Eur. J . Hiocheni. "3 501 -509 (1978)

The 5-S RNA - Protein Complex from an Extreme Halophile, Halobacterium cutirubrum Purification and Characterization

Nancy SMITH. Alastair T. MATHESON. Makoto YAGUCHI, Gordon E. WILLICK, and Ross N . NAZAR

Division of Biological Sciences, National Research Council of Canada, Ottawa

(Received Dcccniber 5 . 1977, March 6, 1978)

A 5-S RNA . protein complex has been isolated from the 50-S ribosomal subunit of an extreme halophile, Hulobacterium cutirubrum. The 50-S ribosomal subunit from the extreme halophile requires 3.4 M K' and 100 mM Mg" for stability. However, if the high K' concentration is maintained but the Mg2' concentration lowered to 0.3 mM, the 5-S RNA . protein complex is selectively extracted from the subunit. After being purified on an Agarose 0.5-m column the complex had a molecular weight of about 80000 and contained 5-S RNA and two proteins, HL13 and HL19, with molecular weights (by sedimentation equilibrium) of 18 700 and 18000, respectively. No ATPase or GTPase activity could be detected in the 5-S RNA . protein complex. The amino acid composition and electrophoretic mobility on polyacrylamidc gels indicated both proteins were much more acidic than the equivalent from Escherichia coli or Bacillus steurotliermoI)hilus. Partial amino acid sequence data suggest HL13 is homologous to EL18 and HL19 to EL5.

The 5-S RNA, a component of the 50-S ribosomal subunit of all prokaryotes, is believed to play a key role in the structure and function of the ribosome. Studies by Erdmann and his co-workers (see [1] for review) have indicated that the 5-S RNA forms part of the aminoacyl-tRNA binding site on the ribosome specifically interacting with the T-Y-C-G sequence in the tRNA through a G-A-A-C nucleotide sequence. The 5-S RNA is also thought to be involved in the translocation step in protein synthesis [2].

In Eschericliiu coli, two proteins, ELI8 and EL25, specifically bind to the 5-S RNA [3] while in the therniophile Bacillus stcarotlicrrnc~~~hilus BL5 and BL22 have a similar role [3]. There is also evidence in E. coli that a third protein EL5 is also part of the 5-S RNA . protein complex [4]. The sequence of the 5-S RNA from a large number of prokaryotes has been determined [5] and the complete amino acid sequences of the ribosomal proteins EL5 [ 6 ] , EL18 [7] and EL25 [8,9] are now known. .. -~

This papcr is NRCC publication No. 16917. Some of thc ex- periments reportcd in this paper are from the thesis of N.S. sub- mittcd to thc Dcpartment of Biology. Carleton LJnivrrsity in piirlial fulfillment of thc rcquircmcnt of thc M. Sc. degrcc.

Abhreik7rion. Buffcr Tris/K/Mg, buffcr containing Tris, K' and Mg4..

Dc/irririon. A AZ6() unit, the quantity of material conpained in 1 nil of a solution which has an absorbance of 1 at 260 nin, when measured in a I-cm pathlength ccll.

Not only is the 5-S RNA . protein complex o f interest in rcgard to its role in protein synthesis but i t is also, because of its size, an ideal model system to study the specific interactions between RNA and protein. As part of an investigation into the properties of the 5-S RNA . protein complexes from bacteria that grow under extreme conditions we have been studying the 5-S RNA . protein complex from the extreme halo- phile Halobacterium cutiruhrum. The ribosomes from this bacterium have several unusual properties. They require a high salt concentration (3.4 M K ' and 0.1 M Mg") for optimal stability and the ribosomal proteins from this extreme halophile are much more acidic than those from other bacteria [lo, 1 I ] . Because of these unusual properties it was of interest to isolate and characterize the 5-S RNA . protein complex from the extreme halophile and compare its properties to those of the equivalent complex in E. coli and B. stearo- thermopliilus.

