inaddition, the NHz terminally truncated mutants were subjected to protein sequencing.
Ribosomal Stalk Proteins of Archaebacteria
1386
FIG.5. Analysis of the purified (following size exclusion) mutant proteinson a 16% high-Tris-SDS-polyacrylamide-gel. Lanes (from left to right): 1 , Rainbow Markers (Amersham, United Kingdom) from bottom to top: 6,500,14,300,21,500,30,000, and 46,000 Da; 2, SsoL12‘; 3, SsoL12MNl; 4, SsoL12MN2; 5, SsoL12MCl; 6, SsoLl2 MC2; 7, SaoL12MC3. TABLE I Amino acid analysis data of the purified, overexpressed protein L12 mutants SsoLl2’ SsoL12MNl SsoL12MN2 SsoL12MCI SaoL12MC2 acid Coded Found Coded Found Coded Found Coded Found Coded Found
Asx Glx Ser Gly His Arg Thr Ala Pro Tyr Val Met Ile Leu Phe
Lvs
5 20 6 7 1 1 4 18 3 2 7 2 7 9 1 12
5 4.7 18.7 19 5 5.0 7 6.5 1.0 0 1.4 1 3.6 4 18.8 16 3 2.1 0 2.0 7.6 7 1.2 1” 7.3 6 10.5 6 1.3 1 12.6 12
5.1 2 2.1 19.4 15 14.8 3 4.7 2.5 7.9 6 5.6 0 0 0 1.0 0 0 3.8 3 2.8 16.0 10 9.8 3.2 3 2.7 0 0 0 7.1 3 3.0 0.5 1” 0.7 5.0 3 2.9 5.7 3 2.9 1.0 1 1.0 10.2 8 8.7
5 17 4 4 1 1 4 19 3 2 7 2 6 7 0 12
6.1 18.0 3.8
4.5 1.0 1.2 4.0 18.7 2.3 1.8 7.0 1.9 5.7 7.2 0.3 11.2
5 7 3 2 1 1 3 16 2 2 7 2 6 7 0 6 ~
6.0 7.1 2.8 3.0 1.0 1.3 2.4 16.5 2.6 1.7 7.3 2.1 5.8 7.2 0.4 5.4 ~~
~~
The coded NHZ-terminal methionine is removed by post-translational modification in E. coli as shown by NHZ-terminal sequencing of the purified mutant proteins.
Analysis of the Overexpressed Proteins-The SsoL12 protein and its mutantsshow a strange behavior on SDS-polyacrylamide gels, two-dimensional polyacrylamide gels, and on RP-HPLC columns. As previously described for L12 preparations from other organisms (Kopke al., et 1991), broad peaks and double peaks were found in thereverse-phaseHPLC profile for pure preparationsof the Sso proteins (as judged by amino acid analysis, protein sequencing and polyacrylamide gels). InadditiontheNH2terminallyshortenedmutants exhibit a lower migration speed in SDS gels than we expected based on their low molecular weight, while the COOH-terminal-truncated mutants were migrating as expected. All L12 proteins, and especially the COOH terminally shortened mua tail tant proteins, migrate sometimes as concave bands with in SDS-polyacrylamide gels. If the, samples were absolutely free of salts, present in small amounts and not boiledin loading dye prior to their application on the gel, they ran as a normal single band (Fig. 4). In two-dimensional gels even the homogeneous proteinpreparation of SsoL12MC1 (as proven by amino acid sequencing and amino acid analysis) migrated as a double spot, where the second spot had much lessintensity,appeared aslittle morepositively charged (shifted to the right), and of slightly smaller size. Sequence Analysis of the Overexpressed Proteins-Sequenc-
ing of the purified, overexpressed proteins showed that the NH, terminusof SsoLlP’was totallyblocked when the protein was produced in the late phase of exponential growth in the E. coli host. Cleavage of the blocked protein with cyanogen bromide (as described in Kopke et al., 1990) resulted in two sequenceablepeptideswiththe expected protein sequence. Expression in the early growth phase produced only partial NH,terminally blocked SsoLlP’ protein together with an excess of a smaller overexpression product. This additional product was purified by reversed-phase HPLC andsequenced. It was identified as an unblocked, NH, terminally shortened SsoL12proteinstartingwith a methionine in amino acid position 22 and continuing with the