Threonyl-tRNA synthetase from Thermus thermophilus: Purification and some structural and kinetic properties

Biochimie (1994) 76, 71-77 © Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Paris

71

Threonyl-tRNA synthetase from Thermus thermophilus: Purification and some structural and kinetic properties J Zheltonosova a, E Melnikova a, M Garber a, J Reinbolt b*, D Kern b, C Ehresmann b, B Ehresmann b .~ alnslitute of Proteh, Research, Departmem of Sa'ucture and Function of Ribosomes, Russian Academy of Sciences, 142292 Pushchino, Moscow region, Russia; bUPR No 9002. Institut de Biologic Mol&wlah'e et Ceihdab'e du CNRS, 15, rue Rend Descartes, 67084 Strasbourg Cedex, France

(Received 22 April 1993; accepted 8 December1993) Summary - - Threonyl-tRNA synthetase (ThrRS) has been isolated from an extreme thermophile Thermus thetwwphilus strain HB8. The enzyme was purified to electrophoretic homogeneity by combinations of column chromatographies on DEAE-Sepharose, S-Sepharose, ACA-44 Ultrogel and HA-Ultrogel. Seventeen mg of purified enzyme were obtained from 1 kg of biomass. In parallel, purified aspartyi- and phenylalanyl-tRNA synthetases were obtained. The purified ThrRS is composed of two identical subunits with a molecular mass of about 77 000 (virtually the same as E coli ThrRS). The N-terminal sequence has been determined. The homology between the first 45 amino acid residues of ThrRS from T thermophih¢s and E co# is about 29%, A coml3arative study of tRNATM charging by ThrRS from E ct li and T thermophilus reveals a similar efficiency of the reaction in both homologous systems. This efficiency remains unchanged for aminoacylation of tRNAThr from T thermophilus by the heterologous ThrRS from E coil, but decreases 700 times for aminoacylation of E coil tRNA"rhrby ThrRS from T thermophilus, aJ

~

¢'

ThrRS / protein purification / N.t sequence I T thermophilus / tRNA aminoacylation / tRNA mischarging Introduction Purification of various components of the proteinsynthesizing machinery from extreme thermophilic microorganisms offers new possibilities for structural investigations of the translational apparatus. During the last decade substantial progress has been reached in crystallization and in structural investigations of ribosomes, ribosomal proteins, elongation factors and several aminoacyl-tRNA synthetases [1--4]. ThreonyltRNA synthetase (ThrRS) from E coli is being investigated very intensively, Besides its function in tRNA aminoacylation, it has been shown that this enzyme autoregulates negatively its own synthesis by binding to the leader region of its m R N A [5-7]. The spatial structure of this protein attracts great interest, but attempts to crystallize E coli ThrRS have been unsuccessful up to now. This stimulated us to purify ThrRS from an extreme thermophile T thermophilus for structural investigations. The first attempt to purify ThrRS from T thermophilus was undertaken several years ago by Yaremchuk et al [8]. The protein purified

*Correspondence and reprints

by this group was crystallized and crystals were described as crystals of ThrRS [9]. Unfortunately, N-terminal sequencing of the protein material from these crystals revealed 64% identity with aspartyl-tRNA synthetase from E coli and no significant sequence analogy with ThrRS fi'om E coli (result not shown). The present paper deals with the first real description of the purification of ThrRS from T thermophilus and reports some structural and kinetic properties of this enzyme.

Materials and methods Materials

DEAE-Sepharose Fast Flow and S-Sepharose Fast Flow were purchased from Pharmacia Fine Chemicals (Sweden), DEAEcellulose from Whatman (UK), ACA-44 Ultrogel from LKB (Sweden), HA-Ultrogel and BD-cellulose from Serva (Germany), and butyl-toyopearl 650S from Toyo-Soda Oapan). [14C]- and [3H]-labelled amino acids were purchased from Amersham International (UK). DNAsel was purchased from Serva (Germany). Glass microfiber filters GF/C were purchased from Whatman (UK). All the reagents were purchased from Fluka (Switzerland) or from Serva. The protein markers for molecular mass determinations were from Serva (protein test mixtures 4 and 5). All chemicals used for the Edman

