Cloning and complete nucleotide sequence of the Bacillus stearothermophilus tryptophanyi tRNA synthetase gene

Gene, 46 (1986) 37-45 Elsevier

37

GENE 1710

Cloning and complete nucleotide sequence of the Buciffus s#etuwthermophiZus tryptophanyl tRNA synthetase gene (Recombinant DNA; oligonucleotide probe; molecular cloning; transcription terminator; low-copy-number plasmid vector)

David A. Barstow l, Andy F. Sharman, Tony Atkinson and Nigel P. Minton Microbial Technology Laboratory, PHLS Cenne for Applied Microbiology and Research, Porton Down, Salisbmy, Wilts. SP4 OJG (U.K.) Tel. (0980)610391 (Received April 16th, 1986) (Accepted June 26th, 1986)

SUMMARY

The Bacillus stearothemophilus NCAl503 tryptophanyl tRNA synthetase (WTS ; EC 6.1.1.2) gene has been cloned in Escherichia co/i and the amino acid (aa) sequence of the enzyme deduced unequivocally from the DNA sequence of the cloned gene. The predicted aa sequence of the WTS enzyme agrees with the previously determined aa sequence except that the DNA sequence indicates a third Arg residue at the C terminus of the enzyme over the two Arg residues indicated by sequencing the protein itself. The trpS gene consists of a 984-bp open reading frame commencing with an ATG start codon and ending with a TAA stop codon. Putative transcriptional promoters, a Shine-Dalgamo sequence and a transcription terminator have been idemiSed. Thus the ?rpS gene probably constitutes a single transcriptional unit.

INTRODUCTION

Bacterial aminoacyl tRNA synthetases are a diverse group of enzymes and have been the subject of much investigation with a view to solving their structure-function relationships (Schimmel and SW, 1979; Ofengand, 1977). The tryptophanyl * To whom correspondence

and reprint requests should be

addressed. Abbreviations: aa, amino acid(s); Ap, ampicillin; BCIG, 5-bromo-4-chloro-indolyl-gDgalactopyrano&de; bp, base

tRNA synthetase (WTS) of the mesophile E. coli and the thermophile B. stearothermophilus are functionally homologous (Neidhardt et al., 1975 ; Schimmel and So& 1979; Atkinson et al., 1979) and are both cc2dimers of approx. 37 kDa. The enzymes catalyse the production of tryptophanyl tRNATw (Ofengand, 1977; Schimmel and Soil, 1979) binding two molepair(s); kb, 1000 bp; nt, nucleotide(s); oligo, oligodeoxynucleotide; ORF, open reading frame; R, resistant; RBS, ribosomebinding site; Tc, tetracycline; T,, dissociation temperature; WTS, tryptophanyl tRNA synthetase; trpS, gene coding for WTS; YT, yeast tryptone.

0378-l119/86/$03.500 1986Elsevier Science Publishers B.V. (BiomedicalDivision)

38

cules of each of the substrates tryptophan, ATP and tRNATq per native dimer (Joseph and Muench, 1971). The E. co& trpS gene has been cloned (Hall and Yanofsky, 1981) and its primary sequence deduced from the nt sequence of the cloned gene (Hall et al., 1982). This sequence is homologous to the primary sequence of the B. stearothermophilus enzyme (Hall et al., 1982; Winter et al., 1977) displaying an overall sequence homology of 60 % with regions of 80-90 % homology. However, for the aa sequence of the B. stearothermophilusenzyme aa 32 and 42 have been assigned only as Glx and aa 41 as Asx (Winter and Hartley, 1977). Studies on the mechanism of action of both the E. coli (Andrews et al., 1985) and B. stearothennophilus(McArdell et al., 1982) WTS enzymes are in progress and in addition work on the crystallographic structure of the B. stearothermophilus enzyme is underway (Coleman and Carter, 1984). To resolve the aa residue ambiguities and ultimately to carry out aa replacements by site-directed mutagenesis we have cloned the B. stearothermophilusQS gene in E. coli and determined its complete nt sequence.