normal SsoL12 protein sequence. Position 22 is an isoleucine in the genuine SsoL12 protein, coded by an ATA codon. Sequencing of the overexpression plasmid containing the SsoL12gene showed that no point mutation had occurred during the cloning procedures and the ATA codon was still present. Further results about this findingwill be published elsewhere (Kopke and Leggatt, 1991). The NH,-terminal methionine coded in the SsoL12MN1 gene wasnot found inthesequence of the overexpressed proteinmutant.Insteadthisproteinstarted with anunblocked NH,-terminalalanine, which is the second coded amino acid, and continuescoded as by the gene. This indicates that the protein wasprocessed by the E. coli enzyme that removes NH,-terminalmethionines. The SsoL12MN2mutant protein was processed in the same manner. When 1.6 nmol of SsoL12MN2 were sequenced, alanine was found as thefirstamino acid(coded by the secondcodon of this artificial gene) and no otherresidues were observed. Selective Extraction of SsoLl2 from Total Ribosomes of S . solfataricus-Ribosomal coreslackingthe SsoL12 protein were produced as described under “Materials and Methods,” similar to Hamel etaf. (1972) and Casiano et al. (1990). I t was found that use of high salt concentrations removed many additional proteins, possibly from the 30 S subunit of the S. solfataricus ribosome (data not shown). The two-dimensional gel pattern of ribosomal core particles derived from the extraction procedure described under “Materials and Methods” showed no SsoL12 protein spotwhen loaded with the appropriate amount of total ribosomal proteins (Fig. 6). However, when the two-dimensional gels are heavily overloaded (double concentration) the SsoLl2 protein spotis visible at low concentrations. This finding suggests that most of the protein has been extracted by the describedprocedure. A second protein spot, referred to as “A” in the original two-dimensional polyacrylamide gel nomenclature by Schmid and Bijck (1982), was also present in the ethanol extracts. This protein was described as not always present in the ribosomal preparations of S. solfataricus. The proteinwas purified by reversedphase HPLC, NH, terminally sequenced, and identified by sequencesimilarityasanarchaebacterial flagellin protein (data not shown). Incorporation of Overexpressed Ribosomal Protein SsoLl2 and Its Deletion Mutants in S. solfataricus Ribosomal Cores and the Suggested Ribosomal-binding Site-Ribosomal core particles were incubated with theoverexpressed SsoL12 protein. The reconstituted particleswere purified through a high salt sucrose cushion to separate the unbound or loosely attached proteins from the ribosomal particles (see “Materials and Methods”). The ribosomal pellet was analyzed by twodimensional polyacrylamide gel electrophoresis. TheSsoL12’ proteinspotis clearly visible in thetwo-dimensional gel pattern of these reconstituted ribosomes (Fig. 6). In addition, the mutant proteins SsoL12MC1 and SsoL12MC2(Fig. 6)
Ribosomal Stalk Proteins of Archaebacteria
.~
1387
111
FIG. 6. Analysis of S . solfataricus ribosomal proteins on two-dimensional polyacrylamide gels. First dimension from left to right, second dimension from top to hottom. The total proteins of ahout 5 A,,,, of 7 ) S were loaded on to mini two-dimensional acrylamide gels (Rio-Rad Mini Protean 11). The SsoL12 and the overexprenned. purified and reconstitutedprotein mutants are indicated by arrows. The lack of Sso1,l'L in ethanol-treated rihosomes is indicated by the brackets.