72 degradation as well as for the sequencer apparatus were from Applied Biosystems (Roissy, France). Unfractionated tRNA from E coil was purchased from Serva; unfractionated tRNA from T thermophilus was kindly provided by Dr G Yusupova (Pushchino, Moscow, Russia). Pure tRNA'rhq from E coli (acceptance capacity: 25 nmol/mg) was obtained from Subriden (USA). tRNATM from T thermophih~s was partially purified by Dr G Keith (IBMC, Strasbourg) by two successive chromatographies on DEAE-cellulose and BD-cellulose of bulk RNA obtained by phenol extractions of the cells. Two active fractions containing tRNATM were obtained by BDcellulose chromatography: F3 eluting at 0.63 M NaCI (acceptance capacity: 1.42 nmol/mg) and F6 eluting at 0.72 M NaC! (acceptance capacity: 1.45 nmol/mg). These tRNAs correspond probably to the tRNA TM isoacceptors defined by sequencing of the genes I101. The ThrRS from E coil was obtained according to a derived procedure described in [ 111.

from T thermophilus (0.12 to 1.2/ag/ml). The Kms were determined from initial rates according to the double reciprocal plot representation ( I/V= f (I/S)). The k~..,tvalues for tRNA~r charging were determined in the above described reaction mixture with 0.050 mM L-[14C]threo nine (specific activity: 62.5 cpm/pmol), a saturating concentration of tRNA TM (50-100 Km) and an enzyme concentration allowing initial rate measurements. The labelled aminoacyltRNA formed as a function of incubation time was measured as described above. The reactions were conducted at 37°C unless otherwise indicated.

Cells

Electrophoresis

T thermophihcs HB8 and VKI strains were kindly provided by

SDS slab gel electrophoresis was performed according to the method of Laemmli 1131 in a 9-25% linear gradient of polyacrylamide.

Prof T Oshima (Tokyo, Japan). Cells were grown at 75°C and then harvested by centrifugation near the middle of the logarithmic phase of growth and stored at -70°C.

Buffers Buffer A contained 20 mM Tris-HCI (pH 7.6), 25 mM NaCI and 10 mM MgCI.,. Buffer B contained 15 mM MES-KOH (pH 6.5) and 20 mM KCI. Buffer C contained 20 mM Na.,HPOd KH.,PO4 (pH 6.5) and 50 mM KCi. Buffer D contained 5 mM K.,HPOdKtt.,PO~ (pH 6.5). All buffers contained in addition 0.003 % NaN~ and, except buffer C, 5 mM 2-mercaptoethanol.

Enzyme activiO' measurements ~'ThrRS and other aaRS from T thermophilus The activity of ThrRS was measured by the rate of lbrmation of threonyl-tRNA at 65°C. The reaction mixture contained 0.1 mM of unfractionated tRNA from T thermophilus, 0.025 mM L-[14CIthreonine (228 mCi/mmol), 2 mM ATP, 50 mM KCI, 10 mM MgCI2, 10 mM Bicin-KOH (pH 8.0 at 65°C) and an adequate amount of enzyme. After incubation at 65°C tbr a time allowing initial rate measurements, the reaction mixture was transferred onto a GF/C disc and washed three times in 5% trichloroacetic acid at 0°C. The disc was dried and then placed in 5 ml of a scintillator (40 mg of 2,5-diphenyloxasol and I mg of i,4-bis-2-(5-phenyloxasolyl)benzene in 10 ml toluene), and the [J'~Ciradioactivity was counted in a Beckman liquid scintillation system. The unit of ThrRS catalyzes the formation of I nmol threonyl-tRNA per min under the reaction conditions described above. The activities of AspRS, AsnRS, PheRS, ValRS, LeuRS, lleRS, GlyRS, MetRS and LysRS were measured by the same procedure using [t4CIlabelled amino acids.

Determimttion of the kinetic constants of ThrRSfor charging of homologolts and hetelv~logous tRNA TM The Kms of ThrRS for tRNA'rhr were measured in a reaction mixture containing 100 mM Hepes-NaOH buffer (pH 7.2), 30 mM KCI, 10 mM MgCI.,, 2 mM ATE (I.020 mM L-I3HIthreonine (specific activity: 1500 cpm/pmol), either tRNAThr~ from E coli (in the concentration range of 0.06 to 1 /aM) or tRNA TM from T thermophihrs (in the concentration range of 0.06 to 4 laM) and pure ThrRS from E coil (0.05 /ag/ml) or

Protein concentration The concentration of protein solutions was determined by the method of Bradford !121, using bovin serum albumin as standard.