METHODS, RESULTS AND DISCUSSION

(a) Genomie probii

for the trpS gene

From the previously determined aa sequence of the B. stearothermophilus NCA 1503 WTS enzyme (Winter and Hartley, 1977), a synthetic oligo mixture CAT/C CAT/C TGG ATG GA, representing all possible coding sequences for aa 288-292, His-HisTrp-Met-Glu, was synthesised, by the phosphite triester method (Atkinson and Smith, 1984) with an Applied Biosystems 38OA DNA synthesiser. These oligos, a mixture of four 14-mers, are specific for the B. stearothemophilus npS gene because the aa sequence of the E. coli enzyme difkrs from the B. stearothermophilus enzyme in this region (Winter and Hartley, 1977; Winter et al., 1977). This specificity was subsequently confirmed by Southern blot experiments. Aliquots (10 pg) of B. stearothermophilusNCA1503 genomic DNA isolated as

described by Barker (1982) were analysed by Southern hybridisation (Southern, 1975) using the oligo mixture end&belled with 32P, as a probe (Wallace et al., 1979). Using the formula 2°C per A/T bp and 4” C per G/C bp (Suggs et al., 1982) the T, of the oligo mixture was calculated to be between 40°C and 44°C. A hybridisation and filter-washing temperature of 30°C (Tr, -1O’C) was used and under these conditions the oligo mixture hybridised to restriction endonuclease digests of B. stearothermophilusbut not E. coli genomic DNA.

Among the genomic restriction fragments shown to hybridise to the oligo probe were a 4.2-kb EcoRI fragment, a 4.1-kb SaZI fragment and a 2.2-kb SaZI-EcoRI fragment. It was decided to determine whether this hybridisation was to the B. stearothermophilus ttpS gene by cloning and obtaining the nt sequence of the cloned 2.2-kb SalI-EcoRI fragment. In addition, if the nt sequence revealed that the rrpS structural gene had an internal SaZI site then the entire gene could be cloned as an EcoRI fragment and vice versa. Thus 500 pg of B. stearothemophdlus genomic DNA was digested with EcoRI, electrophoresed on a 1y0 agarose gel and fragments of 4.2 kb in size were excised from the gel and purified by electroelution. A sample of this DNA was digested further with SalI and fragments of 2.2 kb in size purified in a similar manner. This gave an estimated lOO-fold purification of the trpScontaining fragment thus simplifying cloning into the bacteriophage Ml3 for nt sequence analysis. The EcoRI-SaA fragments were ligated to EcoRI + SalIdigested M13mp8 and M13mp9, and used to transfeet E. coli JM105 (Messing and Vieira, 1982) and recombinant phage detected by their inability to cleave the &galactosidase chromogenic substrate BCIG. Single-stranded DNA was isolated from recombinant phages and dot-blots probed with the 32Plabelled oligo mixture. A total of ten out of 70 of the M13mp8 clones and zero out of 70 of the M13mp9 clones gave a positive hybridisation signal. This was predictable as the oligo mixture was specific for one strand of the ?rpS gene and directional cloning into the Ml3 vectors was employed. Analysis of the cloned EcoRI-Sal1 fragment using both the oligo

t 00

1

1.0

I

2.0

I 3.0

I 4.0

1 4.2 kb

Fig. 1. Restriction sites in the trpS region of the B. stearo:hermophilm chromosome. The position and extent of the trpS gene was located by sequencing across the Sal1 site which lies within the hpS structural gene. The nt sequence of the 2.2-kb SalI-EcoRI fragment located the position of the two PvuII sites 3’ of the rrpS gene whilst Southern blot experiments using the 2.2-kb SalI-EcoRI fragment as a hybridisation probe identified the 4.5-kb, 1.5-kb and 0.19-kb PVUII fragments.

mixture and universal sequencing primer (AGTCACGACGTTGTA) as primers for DNA sequencing revealed that this fragment contained part of the tr_nSgene. However the SalI site was within the trpS structural gene corresponding to aa 187 and 188 (Val-Asp). Therefore, the entire crpS gene lay within the previously identified 4.2-kb EcoRI fragment (Fig. 1). Initially attempts were made to clone the 4.2-kb EcoRI trpS-containing fragment into the cloning vector pUC8 (Vieira and Messing, 1982). Using the purified fragments it was estimated that about 1% of the clones should contain the desired fragment. However, screening of 5000 recombinant clones failed to detect the trpS-containing fragment. In addition the trpS gene was not detected in gene banks constructed by inserting B. stearothemophilus genomic DNA, partially digested with Sau3A, into the plasmid pAT153 (Twigg and Sherratt, 1980) and bacteriophage 121059 (Kam et al., 1980) (not shown). As several aminoacyl tRNA synthetase genes (Barker, 1982; Dardel et al., 1984) including the E. coli trpS gene (Hall and Yanofsky, 1981), have been cloned and expressed at high levels in E. coli, it was thought unlikely that the failure to clone the 4.2-kb EcoRI fragment was due to lethal expression of the B. stearothermophilus tqv.9 gene. However, nt sequences 5’ of the trpS gene may be deleterious to E. coli cells. This could result from unstable nt sequences per se or the high level synthesis of a toxic gene product. If the latter of these was the reason for the failure to clone the rrpS gene it should be possible to clone the 4.2-kb EcoRI fragment into