were found reconstituted into ribosomal particles following observed in two-dimensional polyacrylamide gels. The intensity of the spots of the incorporated protein was the described procedure. somewhat different for the different mutants and within the SsoL12MN1 was found once in these particles when an extremely high excess of this protein was used for the recon- various preparations, but all incorporated proteins were restitution.Thisresultwasnot reproducible, so that Sso- producibly ohserved in multiple (more than three) reconstiLl2MNl cannot be regarded as a core-binding protein. It is tution experiments. Summarized, these results show that the possible that part of theproteinprecipitatedinthisone SsoNH2terminallytruncatedmutants,SsoL12MNIand experiment due to its high concentration.Mutantprotein L12MN2, are not capahle of binding to the core particles, SsoL12MN2 was always absent in the two-dimensional gel while theCOOH-terminal-truncatedmutants were hound. patterns of reconstituted core particles incubated with this ThefindingthatSsoL12MC2can bind to ribosomes and apparently dimerizes suggests that an amino acid sequence mutant. necessary for rihosome binding and dimerizationis located in SsoL12MCl was found as a clear spot in the two-dimensional-gelpatterns of thereconstitutedcoreparticles. As the first 70 residues of the archaehacterial protein (theregion already described for the pure SsoL12MCl protein, an extra covered by SsoL12MC2). The inahility of SsoLl2MN1 and a lesser intensitywas SsoL12MN2 to bind to ribosomal core particles supports that spot adjacent to the main spot but with also found for the reconstituted protein. The two-dimensionalobservation andsuggests that the first12 amino acids missing polyacrylamide gels of the particles reconstituted with SsoL12in SsoL12MN1 are already essentialfor rihosome hinding or MC2 show two extra spots when compared to the pattern of proper protein folding. It is possible that, in addition to the deleted regions (1-46), residues 46-70 are also required for the ribosomal cores. The top spot is likely representing a SsoL12MC2dimer while thebottomspotmight be the rihosome binding, since theseresidues were never deleted and SsoL12MC2 monomer. Intwo-dimensional polyacrylamide are present in SsoL12MC2 which was successfully reconstigels of pure (as proven by amino acid analysis) SsoL12MC2 tuted into rihosomal cores and dimerized. Thus, the NH,mutant protein the same two spots are visible (not shown), terminal partof the protein SsoLl2is essential andsufficient suggesting partial stabilityas a dimer under these conditions for ribosome binding. (pH 4 , 6 M urea). Thus, the mutant lacking the entire COOH- As a comparison, theE. coli L12 rihosome-hinding sitewas terminal region appearsto produce dimers that are more located by Gudkov et 01. (1980) hetween residues 1 and 74. A stable than the intact L12 protein, where no dimers were fragment comprising these residues was ahle to hind to riho-
1388
Ribosomal Stalk Proteins of Archaebacteria
somes depleted of EcoL12, dimerized in solution and competed with the original EcoL12 protein, while a fragment of theprotein from residue 27 to 120 could notrestorethe functional activity of these core ribosomes, did not dimerize and could not compete with the entire EcoLl2 for its ribosome-binding site (Gudkov et al., 1980). Therefore, the archaebacterial/eukaryotic and eubacterial L12 proteins seem to share the location of their ribosomal binding regions and dimerizationsite. Functionalprimary sequences in this region therefore seem to be of great variability. FIG.I. Activity of 5 ' . solfataricus native and reconstituted Poly(U) Activity of the Reconstituted Ribosomes-The activ- ribosomes in the poly(U) assay. Data were collected and reconity testswere found to he very sensitive to higher monovalent firmed in multiple independentexperimentseachcontainingthe ion concentrationsas previously reported (Londei et ul., 1986). controls as an internal standards. The dataset shown derived from This sensitivity prevented the direct use of the reconstituted one duplicate experiment for each setup with the error bars repreribosomes made for two-dimensional acrylamide gel analysis, senting the differencebetween these twovalues. The background since they were precipitated in high salt cushion buffer. In activity for the assay without ribosomes of about, 1000 cpm was not substracted from these values. Abbreviations: IZibos, untreated riboaddition slight changes in the pH of the assay buffer resulted somes; Cores, ethanolextracted ribosomes; L12, ethanol-extracted in total loss of activity. The introduction of HEPES buffer, ribosomes reconstituted withSsoL12'; N1, ethanol-extracted ribothe optimization of the S150 fraction concentration and tRNAsomes reconstituted with SsoL12MNl; N2, ethanol-extracted riboconcentration and the dialysis of pure mutant proteins againstsomes reconstituted withSsoL12MN2; C I , ethanol-extracted ribowater resulted in reliable translational activity. About 35,000 somes reconstituted with SsoL12MCl; C2, ethanol-extracted ribosomes reconstituted with SsoL12MC2. cpm were found per 0.2 A,,, of S. solfataricusribosomes utilizing the assay conditions given under"Materialsand Methods." For up to 0.5 A,,,,of ribosomes/50 pl of assay buffer, the activity had a linear correlation to the amountof ribosomes (results not shown). Theactivity experiments were always prepared induplicate. The variation within one experiment was usually less than 5%. The variation between replications of activity determinations a t different times was up to 30%, although identical ribosome preparations and assay conditions were