N-telw~inal sequencing Protein was sequenced by automated Edman's degradation, using an Applied Biosystems 470A Protein Sequencer equipped with a PTH 120A Analyser i!41.

Results Purification (~'threonyl-tRNA .Lvnthetase Step !: Cell lysis I kg o f frozen T thermophilus HB8 cell paste was t h a w e d overnight at 4 ° C and suspended in l ! o f buffer A containing 20 m g o f p h e n y l m e t h y l s u l p h o n y l fluoride. The ceil suspension (1.5-2 l) w a s passed through a French press ( M a n t o n - G a u l i n ) u n d e r a pressure o f 8 000 psi. D N A s e 1 (200 I.tg) w a s added, the lysate was stirred 30 rain at 4°C, and c e n t r i f u g e d at 30 000 g to r e m o v e the cell debris. T h e supernatant was finally centrifugated at 1130 000 g in a Ti-15 zonal rotor ( B e c k m a n ) to r e m o v e the ribosomes.

Step 2: DEAE-Sepharose fractionation T h e postribosomal extract was applied o n a D E A E Sepharose c o l u m n (20 x 5 cm, flow rate: 5 0 0 ml/h) equilibrated with buffer A. After w a s h i n g with I I o f the same butler, elution was p e r f o r m e d with a linear gradient of two t i m e s 3 I o f NaC! f r o m 25 m M to 250 m M in buffer A (flow rate: 200 ml/h), and fractions o f 20 ml were collected. T h e fractions containing T h r R S activity (see fig I) were pooled, the proteins precipitated with a m m o n i u m sulphate at 70% o f saturation, and the precipitate collected b y centrifugation.

73 1.2

Step 3: S-Sepharose fractionation The precipitated proteins were dialyzed against buffer B and applied on a S-Sepharose column (20 x 5 cm, flow rate: 60 mi/h) equilibrated with buffer B. After washing with 300 ml of this buffer, the elution was performed with a linear gradient of two times 1 i of KC! from 20 mM to 200 mM in buffer B, and fractions of 10 ml were collected. The active fractions were pooled, and the proteins were precipitated and collected as in step 2.

Phel~ o ¢o t~ 0.8

¢1 ~0.4
Step 4: Gel filtration on ACA-44 Ultrogel The precipitate was dissolved in 10 ml of buffer C, centrifuged, and the supernatant was applied on a ACA-44 UItrogel column (270 x 2.5 cm) equilibrated with buffer C. The gel filtration was performed at a flow rate of 30 ml/h and fractions of 10 ml were collected. The fractions containing ThrRS were detected by SDS-PAGE (fig 2), pooled and directly applied on a HA-Ullrogel column.

0.0

i

0

10

20

Fraction

t'~

30

|

40

5

number

Fig 2. Separation of ThrRS and PheRS by gel filtration on ACA-44 Ultrogel. ThrRS was not identified by activity measurements; since its purity was about 70%, analysis by SDS-PAGE allowed to localize the fractions containing the protein; the total activity of ThrRS appears in table I.

Step 4": Butyl-toyopearl fi'acu'onation In some purifications, the gel-filtration was substituted by a hydrophobic interaction chromatography. The protein precipitate obtained in step 3 was dissolved in ammonium sulphate at 30% of saturation, centrifuged and the pellet resuspended in ammonium sulphate saturated at 20% and centrifuged, Both supernatants were pooled and applied on a butyl-toyopearl column equilibrated with 100 mM potassium phosphate buffer (pH 7.0) and ammonium sulphate at 30% of saturation. Elution was performed with a decreasing linear NaCI eluh'on profile - - ipmment p r o m e . . . . . activit~

M .0,3

A~ S

o 157 s

.0,2

S S S

gradient of two times 0.25 i of ammonium sulphate from 25% to 5% of saturation in the same buffer (flow rate: 30 ml/h). ThrRS fractions were pooled and the proteins precipitated with ammonium sulphate at 70% of saturation, collected by centr|fugation and dialyzed.