Most of the studies on the 5-S RNA complexes from other prokaryotes have been carried out on complexes that have been rcconstituted from the purified RNA and proteins [l]. However, since pro- teins isolated from the extreme halophile are often irreversibly denatured at the low ionic conditions used during purification [32], it was questionable whether a reconstituted 5-S RNA complex could be obtained from the extreme halophile. In addition, it

502 Ribosoiiial 5-S R N A . Protein Complex: Purification and Characterization

was felt that a reconstituted complex might show properties that differ from those of the native 5-S RNA complex. This paper, therefore, describes the isolation and characterization of a native 5-S RNA . protein complex from the extreme halophile H. cuti- rubrum.

MATERIALS AND METHODS

Isolation of 50-S Subunits

H . curirubruni cells were grown in the complex medium of Seghal and Gibbons [13] in a 120-1 fer- menter with vigorous aeration of 42 C and harvested in mid-log phase growth. The isolation of the 70-S ribosomes [ 1 1 ] and the separation of the ribosomal subunits 1141 were as previously described and the purity of the 50-S ribosomal subunit was verified on a Spinco model E analytical ultracentrifuge.

Extruction and Purifi'cution of the Native 5-S R N A . Protein Complex

The SO-S ribosomal subunit of H . cutirubrum is stable in buffers containing 3.4 M K4- and 100 mM Mg2'~ [lo]. Previous studies [14] had indicated that considerable 5-S RNA and protein were released when the SO-S subunit was extracted with buffers con- taining low K': bufferA = 50 niM KCI, 100mM MgC12, 10 mM Tris-HCI pH 7.6, 6 mM 2-mercapto- ethanol; low Mg".: buffer B = 3.4 M KC1, 0.3 mM MgCI2, 10 mM Tris-CI pH 7.6, 6 mM 2-mercapto- ethanol or buffer C = 3.4 M KCI, 1 mM sodium EDTA, 10 mM Tris-C1 pH 7.6, 6 mM 2-mercapto- ethanol; low K'. and low Mg": buffer Tris/K/Mg = 50 mM KCl, 10 mM MgC12, 10 mM Tris-C1 pH 7.6, 6 mM 2-mercaptoethanol. To determine whether the 5-S RNA was released as a complex or as naked RNA, the 50-S subunits (5 mg/ml) were dialysed against the appropriate buffer (three changes of 6 1). The suspension was centrifuged for 24 h at 165000 x g to remove the 23-S RNA-core particle and 10 ml of the supernatant, containing about 1500 Az60 units, was applied to an Agarose 0.5-m column (2.5 x 90 cm). The various components present in the supernatant were eluted from the column with the same buffer used for the initial extraction. The flow rate was 12 ml/h and 5-ml samples were collected. RNA was detected by scanning the eluate at 260 nm on a spec- trophotometer and the RNA species was identified by electrophoresis on polyacrylamide gels [15]. Pro- tein was detected and partially identified by poly- acrylamide gel electrophoresis, pH 8.7 [ l l ] or by sodium dodecyl sulphate gel electrophoresis [I61 of the various fractions. The column was calibrated for molecular weight determinations using 5-S RNA and the monomer and dimer of bovine serum albumin as markers. In each experiment a trace of 32P-labelled

5-S RNA, usually from yeast, was used as a marker for 5-S RNA.

Identification of the 5-S RNA-Binding Proteins

The proteins present in the 5-S RNA . protein complex were identified by two-dimensional poly- acrylamide gel electrophoresis using the Strom and Visentin system [17] for acidic ribosomal proteins and the Kaltschmidt and Wittmann system [IS].

ATPase and GTPase Activity of the 5-S R N A . Protrin Comp1t.s

ATPase and GTPase activities were determined by a modification of the method of Gaunt-Klopfer and Erdmann 1191. Freshly prepared 5-S RNA . protein complex (0.15 Azho unit) was incubated at 42 C in a buffer containing 3.2 M KC1, 0.7 M MgC12, 0.25 M NH4C1, 0.01 M Tris-HCI at pH 7.6 in the presence of either 0.12 mM [1'-32P]ATP (specific activity S Ci/ mol) or 0.027 mM [1'-32P]GTP (specific activity S Ci/ ymol) purchased from New England Nuclear.