a low-copy-number cloning vector. Therefore the plasmid pSCLAC5 was constructed (Fig. 2) and the 4.2-kb EcoRI fragments were cloned into the unique EcoRI site of this vector. One thousand recombinant clones were screened for the presence of the trpS gene but even in this vector the bps gene was not detected. Therefore, a strategy to overcome this cloning problem was devised. The nt sequence of the entire 2.2-kb EcoRI-SalI @S-containing fragment was determined and endonuclease cleavage sites 3’ of the trpS gene but not within the sequenced 3’ end of the trpS structural gene identified (Fig. 1). The eventual aim was to identify restriction enzymes which cleaved 5’ of the trpS gene thus removing any ‘lethal’ DNA that may be present. B. stearothermophilusgenomic DNA was then digested with the appropriate enzymes (DdeI, HaeII, PvuII and SphI) and probed with the 32P-labelled 2.2-kb EcoRI-SalI fragment. The PvuII digest, which gave fragments 4.5 kb, 1.5 kb and 0.19 kb in size, was chosen for further analysis. B. stearothennophilusgenomic DNA and a sample of the previously purified 4.2-kb EcoRI fragments were digested with PvuII and subjected to Southern-blot analysis. This identified a PvuII fragment about 1.5 kb in size which was deduced to contain 141 bp 3’ of the trpS gene, 984 bp of the trpS structural gene and only about 400 bp 5’ of the gene (Fig. 1). In a similar experiment using the 32P-labelled oligo mixture as a probe, a fragment 1.5 kb in size, in both cases, lit up confiig that this fragment contained the trpS gene (not shown). Thus possibly lethal sequences 5’ of the gene would have been largely removed.

blunt end wilh’klmow’

&m~raao

Fig. 2. CoWr~ction of the low-copy-number cloning vector pSCLAC5. The vector was isolated by wlectiq for Tc-resistaot, blue colonies tier plating onto YTx2 agar contaiaing 12 ~g Tc/ml and BCIG. It has unique restriction sites for the enzymes EeoRI, &/I, SmI, Xbul, and Pd. ’ Stoker et al.. 1982.

41

A sample of the 4.2-kb EcoRI fragments purified previously, was digested with PvuII and cloned into pUC8 digested with SmaI. Using the puri&d 32P-labelled2.2-kb EcoRLWI f@ment as a probe_ twelve strongly hy~~s~g clones were detected out of 1200 colonies screened. DNA was isolated from one of these and the complete nt sequence of the cloned npS gene was determined (Fig. 3). Since the npS gene was cloned as a PwXI fragment tbis suggested that there were sequences 5’ of the h;Dsgene which hindered the cloning of the 4.2-kb &oRI fragment. The nature of these sequences is unknown. (c) Featms of the WTS-c&g

region

The 3. ~t~urothe~~hil~ trpS gene consists of an ORF of 984 bp commencing with an ATG start codon and ending with a TAA stop codon. The deduced aa sequence of the enzyme agrees with the previously determined primary sequence (Winter and Hartley, 1977) except that our sequence has an additional (third) Arg residue at the C-terminus of the enzyme. However it is possible that this aa is

removed by proteolytic cleavage in the mature protein or is removed by proteolytic ‘nicking’ during enzyme purification. The aa 32 and 42 are Glu and G&I,~~~ti~ei~, whilst aa is Asp. The codon u~sation pattern of the 3, stearothemophilus trpS gene (Table I) more closely resembles that of the B. stearothemophilus tyrosyl tRNA synthetase (tyrs) gene than the E. cob tqd gene. The latter shows strong preference for the cwions CTG (Leu), GTG (Val), CGG (Gin) and GAA (Glu) and does not use the codons ACA (Thr), CGA and CGG (A& and GGA and GGG (Gly) whilst the B. stearothemophilustrpS and &rS genes show a preference for the c&dons GTC (Val), CGA (Gin) and GAC (Asp). The codon utilisation pattern of the B. stearothemophilus tq& gene is more similar to that of B. coliweakly expressed genes (Grosjean and Fiers, 1982) in showing preferences for the codons TIT> ?TC (Phe), GCC > GCT (Ala>, TAT r TAC ,f-l”yrj,CGC > CGT (A& GGC > GG.T (Gly and CCC > CCT (Pro) (although codon preferences ATC > ATT (Ile) and AAC > AAT