Step 5: Hydroxyapatite fi'actionation The active fractions (30 ml) were applied on a HAUItrogel column (30 ml) equilibrated with buffer D. After washing with 30 mi of 50 mM potassium phosphate buffer (pH 6.5), elution was performed with a linear gradient of two times 0.3 i of potassium phosphate from 50 to 200 mM (pH 6.5) and fractions of 5 ml were collected. The fractions containing pure ThrRS were detected by PAGE, pooled and the protein was then precipitated with ammonium sulphate at 70% of saturation. The protein precipitate was stored at 4 °C.

t Ss

S

Summary of purification

¢Q -0.1

!

-- -iiii i

0

~

i

I i

50

i

IIiii

100

I

!

IJ ........................

ii i I I II I I I ~ I

150 Fraction

2OO

250

300

0.0 350

number

Fig 1. Chromatography of the crude protein extract from T thermophilus on DEAE-Sepharose CL-6B. The conditions are described in Materials and methods. The various aaRS were identified according to activity measurements effected with ! Ill of each tenth fraction. They eluted in three groups: group 1 contains ValRS, LeuRS, IleRS and MetRS, group 2 contains AspRS, GiyRS, ThrRS and PheRS, and group 3 contains AsnRS and LysRS.

Table I summarizes the purification procedure of ThrRS from T thermophilus. The analyses by SDSPAGE of the protein fractions obtained after each purification step are shown in figure 3. The most important step of the purification is S-Sepharose chromatography (step 3). Binding of ThrRS to the sulphogroups appears specific and allows to purify the enzyme by a factor of 15. Gel filtration and hydrophobic interaction chromatography are interchangeable. At step 4, ThrRS is separated from PheRS (fig 3, lane 5). Minor impurities can be eliminated only after hydroxyapatite fractionation (fig 3, lane 6). Purity of final preparation of ThrRS is usually better than 95% and its specific activity is at least 350 units/ mg (see table 1).

74 Table !. Purification of ThrRS from 1 kg of T thermophilus HB8.

Purification step

Total protein (rag)

Total activi~ (units)

Specificactivity (units/rag)

Purification factor

Yield (%)

4608

29520

6.4

1

1O0

210

20760

98.8

15

70.3

ACA-44

22

6969

316.8

50

23.6

Hydroxyapatite

17

6013

353.7

55

20.4

Crude extract" DEAE-Sepharose S-Sepharose

"It was not possible to determine accurately the total activity in the crude extract, therefore the activity after e~ch step was compared to that of the DEAE-Sepharose fraction (total activity = 100%). The purified enzyme was shown to compete with ribosome for binding to the translational operator of the ThrS mRNA with the same efficiency as ThrRS from E coli (P Romby, personal communication).

Isolation of aspartyl- and phenylalanyl-tRNA synthetases As shown in figure 1 there are several groups of aaRS in the elution profile from DEAE-Sepharose. Group 2

kOa 92.567.045.029.0 -

also contains AspRS, GIyRS and PheRS besides ThrRS. PheRS binds to S-Sepharose under the same conditions as ThrRS and is present in the ThrRS preparation after S-Sepharose chromatography (fig 3, lanes 4 and 16). These two synthetases can be separated by gel-filtration (fig 3, lanes 5 and 17), or by salting out PheRS at 30% saturation with ammonium sulphate (steps 4 and 4'). Unfortunately the small subunit of PheRS is rather sensitive to proteolytic activities present in the crude extract, and probably corresponds to the fragment of molecular mass of about 29 000 visible in figure 3 (lane 17). As for AspRS, it does not bind to S-Sepharose in the same conditions as ThrRS but it binds in the presence of 5 mM MgCl2 in buffer B. To purify this enzyme after S-Sepharose chromatography, we used gel-filtration on ACA-44 Ultrogel and hydrophobic interaction chromatography on butyl-toyopearl under the same conditions as for ThrRS (steps 4 and 4'). Purity of AspRS preparation after hydrophobic chromatography was better than 95% (fig 3, lane 12).