Purification of the 5-S-Binding Proteins

Initially the 5-S RNA . protein complex from several Agarose runs were pooled, dialysed against Tris/urea buffer (6 M urea, 0.01 M Tris-HCI, pH 6.0, 9 mM methylamine and 0.1 mM dithiothreitol) and concentrated on a Diaflo filter (UM2). The sample was titrated to pH 8.0 with solid Tris just prior to its addition to a DEAE-cellulose column (DE-52, Whatman, 1.5 x 90 cm) previously equilibrated with the pH-8.0 buffer. The proteins were eluted with the Tris/urea buffer, pH 8.0, using a linear KCl gradient of 0-0.3 M. The flow rate was 20 ml/h and 10-ml samples were collected. The proteins were detected by scanning the column fractions at 230 nm and their location and purity verified by polyacrylamide gel electrophoresis.

For large-scale purification, the 50-S ribosomal subunits were extracted with buffer A and the extract, containing the two binding proteins, was fractionated on DEAE-cellulose as described above.

C'liuructerizutioti of tlir ~ - S - R N A - B I I I L I I I I ~ Pi~)tc>iri.\

The molecular weights of the 5-S-RNA-binding proteins were determined by sodium dodecyl sulphate/ polyacrylamide gel electrophoresis 1161 and by sedi- mentation equilibrium. For the gel electrophoresis, lysozyme, myoglobin (monomer and dimer), chymo- trypsin, ovalbumin and bovine serum albumin were used as the calibration proteins. The sedimentation equilibrium experiment were carried out with a modi- fied meniscus-depletion technique 1201 in 0.5 M KCl, 0.01 M magnesium acetate, 0.01 M Tris, pH 7.4 and 0.01 M 2-mercaptoethanol.

N. Smith. A. T. Mathcson. M. Yaguchi, G . E. Willick. and R. N. T

0.9

0.8

0.7

6 0.6 z z 0 . 5 s: a a 0.4

0.3

0.2

503

-

-

-

-

-

-

-

-

1 2

1 1 I I , J O 100 200 300 400

0.1 ' Volume (ml)

Fig. I . / .wh/ ioi i 0 / 5 - S R N A . protc+i cor~~pl,les,fkom H. cutirubrurn. Thc 5 0 4 ribosomal subunits were extracted with buffer D. the 50-S core rcmovcd by centrifugation and the supernatant was fraction- ated on a n Agarose 0.5-m column (25 x 90 cm) at a flow rate of 12 ml!h; 5-nil fractions were collected. (A-A) Absorbance at 260 nm; (.-----a) 3zP-labelled 5-S RNA marker from yeast

The amino acid analysis was determined on a Durrum D-500 amino acid analyzer. The protein fractions were hydrolysed for 20 or 70 h at 110 C in 6 M HC1. Tryptophan was estimated by the method of Edelhoch [21] using 6-M guanidine hydrochloride.

The first 36 residues of HL13 and HL19 were automatically degraded [22] with a Beckman model 890C protein sequenator with a 0.5-M quadrol pro- gram (no. 122974). The thidzolinone or phenylthio- hydantoin derivatives were hydrolyzed with 6 M HCI in the presence or absence of 0.1 SnClz [23] at 130 C for 20 h, and the amino acids formed were analyzed with the Durrum D-500 amino acid analyzer. The identification of some phenylthiohydantoin de- rivatives (Asp, Asn, Glu, Gln) was made by thin-layer chromatography on silica gel plates [24,25].