TABLE f A

comparison

of the codon u~s~t~o~ of the 3. ~~e~~~rhe~~~~I~~~ph~~yl

tRNA synthetase gene (E @S,

this report) with that

of the E. coii tryptophanyl tRNA synthetase gene (E trp5, Hall et al., 1982) and the B. srearotiemaphilus typrosyl tRNA synthetase gene (B f@,

uuu

Winter et al., 1983)

1 Phe

uuc

5

7

12

3

5

7

1

3

7

4

12

6

1

fO

AUC

1 fe

AUA 1 AUG

Met

6

2

Terd

0

0

Trp

2

6

UGU UGC

CYS

1

1

1

UGA

0

a

0

UGG

4

5

8

0 7 2

8

3

8

I9

0

1

11

0

5

;

1;

22

7

x1

6

13

2

t

16

II

14

4

7

9 AUU

2

Cl

2

4 10

6

7

2

6

8

12

4

12

6

16

5

CGG

0

0

i

0

0

10

10

6

0

1

4

6

4

12

0

10

13

$3

0

a Ter, stop cadon,

6

22

GGG

1 11

10

0

10

19

3

0

4

5

D

13

42

w

Mm Lys mr Ik Phe SW GATCG ATG AAA ACC ATT l-l-l XC 360

ciy II_ Gk Pm Ser G1y ml IIC nlr CCC ATT CAG CCA AGC CGC GTC ATC ACC S60

IlC GIY Am TF 118 CIY Ala Lw Ar) Gin Phe ATT CCC AAC TAT ATT GGG CCC CTG CCC CAG m 360 600

Val Clu Leu Cln His Clu Tyf GTC GAG CTC CAC CAT GAA TAC 620

Asn Cys Tyr Phe Cys lb Vd Asp Cb His Ala Ila lhr WI Trp GA Asp Pro AAC TGC TAT TTT TGC ATC CTT CAC CAA CAC GCC ATT ACC CTT TCG CAA CAC CCA 660 NO 460 His Glu Leu Arg Cln An UC Aq CAC GAA CTC CCC CAA AAC A;$CGC

Arg Lau Ala Ala Leu Tyr Leu Ah Vsl Gly CCC CTC CCC CCT TTC TAT TTC CCC GTC CCC 520

IIC Asp Pm Thr Cln Ah Thr Leu Phe Ile Cln Ser Glu Val Pm Ala His Ah ATC GAC CCC ACG CAA GCG ACG TTC TX ATC CAG TCA GAA GTG CCC GCG CAC CCT 560 560 SaO Clq Ala Ab Trp Met Lw Cln Cg Ile Vd Tyr Ilc Gly Clu Leu Glu Ar6 Met CAA CCC GCT TGG ATC CTC CAG TCC ATC GTT TAT ATC CCC CAA Cl-l GAG CGG ATG 660 600 620 Thr Cln Phe Lp Glu Ly, SIX A,. Gly Lys Clu Ala Vd Sn Ah Cly LeU LeU ACG CAA TTT AAA GAA AAA TCG GCC GGC AM GAG CCC CTC AGC CCC GGC CTG CTC 660 660 Thr Tyr Pm Pm Leu Met Ah Ala Asp Ile Leu Lou Tyr An Thr Aq ACG TAC CCC CCC CTC ATG CCC GCT GAC A-I-T TTC CTT TAT AAC ACC WC 700 720 740

Ue Vd ATC GTC

Pro Vd Gb GIU Asp Cln Lyr Gin His Ilc Clu LeU Thr Ar6 ASP L*l Ala ClU CCC CTC CCC GAA GAC CAA AAG CAG CAT ATC GAA TTG ACG CGC CAT CTC CCC GAG 600 760 760 Pro CIU Ala Arg Ik Pro Arg Phe Atn Lys Alg Tyr Gly CIU LCU Phc Thr Ilc CCC TTC AAC AAA CGG TAT GGC GAG CTC ‘l-IT ACC ATC CCT GAG GCG CCC ATT CCG 600 62.0 Met SW Leu Vd Asp Pro Thr Lyr Lys 6&t Sar Lys Lys Val Cly Ala Ar6 Ilc AAA GTC CCC CCC CCC ATC ATG TCG CTT GTC GAC CCC ACG AAA AAA ATG AGC AAA 900 660 660 WI ‘tlv Leu Leu Asp Asp Ala Lya Thr IIC Ser. Asp Plo Am Pro Lys Ala Tyr IIs TCC GAC CCC AAC CCG AAA GCG TAC ATC ACA CTG CTT GAC CAT GCG MA ACC ATC 960 960 920 GlU Lys Lyr lk Lp SW Ala Val Thr Aq Ser Glu Gly Thr lie m Tyr Atp GAG AAG AAG ATC AAA ACC GcG GTC ACC GAC TCG GAA GGA ACC ATT c~c TAC GAC 1020 IO00 960 SW A,” Lw Lys Clu Ala Lys Pm Gly Ile AAA CAA GCG AAC CCA CCC ATT TCG AhC TTC 1060 Cly Cln Sa GGT CA;o;;G