Molecular mass and subunit structure of threonyltRNA synthetase

21.012.56.512345

6 7 8 910 II

12 13141516 17

Fig 3. Analyses by SDS-PAGE of ThrRS, AspRS and PheRS fractions after the various purification steps. Lanes : I, 7, 13, marker proteins; 2--6, purification of ThrRS; 812, purification of AspRS; 14-17, purification of PheRS; 2, 3, 8, 9, 14, 15, protein tractions after chromatography on DEAE-Sepharose; 4, 16, protein fractions containing ThrRS and PheRS after chromatography on S-Sepharose; 10, AspRS fraction alter chromatography on S-Sepharose; 5, I1, 17, ThrRS, AspRS and PheRS fractions after gelfiltration on AcA-44 Ultrogel; 6, purified ThrRS after chromatography on hydroxyapatite; 12, purified AspRS after hydrophobic interaction chromatography.

The molecular mass of ThrRS from T thermophilus determined under denaturing conditions by SDSPAGE has been estimated to be 77 000. The electrophoretic mobilities of ThrRS from T thermophilus and from E coil are very similar (data not shown), Gelfiltration on ACA-44 Ultrogel effected under nondenaturing conditions revealed an apparent molecular mass of about 150 000 for the enzyme; this value is similar to that found for ThrRS from E coil [ 151 and allows to establish an or2 dimeric structure for ThrRS from T thermophih~s.

N-terminal sequencing We could unambiguously establish the N-terminal sequence of ThrRS from Met I to Asp45 from T ther-

75 Table H. N-terminal sequence of ThrRS from T thermophi-

lus stre~ HB8. 1 HTVYL 21 DVARA 41 GTLYD

i0 20 PLEL P E G A T A K 30 40 L G E GW E R RAVGA I VD PDGK

45

mophilus strain HB8 (table II). The sequence results are becoming quite uncertain and ambiguous after degradation step 45, even when increasing markedly the amount of ThrRS submitted to the sequencing technique. Such a phenomenon of an unexpected rapid stop of an unambiguous sequence alignment is usually observed with proteins of high molecular mass. This is most probably induced either by some conformational change of the protein during sequencing or by accumulation of overlapping residues. We also sequenced the N-terminal part of ThrRS from T thermophilus strain VKI in order to compare it with that of strain HB8. Unfortunately for ThrRS from strain VKI the sequencing results appear relevant only up to residue Trp30. This could be due to the preparation conditions used and to the presence and some impurities rendering the sequencmg and the interpretation of its results more difficult. Nevertheless, the N-terminal sequences of ThrRS from both strains HB8 and VKI are strictly identical at least up to residue 30, suggesting strongly that the primary structures of both enzymes are identical; this presumption is reinforced by the fact that the protein sequences of the AspRS of these strains were found identical as deduced from nucleotide sequences of their genes [ 16]. The N-terminal sequence of ThrRS from T thermophilus was compared with that of ThrRS from E coli (results not shown). Alignment of the proteins is based on the BestFit program [ 17]. The percentage of

identity may be considered as relatively low, as both enzymes share about only 29% of identity. However, when using the FASTA program this result appears quite significant, since the N-terminal sequence of ThrRS from T thermophilus exhibits about the same percentage of identity with the N-terminal sequences of all the ThrRS from different sources studied so far. The N-terminal sequence of ThrRS from T thermophilus was aligned with these homologous proteins from other sources using the Pileup program of the University of Wisconsin Genetics Computer Group (UWGCG) [17-19]. There are some consensus sequences between the N-terminal sequence of ThrRS from prokaryotes and also from an eukaryote (table Ill), suggesting an evolutionary relationship, since it is well known that eukaryotic aminoacyl-tRNA synthetases are larger than the prokaryotic ones by an extension at the N-terminal domain.

Determination of the kinetic constants of aminoacyiation of tRNA rl'"fi'om T thermophilus and E coli by homologous and heterologous ThrRS We determined the/(ms and the keel values of ThrRS from T thermophilus and E coil tor tRNA TM of both types of bacteria (Table IV). The E coil system was studied at 37°C, the T thermophilus system at 37°C and 70°C, and heterologous systems were studied at 37°C. As shown by the k~JKm ratios, both ThrRS aminoacylate their homologous tRNA TM with a comparable efficiency at optimal temperature (8.2 x 106 s-~ M-! for E coli at 37°C, 6.2 x 100 and 3.1 x 106 s-i M-~ for T thermophilus at 70°C). In addition, ThrRS from T thermophilus aminoacylates both tRNA TM isoacceptors from T thermophilus with the same efficiency at comparable temperature (1.0 × 106 and 0.43 x 106 s-l M-1 at 37°C, 6.2 x 106 and 3.1× 100 s-I M-~ at 70°C). As expected, the increase of the temperature from 37°C to 70°C increases the kca, of tRNA charging by ThrRS from T thermophilus about 10 times without affecting the Km and therefore increases by a same extent the factor of efficiency.