RESULTS

Extruction q f u Native 5-S RNA . Protein Complex

When thc 50-S ribosomal subunit was extracted with buffers containing a high K' concentration and a low or zero concentration of Mg" (buffers B and C), the 5-S RNA was eluted as a complex with a rnolec- ular weight of about 80000 on an Agarose 0.5-m column as shown in Fig. 1. The initial peak of ab- sorbance at 260 nm, in the breakthrough volume, was composed of the 50-S ribosomal core particle while the main peak (eluted around 290 mi) contained only

A Fig. 2. I(len!~/ication of' R N A atid pro!(,in c o n q ~ o t i ~ ~ n i , \ in cotnples isolcued on an Agarosv 0.5-m column. (A) Electrophvresia of R N A sample on an 8Y.i polyacrylaniide gel at 35 mA for 3.5 h. The gel was stained with methylene blue. ( I ) 5-S marker from H . cufiru- hnm. (2) Fraction from Agarose 0.5-m column (Fig. 1) containing complex. (B) Dodecyl sulphateipolyacrylamide gel clcctrophorcsis of complex isolated on an Agarose 0.5-m column

5-S RNA (Fig.2A) as well as two proteins dctccted on dodecyl sulphate gels (Fig. 2B) with molecular weights estimated as 24800 and 21 600 from these same gels.

When the ribosomes were extracted with buffers containing low concentrations of K' (buffers A or Tris/K/Mg), the 5-S RNA was eluted from the Agarose column in the same location as the 5-S RNA marker, indicating the absence of a complex under these extraction conditions.

Identification of the 5-S- RNA-Binding Proteins

When the proteins present in the 5-S RNA ' protein complex were separated on the two-dimensional poly- acrylamide gel system of Strdm and Visentin [I71 two major proteins, HL13 and HL19, were detected (Fig. 3). In addition traces of HL20, which is thought to be equivalent to E. coli L7/L12, and an unidentified pro- tein (possibly L3/4) were also present in the complex fraction in small amounts which varied somewhat depending upon the preparation.

Further evidence that HL13 and HL19 were part of the 5-S RNA . protein complex is shown in Fig. 4. In this experiment the proteins in each fraction from

504 Ribosomal 5-S R N A . Prolein Complex: Purification and Characterization

the Agarose column were separated and identified on dodecyl sulphate/polyacrylamide gels. The profiles of HL13 and HL19 completely parallel the 5-S RNA profile and are the only ribosomal proteins to show this property.

ATPase und GTPase Activity of5-S R N A . Protein Complex

Repeated attempts to detect GTPase activity in the purified native 5-S RNA . protein complex of H . cutii~ibrum were unsuccessful (Fig. 5) , although GTPase

Fig. 3. ItkmtiJicarion q/ prolrins in the 5-S R N A . prorein c u n i p l c ~ in H. cutirubrum using ta.o-dinien.siorial gel eltctroplinre.sis [ 171. (A) Total proteins of H . cutiruhrum 5 0 3 ribosomal subunit. (B) Proteins present in 5-S RNA . protein complex (Strom-Viben- tin system). (C) Proteins present in 5 - S RNA . protein complcx (Kaltschmidt-Willinann system). A purified sample of EL7/EL12 was included as a marker

activity could be detected in the cytosol fraction. The 50-S ribosomal subunits were also inactive but ad- dition of the subunits to the cytosol fraction greatly enhanced the GTPase activity. When the 5-S RNA . protein complex was added to the cytosol fraction it had no effect.

Increased concentrations of the complex, changes in Mg2' and NH,' concentrations and isolation of the ribosomes at various stages of growth all gave 5-S RNA . protein complex samples with no GTPase activity.

Similar results were obtained when the samples were assayed for ATPase activity (results not shown). The cytosol fraction showed a great deal of activity but no ATPase activity could be detected in the isolated complex.