used. Therefore, every set of experiments included the desired controls to set the activity values into relation.Whenthe specific values were compared tothe FIG.8. Activity of S . solfataricus reconstituted ribosomes standards in the same set of experiments the variation bein the poly(U) assay.The core particles were first incubated for 15 tween experiments was less than 10%. min a t 60 "C with the mutant protein to reconstitute intoribosome the Ethanol-extracted ribosomal core particles have a residual and this was followed by incubation for further 15 minwith an equal (SsoL12"). Data were collected and translational activity of about 40%. This had been expected amount of theentireprotein since the extraction of SsoLl2 from the ribosomal cores is reconfirmed in multiple independent. experiments each covering core not complete as determined by overloaded two-dimensional particles and SsoL12" reconstitutions as an internal standard. The error burs represent the estimated accuracy of the specific values polyacrylamide gels. In addition, thecytosolic pool of SsoL12 when multiple results were compared. Abbreviations: Cores, ethanolpresent in the S150 fraction which is added to the transla- extracted ribosomes; L12, ethanol-extracted ribosomes reconstituted tional assay will reconstituteinto ribosomes and partially with SsoL12'; NI/L12, ethanol-extracted ribosomes incubated with SsoL12MN1 first and subsequently with SsoL12'; N2/L12, ethanolrestore activity. The overexpressed entire SsoL12" protein can restore the extracted ribosomes incubated withSsoL12MN2 firstand subsequently with SsoLl2'; C l / L f 2, ethanol-extracted ribosomes incubated activity of ribosomal cores (Fig. 7), proving that the overex- withSsoL12MClfirstand subsequentlywith SsoL12'; C2/L12, pression, purification, and reconstitutionof the entire protein ethanol-extracted ribosomes incubated withSsoLlBMC2 firstand yields an active ribosomal constituent. In contrast, none of subsequently with Ss0L12~. the overexpressed mutant proteins were able to restore the ribosomal core activity toany significant degree (Fig. 7). with this mutant proteinwere not pursued further. Based onthe knowledge of the L12 proteinfunctionsin Interestingly,the COOH terminallytruncatedmutants ribosome binding and factor interaction in E. coli two conclu- which were found to bind to ribosomal cores in the reconstisions can be drawn: 1) the deficiency of ribosome binding tution experimentscould not significantly prevent thehinding found for the NH2 terminally truncated mutants prevents of the SsoL12" protein. In experimentswhere the core partitheir action in protein synthesis;2) for the COOH-terminally cles were first incubated with the inactive COOH-terminal truncated mutants, the factor interaction domain which is truncated mutants and then the SsoLlae proteinwas added, essential for ribosomal activity is likely missing or distorted. the cores were restored to almost full translational activity This shows that the entire molecule is needed for ribosomal (Fig. 8).A possible reason for the lack of competition between activity. the binding of the L12 mutants and the intact protein might Preliminary reconstitution experiments with SsoL12MC3 bea steady exchange of L12 molecules occurring on the indicated that this protein was reconstituted into ribosomal archaebacterial ribosomes in which the affinity of factor cores but showed no translational activity as already shown bound L12 dimers for the ribosomal-binding site might be for the much smaller SsoL12MC2. Therefore, these results significantly higher than that of "empty" dimers. This would provided no additional or significant information since the be quite distinct from the situationin E. coli where an NH2protein sequences in SsoL12MC3 additional to SsoL12MC2 terminal fragment (residue 1-74) was able to compete with the intact protein. Recent resultsfrom yeast ribosomes indimight simply be misfolded; consequently, the experiments
Ribosomal Proteins Stalk cate that there is an extensive exchange of the various L12 protein versions on the yeast ribosome (Tsurugi and Ogata, 1985). It is possible that the mechanism of L12 protein exchange on the ribosome is important for the translational process and is differentineubacteria and archaebacteria/ eukaryotes. The role of the highly charged COOH-terminal regions of the archaebacterial and eukaryotic L10 and L12 proteins have to be investigated further. The truncation of these sequences (SsoL12MC3) resulted in atotal lack of activity in preliminary experiments, which could be due to the lack of this essential region, or a different folding of the residual protein. This region is of great interest since it appears to be the only major structural difference between the L12 proteins from the eubacterial group and theL12 proteins from the archaebacterial and eukaryotic group. Activity of Ribosomal L12 Proteins and TheirMutants, Overexpressed in E. coli, and Purified by HPLC-Ribosomal L12 proteins from different archaebacterialorganisms (Kopke et al.,1991) as well as the SsoLl2protein mutants presented in thispaper have been overexpressed in E. coli and purified on reverse-phase HPLC. These proteins are active as ribosomal constituentsafter renaturationin an appropriate buffer a t temperatures optimal for their host organism. This means that neither their small modifications in the E. coli host, e.g. the artificial NHp-terminal acetylationfor the MvaL12" protein (Kopke et al., 1990), nor their purification utilizing 2propanol or acetonitrile as theelution buffer for the reversedphase chromatography, has destroyed their ability to refold into theirfunctional tertiary structure.