Table !!!, Sequence alignment of ThrRS from different sources. The consensus sequences are in bold letters. Trs 1Bs and Trs 2Bs: the two ThrRS from Bacillus subtilis, Trs Ec: ThrRS from Escherichia coli; Trs Tt: ThrRS from Thermus thermophilus; Trs Sc: ThrRS from the cytoplasm from Saccharomyces cerevisiae. TrslBs

1

MSDMVKITFP

DGAVKEFAK.

GTTTEDIAAS

ISPGLKKKSI

AGKLNGKEID

Trs2Bs

1

MSKHVHIQLP

DGQIQEYPK.

GITIKEAAGS

ISSSLQKKAA

AGQVNGKLVD

TrsEc

1

...MPVITLP

DGSQRHYDH.

AVSPMDVALD

IGPGLAKACI

AGRVNGELVD

TrsTth

1

.... M T V Y L P

DGKPLELPE.

GATAKDVARA

LGEGWERRAV

GAIVDGTLYD

71

PRVPLKIVLK

DGAVKEATSW

ETTPMDIAKG

ISKSLADRLC

ISKVNGQLWD

...... I - L P

DG---EY

TrsSc Consensus

. . . . .

T--D-A--

I---L

. . . . . . . .

VNG-L-D

76 Table IV. Kinetic constants of amuinoacylation of tRNATM from E coil and T thermophilus by the homologous and heterologous ThrRS. F3 and !::6refer to BD-cellulose fractions containing tRNA~r (see Materials and methods). tRNATh,•

ThrRS T thermophilus Km (~M)

k,,,,(.v-l)

E coli Thr~

k,,,/K,, (106 s-! M-I)

0.037"

0.037"

1

0.037 b

0.23 b

0.060a

0.026 a

6.2 0.43

T thermophilus F3 T thermophilus 1:6

E coil K,,, (IMVI)

k,,,, (s-l)

k, JKm (!06 s-I M-i)

0.085 ~

0.7(P

8.23

nd

0.52a

-

0.050a

0.53a

10.6

o

0.070b 0.14~

0.22 b 0.2 IO-3a

3. I 1.4 10-3

aDetermined at 37°C; bdetermined at 70°C; nd, not determined. Different behaviours were observed in the heterologous systems. ThrRS from E coli aminoacylates tRNA TM from T thermophilus as efficiently as the homologous tRNA TM (kJi(m = 10.6 X 106 and 8.2 x I(P s-t M-~ respectively), whereas ThrRS from T thermophilus aminoacylates tRNA TM from E coli 700 times less efficiently than the homologous tRNA TM (k~JK,,, = 1.4 x 103 and 1.0 x 10~ s-J M-I respectively). This decrease results essentially from a k~,,, effect: ThrRS from T thermophilus aminoacylates tRNA TM from E coli with less than 1% of the Vm,,~of aminoacylation of tRNA TM from T thermophilus, whereas the Km is increased only 2-4 times. These results agree with the data reported by Kumazawa et al [20] showing that ThrRS from T thermophilus aminoacylates only 5% of tRNA TM in unfractionated tRNA from E coil, whereas ThrRS from E coil aminoacylates tRNA TM in unfractionated tRNA from T thermophilus near to 100%. According to the theory of the plateaus of tRNA aminoacylation by aaRS [21], incomplete aminoacylation extents are related to an equilibrium between aminoacylation of tRNA and deacylation of aminoacyl-tRNA. The decrease of the rate of tRNA aminoacylation without modification of the rate of deacylation of aminoacyltRNA establishes a new equilibrium where the extent of tRNA char~,ing is decreased. These kinetic investigations show that the incomplete charging of tRNAThr from E ~'oli by ThrRS from T thermophihts results mostly from decreased k~.,,, rather than from increased K., values.