Pur$cution of'HL13 und HLlY

The 5-S RNA-binding proteins were fractionated on a DEAE-cellulose column as shown in Fig.6. Protein HL19 was eluted with 80mM KCI while HL13 was eluted with 85 mM KCI. These were the only proteins eluted from the DEAE-cellulose column. As was shown earlier by two-dimensional gel electro-

N. Smith, A. T. Matheson, M. Yaguchi. G . E. Willick. and R. N. Nazar 505

i'\ 1 .o

0.8 6 ._ c

L c c

0.6 0

c a, .- c

0.4 g a, > .- - -

0.2 d

0

Fraction number

Fig. 4. /rlcri/; / ic, trt io,7 of rlw protc4ris prr.rcwt iri / lw S-.S R;VA pro/rin coniplc~.~ eluted jrom the Agtrrose 0.5-m colunin. The absorbance at 260nm was determincd for each fraction which was then dialysed and concentrated prior lo fractionation on dodecyl sulphate gels for identification. After staining with Coomassie blue the gels were scanned a t 570 nm on a Gilford model 240 spectro- photometer equipped with a model 2140 linear transport. Thc amount of each protein was determined by the area under the tracing and thc niiixiniuni conccntration (ix. tube 53) was set at 1.0. (0- 0 ) AhSorhoIlcc 111 260 IIIII. ( A ~ A) HLI3, (0-- -0) HL19, (V - a) 1-1120, (a- ---0) HL3:4('!)

phoresis, thesewere the onlymajor protein components found in the 5-S RNA . protein complex isolated on the Agarose 0.5-m column.

Since the amount of protein obtained from the complex isolated on the Agarose 0.5-m column was small, a method to obtain large amounts of these proteins for characterization studies was required. It was found that extraction of the 50-S ribosomes with buffer A released almost all the HL3 and HL19 along with additional 5 0 4 proteins [14]. These proteins were fractionated on DEAE-cellulose under con- ditions that resulted in the separation of HL13 and HL19 from the other proteins in the extraction.

Molecular Weights qf'HL13 and HL19

The molecular weights of HL13 and HL19 were found to 24800 and 21 600, respectively, by dodecyl sulphate gel electrophoresis and 18 700 and 18 000 by sedimentation equilibrium. Although these proteins were difficult to dissolve from the freeze-dried state, they were quite stable in solution. The ultracentrifugal data indicated no molecular weight heterogeneity; the In c vs r2 plots were linear. In addition, the proteins were mixed in equimolar amounts to check for inter-

u 15 30 45 €0

Time (min) OO

Fig. 5. G'17'nse w / i v i / j . cJf'vnrious ccllu/cr, .f ,crr. i io,u o f H . cutirubrum. The samples contained 0.027 mM [i.-32P]GTP (specific activity 5 Cilmol) in a total volume of 500 pI in a buffer containing 3.2 M KCI, 7 0 m M MgCl2, 250mM NHeCl and 1 0 m M Tris, pH 7.6. Aliquots (100 pl) were removed a t intervals and assayed for in- organic [32P]phosphare as described in Methods. (0 --0)0.15 A2h0

unit of 5-S RNA prolein complex, 10 AZ6" units of 50-S ribo- somal subunit or the control (buffer + GTP alone) gave similar results; (0-j 25 pl cytosol: (A-A) 25 pl cytosol + 0.15 Albo unit of complex: (0 -0) 25 pl cytosol + 10 ,4260 units of 50-S subunits

action between them. There was no indication of this, thus eliminating one potential further complicating interaction in this system.

Amino-Acid Composition of HLl3 and HLlY

The amino acid composilions of HL13 and HL19 are shown in Table 1. Since the composition of HL13 is distinctly different from HL19, they are two differ- ent polypeptides. The amount of alanine and tyrosine in HL13 is two times higher than in HL19 while the phenylalanine of HL19 is almost three times higher than that of HL13. Tryptophan is present only in HL13. Cysteic acid was not detected in either HL13 or HLI 9 after performic acid oxidation.