of Archaebacteria
1389
similarity of these proteinsthat suggested a rearrangementof the L12 proteins duringevolution, so that theCOOH-terminal region of the eubacterial sequences becomes the NHp-terminal region of the eukaryotic and archaebacterial sequences, or models proposing agreatshift between the NHp-terminal regions of the two L12 protein types, are incompatible with our results. A thorough investigation of the structural and functional requirementsfor the L12 proteins seems necessary to completely understand itsrole in the translationalprocess. With thehelp of x-ray crystallography, or two-dimensional NMR, it may also become clear whether the different primary sequences fold into similar tertiary structures. For the aspect of tertiary structurepredictions it seems of general interest if these two different amino acid sequences fold into similar three-dimensional structures.These results might also answer the question why the L12 proteins, which have an essential function in the translationalprocess, have changed so drastically during evolution. Acknowledgments-We thank SandyKielland for protein sequencing and aminoacid analysis and Isolde Gunther for DNA sequencing. The supply of updated information regarding the poly(U) activity tests by Dr. Paloa Londei is gratefully acknowledged. We thank Dr. B. Wittmann-Liebold for all the help and encouragement we received. REFERENCES
Agthoven, A. J., van Maassen, J. A., Schrier, P. I., and Moller, W. (1975) Biophys. Biochem. Res. Commun. 6 4 , 1184-1191 Amons, R., Pluijms, W., and Moller, W. (1979) FEBS Lett. 104,8589 Aquist, J., and Tapia, 0.(1990) Biopolymers 30, 205-209 Beauclerk, A. A. D., Cundliffe, E., and Dijk, J. (1983) J . Bid. Chem. 259,6559-6563 CONCLUSIONS Boublik, M., Visentin, L. P., Weissbach, H., and Brot,N. (1979) Arch. Biophys. Biochem. 198,53-59 In summary, our results suggest that the ribosome-binding siteand dimerization site ofL12 proteins from all three Bullock, W. O., Fernandez, J. M., andShort, J. M. (1987) BioTechniques 5 , 376-379 kingdoms are in comparable locations in the NHp-terminal Casiano, C., Matheson A. T., and Traut, R. R. (1990) J. Biol. Chem. part of the L12 protein sequence, even though they have 265,18757-18761 undergone drastic changes in their amino acid sequences in Fling, S. P., and Gregerson, D. S. (1986) Anal. Biochem. 155,83-88 this region of the protein. The finding that four copies of Geyl, D., Bijck, A.R., and Isono, K. (1981) Mol. Gen. Genet. 1 8 1 , 309-312 SsoL12 bind to SsoLlO (Casiano et al., 1990) is identical to former results that four copies of EcoLl2 bind to EcoLlO Gudkov, A. T., Tumanova, L. G., Gongadze, G. M., and Bushuev, V. N. (1980) FEBS Lett. 1 0 9 , 34-38 (where two EcoL12 copies are NHp terminally acetylated). Hamel, E., Koka, M., and Nakamoto, T. (1972) J. Biol. Chem. 247, The ribosomal binding and dimerization of EcoL7/L12 is 805-814 thought to occur through hydrophobic interactions of the Itoh, T. (1981) Biochim. Biophy~.Acta 6 7 1 , 16-24 EcoL7/L12 protein with EcoLlO (Gudkov et al., 1980). It is Kopke, A. K. E., and Leggatt, P. A. (1991) Nucleic Acids Res. 1 9 , 5169-5172 noticeable that theNH2-terminal half of archaebacterial and eukaryotic L12 proteins comprises a hydrophobic region in Kopke, A. K. E., and Wittmann-Liebold, B. (1989) Can. J. Microbiol. 35,ll-20 the general hydrophilic L12 proteins (see hydrophobicity plot Kopke, A. K. E, Baier, G., and Wittmann-Liebold, B. (1989) FEBS in Strobe1 et al., 1988). The recently suggested bilateral hyLett. 