Discussion Elaboration of a convenient procedurc of purification is the first step in the structural investigation of any

protein. The procedure developed in this work for ThrRS purification allows to obtain a high amount (17 mg) of the protein from I kg of T thermophihls biomass in 2 weeks. This yield of ThrRS compares favourably with yields of different aaRS from T thermophilus purified by other authors [22, 23] who did not use S-Sepharose in their purification scheme. It was found in this work that preparative chromatography on S-Sepharose column is very effective for the purification of at least three aaRS from T thermophii,s: AspRS, PheRS and ThrRS. Presence of magnesium and changing of pH can influence the binding of different aaRS to suiphogroups. As our preliminary experiments show, preparative chromatography on SSepharose column can be useful in purification of ValRS, LeuRS and IleRS from T thermophilus. The use of butyl-toyopearl for hydrophobic interaction chromatography is also very effective and convenient for the purification of ThrRS and AspRS. Previously we successfully used this resin for the purification of elongation factors G, Tu and Tu-Ts from T thermophilus (M Garber and G Zheltonosova, unpublished data). Since ThrRS from E coil aminoacylates tRNA~r from E coil and from T thermophilus with similar efficiency, the identity determinants of tRNA TM [24] seem to be conserved in both mesophilic and thermophilic prokaryotes. The decreased /,'~, of aminoacylation of tRNA~r from E coil by ThrRS from T thermophilus could be related to particular modifications in tRNA from E coil hindering by a steric effect the formation of the productive complex with the synthetase. This will be tested by investigation of the aminoacylation of transcripts of tRNA TM from E coil deprived of the posttranscriptional modifications.

77

This work constitutes the first step of a more general project concerning the intensive study of the structural and kinetic properties of ThrRS from T thermophilus and their comparison with those of ThrRS from mesophilic organisms in particular E coil From sequence of the 45 N-terminal residues of the protein a specific DNA probe can be synthesized by PCR for cloning of the gene. The sequencing of the gene allows the establishment of the protein sequence and its comparison with the known sequences of ThrRS from mesophilic prokaryotes and from eukaryotes. This study will reveal the conserved positions and motifs in the various ThrRS and bring preliminary informations about structural characteristics determining the threonylation function of tRNA; this comparison can also bring a first insight about particularities ensuring the !hermostability of ThrRS. Finally the isolation of the gene will make possible the overproduction of the protein in E coil and the obtention of high amounts of ThrRS, rendering more easy the biochemical studies and facilitating considerably the crystallographic investigation of the enzyme either in its free state or complexed with the tRNA or with the tRNA-like structure of the mRNA. Acknowledgments We are indebted to Drs P Walter and P Romby (IBMC, Strasbourg) for fruitful discussions and alignmem of the N-terminal sequences and to Dr G Keith (IBMC, Strasbourg) for the gift of partially purified tRNATM from T thermophiht,~. We also thank C Licht6 whose technical assistance was greatly appreciated. References I Yusupov MM, Gather MB. Vasiliev VD, Spirin AS (1991) Thermus thermophilus ribo~mes for crystallographic studies. Biochimie 73, 887-897 2 Garber MB. Agalarov SCh, Eliseikina IA, Fomenkova NP, Nikonov SV, Sedelnikova SE. Shikaeva OS, Vasiliev VD, Zhadanov AS, Liljas A, Svens.son LA 11992) Ribosomal proteins from Thermus Ihermophih~s for structural investigations, Biochimie 74, 327-336 3 Gaffer MB, Agalarov SCh, Eliseikina IA, Sedelnikova SE, Tishchenko SV, Shirokov VA, Yusupov MM, Resbetnikova LS, Trakhanov SD, Tukalo MA, Yaremchuk A (1991) Purification and crystallization of components of the protein-synthesizing system from Thermus thermophihcs. J Crystal Growth I !0, 228-236 4 Reshemikova LS, Reiser COA, Schirmer NK, Berchtold H, Storm R, Hilgenfeld R, Sprinzl M 11991 ) Crystals of intact elongation factor Tu from Thermus dwrnwphih,s diffracting to high resolution. J Moi Bio122 I, 375-?"7

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