Amino- Terminal Sequence of'HL.13 und H L 19

About 9 m g of HL13 and HL19 were used for each sequencer run and the first 36 residues were examined. As shown in Table 2, the first 30 residues of HL13 and 28 residues of HL19 were identified. All tryptic peptides corresponding to the first 27 resi- dues of HL13 and 18 residues of HL19 were isolated and their amino acid compositions are consistent with

506 Ribosomal 5-S RNA Protein Complex: Purification and Charactcri/ation

1 Fractions

).lo

- 5

Y c 0

1.05

Fig. 6. t'ur($curion qf the 5-S RNA-hintling proteitis 017 DEAL- wllulosr,. Thc S-S R N A complex, pooled from 1 8 Agarosc column runs. was fractionated on a DEAE-cellulose column (85 x 1.5 cm) in 6 M urea'Tris buffcr pH 8.0 containing 9 m M methylamine and 0.1 M ilithiothreirol using a 0-0.3 M KCI gradient as dcscribed in Materials and Methods. The fractions were scanned at 230 nni and the protein content of each peak was determined using dodecyl sulphate gcl electrophoresis [I61 as indicated in the lower part of the ligurc. The individual proteins werc identitied by two-dimcn- sional gcl electrophorehis [I71

the sequence data (J. Ward, unpublished data). Fig. 7 shows some similarities between the partial sequencc of HL13 and HL19 to those of two 5-S-RNA-binding proteins, from E. coli, EL18 [7] and EL5 [6], respec- tively. The homology between residues 11 - 28 of HLI 9 and residues 25 - 42 of EL5 appears to be highly significant since nine out of 18 positions are identical and one base substitution can explain the amino acid exchange of an additional seven positions (Glu-Val, Ile-Val, Val-He, Val-Leu, His-Asn, Gln-Glu, Gly-Ala).

DISCUSSION

The experiments described in this paper indicated that a native 5-S RNA . protein complex, containing 5-S RNA and two proteins, HL13 and HL19, can be released from the 50-S ribosomal subunit of the extreme halophile, H . cutirubrum, by decreasing the

Table 1. The aniino-wid c,onip~isition q f ih r 5-S RNA bindinx I J W I ~ + ~ . S

$ € I . cutirubrurn The results are an average of four detcrrninations

~ ~ ~~~~

Ammo acid HL13 HL19

mol, 100 mol

Aspartic acid Threonine Serine Glutamate,Glutamine Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phcn ylalanine Histidine Lycine Arginine Tryptophan Cysteinc

11.0 14.0 6.7 7.2 5 .5 5.1

10.0 14.9 5.8 5.4 8.7 x.4

12.7 6.0 5.9 8.4 1.1 1 .S 3.0 4.4 9.0 5 .9 3.5 1.7 1 .6 4.6 3.0 2.2 3.2 2.9 7.8 7.6 1.6" 0.0 0.0 0.0

Total 100.1 100.2

" Determined by the method of Edelhoch [21].

Mg2' concentration down to 0.3 mM or by adding EDTA. The use of EDTA, however, was avoided since there is some evidence to indicate non-specific intcractions between RNA and proteins in the pres- ence of EDTA [31]. Eukdryotic 5-S RNA . protein complexes can also be released by EDTA 1261 and recently a 5-S RNA . protein complex has been ob- tained from E. coli by EDTA treatment (A. Liljas, personal communication). It would appear, therefore, that Mg2+ is involved in the binding of the 5-S RNA complex to the 50-S ribosome.

The presence of Mg2' alone is not sufficient to bind the 5-S RNA complex to the 50-S subunit of the extreme halophile. In buffer A (50 mM K' and 100 mM Mg2+) the 5-S RNA is released but not as a complex. High K' concentrations are required for the integrity of the complex and the release of 5-S RNA in buffer A may be due to the initial release of ribosomal proteins from the 50-S subunit by the low K ', resulting in the subsequent release of naked 5-S RNA.