2 4 7 , 167- 172 drophobic zipper in thevarious L12 protein versions of yeast Kopke, A. K. E, Paulke, C., and Gewitz, H. S. (1990) J. Biol. Chem. 265,6436-6440 (Tsurugi and Mitsui, 1991) can be predicted for all archaebacterial and eukaryotic L12 proteins from multiple align- Kopke, A. K. E., Hannemann, F., and Boeckh, T. (1991) Biochimie 73,647-655 ments in the NHp-terminal region. Leijonmarck, M., and Liljas, A. (1987) J. Mol. Biol. 1 9 6 , 555-580 The hinge region seems to be a conserved property of all Liljas, A. (1991) Znt. Reu. Cytol. 1 2 4 , 103-136 L12 proteins (Liljas, 1991). The high alanine content as well Liljas, A., Kirsebom, L. A., and Leijonmark, M. (1986a) in Structure, as the clustering of the rare prolines in this area are found Function, and Genetics of Ribosomes (Hardesty, B., and Kramer, G., eds) pp. 379-390, Springer Verlag, Berlin, Heidelberg for L12 proteins from all organisms. The hinge region is thought to enable a high flexibility of the L12 dimers in this Liljas, A., Thirup, S., and Matheson, A. T. (1986b) Chemica Scripta 2 6 B , 109- 119 region in order to accomplish conformational changes during Lin, A., Wittmann-Liebold, B., McNally, J., and Wool, I. G. (1982) the translational process. Since it is a conserved element of J. Biol. Chem. 2 5 7 , 9189-9197 all L12 proteins it appears vital for L12 protein function. Londei, P., Teixido, J., Acca, M., Cammarano, P., and Amils, R. (1986) Nucleic Acids Res. 1 4 , 2269-2285 The factor interactingdomain is likely located at the COOH-terminal region of the archaebacterial protein since Matheson, A. T. (1985) in The Bacteria VZZZ:Archaebacteria (Woese, C. R., and Wolfe, R. S., eds) pp. 345-377, Academic Press, New even small COOH-terminaltruncations (SsoL12MCl) totally York inhibit translationalactivity, although these mutantproteins Matheson, A. T., Auer, J., Ramirez, C., and Bock, A. (1990) in The were still found reconstituted into ribosomes. Ribosome: Structure, Function and Evolution (Hill, W. E., Dahlberg, As a consequence, former models based on proteinsequence A., Garrett, R. A, Moore, P. B., Schlessinger, D., and Warner, J.
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Schmid, G., and Bock, A. (1982) Mol. Gen. Genet. 185,498-501 Strobel, O., Kopke, A. K. E., Kamp, R. M., B&k, A., and WittmannLiebold, B. (1988) J. Biol. Chem. 2 6 3 , 6538-6546 Studier, F. W., and Moffatt, B. A. (1986) J.Mol. Bwl. 189, 113-130 Tabor, S., and Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,1074- 1078 Terhorst, C., Moller, W., Laurson, R. A., Wittmann-Liebold, B. (1973) Eur. J. Bwchem. 3 4 , 138-152 Tsurugi, K., and Mitsui, K. (1991) Biochem. Biophys. Res. Commun. 174,1318-1323 Tsurugi, K., and Ogata, K. (1985) J. Biochem. (Tokyo) 9 8 , 14271431 Warner, J. R. (1989) Microbiol. Rev. 53,256-271 Wittmann-Liebold, B. (1986) in Structure, Function and Genetics of Ribosomes (Hardesty, B., and Kramer, G., eds) pp. 326-361, Springer-Verlag, New York Wittmann-Liebold, B., Kopke, A. K. E., Arndt, E., Kromer, W., Hatakeyama, T., and Wittmann, H. G. (1990) in The Ribosome: Structure, Function and Evolution (Hill, W. E., Dahlberg, A., Garrett, R. A, Moore, P. B., Schlessinger, D., Warner, J. R., eds) pp. 598-616, American Society for Microbiology, Washington, D. C. Yaguchi, M., Matheson, A. T., Visentin, L. P., and Zucker, M. (1980) in Genetics and Evolution ofRNA Polymerase, tRNA and Ribosomes (Osawa, S., Ozeki, H., Uchida, H., and Yura, T., eds) pp. 585-599, University of Tokyo Press, Tokyo Zillig, W., Klenk, H.-P., Palm, P., Puhler, G., Gropp, F., Garrett, R. A., and Leffers, H. (1989) Can. J. Microbiol. 3 5 , 73-80