The evidence to indicate that the 5-S RNA eluted from the Agarose column (Fig. 1) is an RNA . protein complex containing 5-S RNA and two proteins HL13 and HL19, is the following. The complex, from its elution profile on Agarose 0.5-m columns, has a molec- ular weight of approximately 80000 which agrees well with the molecular weights of 39000 for 5-S RNA and either 24800 (dodecyl sulphate) or 18700 (ultra- centrifuge) for HL13 and either 21600 (dodecyl sul- phate) or 18000 (ultracentrifuge) for HL19. The only

N. Smith, A. T. Matheson. M. Yaguchi. G. E. Willick. and I<. N. Nazar 507

Table 2. Sequencer unuI.,:vi.v o f HL13 und HLIY Threonine was identified as r-aminobutyric acid and serine as alanine. TLC = thin-layer chromatography

Residue HL13 HL19

6 M HCI TLC assigned 6 M HCI TLC assigned

+ SnCI2 - SnCI2 + SllClZ - SnClz residue rcsidue

nmol nmol

1 Ala 209 Ala Ala Scr 51 - Ser Thr Glx 151 - Glu Glu 2 Tlir 162 -

3 GI)’ 23 1 GlY GIy Thr 80 - Thr Pro 150 Pro Pro Asx 127 Asp A V 4

5 ArE 40 Arg Arg Ser 40 - Ser Thr 6 TYr 41 TYr TY r Thr 76

Ly5 Asx 131 - Asp Asp 7 Lys 62 LYS 8 Val 64 Val Val Phe 63 Phe Phe 9 Pro 85 Pro Pro His 90 His His

10 Met 53 Met Met Glx 117 - Glu Gill 11 Arg 27 Arg A Met 71 Mct Mct 12 Arg 53 Arg A rg Arg 63 Arg Arg 13 Ark7 62 A% A rg Glx 116 OIL1 Glu

12 Arg Arg Pro 104 Pro Pro Glu Glu Arg 46 A rg Arg

14 Arp 15 Glx 48 16 Val 29 Val Val Ile 56 Lle Ile

29 Arl: ArE Glx 105 - Glu Glu - Thr Lys 64 Lys Lys

17 ArB 18 Thr 3 1 19 Asx 36 - ASP ASP Val 52 Val Val 20 TY r 9 TYr TY r Val 63 Val Val 21 His 24 His His Val 68 Val Val 22 Glx 37 - Gln GI11 His 49 IIis 1 lis 23 Arp 15 Arg A rg Met 49 Met Met 24 LCU 35 Leu Leu GlY 86 Gly Cly 25 Leu 44 Leu Leu Val 34 Val Val 26 Lcu 55 Lcu Leu Gly 99 GIY cily 27 Lys 14 Lys Lys Glx 72 - Gln Gin 28 Ala 36 Ala Ala Gly 86 GIY Gly 29 Val 26 Val Val 30 Gly 44 Gly GI4

-

-

-

-

“ N o amino acid detected

9 10 I I Val Pro --- Met Arg

22 EL 18 Met Asp Lys Lys Ser Ala Arg I l e Arg Arg Leu Gln Glu Leu Gly

HL 13 I 5 10

23 Arg Thr _ _ _ _ _ _ Asp Tyr

Pro Arg

Gln Arg Leu Leu Leu Lys

I l e Tyr Ala Gln Val I l e

30 Val Gly

43 Pro Asn

I 10 HL 19 Ser Glu Thr Asp

EL 5 Ala Lys Leu His Asp Tyr Try Lys Asp Glu V a l V a l Lys Lys Leu I 5 10 12

HL 19 _ _ _ _ _ _ 28 29 31 32 34 37 40 42

508 Ribosomal 5-S RNA . Protein Complex : Purification and Characterization

RNA species in the complex is 5-S RNA (Fig.2A) and only major ribosomal proteins present are HLI 3 and HL19 (Fig.2B). The elution profile of these two proteins matches the elution profile of the 5-S RNA (Fig.4) indicating that they are part of the same complex. Except for two ribosomal proteins that are eluted as a broad peak prior to the elution of the 5-S RNA complex, all the ribosomal proteins are eluted well after the 5-S RNA complex and do not contaminate the complex.

As indicated above, HL20, which appears to be equivalent to EL12 [29], is eluted from the Agarose column as a protein complex with another unidentified ribosomal protein, prior to the elution of the 5-S RNA complex. The nature of this protein complex is under investigation and may be equivalent to the protein complex formed between EL12 and EL10 [30]. In addition, HL20 also shows a minor peak at tube 53 (Fig. 4) and it is often evident in the two-dimensional gels (Fig. 3). It is of interest that Erdmann has re- ported the presence of small amounts of the equivalent protein in E. coli and B. stearotliermophilus 5-S RNA complexes [l].

The 5-S RNA complex also contains a second minor component which has an elution profile similar to that of the 5-S RNA complex (Fig. 4). This protein runs close to HL3/4 on two-dimensional gels and has tentatively been identified as HL3/4.

Repeated attempts to detect either ATPase or GTPase activity in the isolated native 5-S RNA . pro- tein complex were unsuccessful. This was surprising since earlier studies had indicated these activities in the reconstituted 5-S RNA . protein complexes from E. coli and B. stearothermrjplzilus [19,27]. However, recent studies (R. Villems, personal communication) also indicate the absence of ATPase and GTPase in native 5-S RNA . protein complexes for E. coli.

When the yeast 32P-labelled 5-S RNA was used as a marker (Fig. 1) there was no evidence of significant exchange between the yeast 5-S RNA and the halo- phile 5-S RNA in the complex. The same was true with chick embryo and Thrrnius aquaticus 32P-labelled 5-S RNA. However, when H. cutiruhrum 32P-labelled 5-S RNA was used as a marker it was entirely eluted with the 5-S RNA . protein complex. This complete incorporation into the complex region could be the result of an equilibrium exchange or it could be due to the interaction of this very small amount of 5-S RNA with free 5-S RNA-binding proteins present in the low-Mg", high-K' extract, to form an additional complex. This could indicate that reconstitution of the H . cutiruhrum 5-S RNA . protein complex can proceed rapidly under the high salt conditions used in these experiments. This aspect is under further investigation.

The 5-S RNA-binding proteins from H . cutiruhrum are much more acidic than the equivalent proteins from E. coli or B. stecrrothrrzophilus as indicated by

their amino acid compositions (Table l j . The ratio of (Asx + Glx)/(Lys + Arg) is 1.9 for HL13 and 2.7 for HL19 while the 5-S-RNA-binding proteins in E. coli give ratios of 0.9, 0.9 and 1.2 for EL5, EL18 and EL25, respectively [6- 91. On a Kaltschmidt-Witt- mann two-dimensional gel [18] the purified HL13 and HL19 run almost as rapidly as EL7/12, the most acidic protein in E. coli (Fig. 3C). On a similar gel system, the 5-S-RNA-binding proteins of E. coli and B. stearothermoplzilus run as basic proteins [27].

Although the equivalent binding proteins from B. .rtearothermophilus and the extreme halophile differ dramatically in their charge properties there is some evidence to suggest that these proteins may have common binding sites on the 5-S RNA. The 5-S RNA from H. cutiruhrum will bind BL5 and BL22. the equivalent proteins in B. stearotliermophilus and the resulting complex can be incorporated into B. stearo- tlzernzophilus 50-S subunits to yield active ribosomes [28] and (Wrede, Matheson and Erdmann, unpub- lished results). Work is now in progress on the sequence of HL13, HL19 and 5-S RNA from the extreme halo- phile. It is hoped that this data, in conjunction with results from current studies on the binding sites for the proteins of the 5-S RNA, will give some insight into the nature of this highly specific interaction.

The authors gratefully acknowledge the expert technical help of F. Rollin, C. Koy and H. Tesier.

REFERENCES

I . Erdmann. V. A. (1976) Progr. Nucleic Acid Res. Mol. Biol.

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N. Smith. M. Yaguchi, G. E. Willick. and R. N. Nazar, Division of' Biological Sciences, National Research Council o f Canada. Ottawa, Ontario, Canada K1A OR6

A. T. Matheson, Department of Biochcmistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada V8W 2Y2