Leu Am Ile Tyr kr TM LeU SW CTC AAT ATT TAT TCC ACC CTA TCC 1060

Ile Glu Clu Lau Glu Arl Gin Tyr Glu GIy Lyr Gly TF CIY Vd ATC GAG GAG TTG GAG C~~o,“^A TAC CAA CGA AAA CGA ;;A& GGC GTC

Glu Thr Leu Ar6 Pro ik Phe Lys Ala Asp Lw A,. Gin Vd Vd Ilc TTC AAA GCA GAC CTC CCT CAA GTC GTC ATC GAA ACG CT? CGA CCG Al-I II60 II60

Gin GIU CAA GAG II10

Ar6 Tyr His His Trp Met Glu kr Glu Glu Leu Asp Ar6 Vd Leu AW GlU GlY ccc TAT CAC CAT TGc ATG GAA ACC GAG GAG err Ghc CGC CTC rrc .GAT GAA GGG I220 1200 Ah Glu GCC fA$

L,‘s Ala Aa Arg V., Ab SW Giu MC, WI Arg Lyt Met GM Glh Ah AAA GCA AAT CGG GTT GCA TCG GAA ATG GTG CGA AAA ATG GAG CAA Gee I260 1260

Wy Leu Cl) ATC GGG CTC FG

Met

T2 Tl A?6 A,6 Arg I) -__CGG CCC CGG TAAATAGCCGCATCCA~CATACAhAAACGGGcA I360 I320

Fig. 3. The complete nt sequence of the B. stearothemophilus trpS gene. The sequence shown has been determined completely on both strands. SD refers to the putative Shine-Dalgarno sequence (RBS). Putative RNA polymeraae recogntion s&&s (promoters PI and PZ), -35 and -10 regions, are indicated by solid lines above the relevant sequence. A, A’ and B, B’ refer to theinverted repeats, and Tl and 72 to putative transcription terminators. * Stop codon of the hpS gene.

43

(Asn) are characteristic of E. coli highly expressed genes). The codon bias seen for weakly expressed genes seen in E. coli may thus occur in B. stearothermophilus since the WTS is produced at only 0.02% of the B. stearothermophifus soluble cell protein (Atkinson et al., 1979). In common with other thermophilic genes for which the DNA sequence has been determined, namely the tyrosyl tRNA synthetase (Winter et al., 1983), a-amylase (Ihara et al., 1985), neutral protease (Takagi et al., 1985) and lactate dehydrogenase (D.A. Barstow, A.R. Clarke, W.N. Chia, D. Wigley, A.F. Sharman, J.J. Holbrook, T. Atkinson and N.P. Minton, manuscript in preparation) genes, the B. stearothemophilus trpS gene shows a bias for G or C residues at the third nt of the codons (Table II). This is seen also with the E. coli trpS gene but to a lesser extent, and it is not a feature typical of E. coli genes (Grosjean and Fiers, 1982). Hence this may be a feature typical of thermophilic genes. (d) Features of the DNA non-coding region

At 6 bp upstream from the ATG start codon is a weak potential RBS with a predicted (Tinoco et al., 1973) AG of -9.4 kcal. This lies below the limit of -14 to -23 kcal which Moran et al. (1982) found for other Bacillus genes. This RB S may not function very efficiently in B. stearothemophilus which could in part explain the low level synthesis of the WTS

in B. stearothemophilus cells. Upstream of this sequence there are two putative transcriptional promoters PI and P2 with overlapping inverted repeat sequences that if transcribed could give hairpin loop structures A,A’(AG = -18.4 kcal) and B,B’(AG = -10.6 kcal) (Fig. 2). The latter of these hairpin structures resembles E. coli Rho-independent transcription terminators since it is followed by a stretch of T residues (eleven out of twelve). It is not known what role, if any, these sequences play in expression of the trpS gene but they may clearly a&& the level of transcription of the gene and may also, in part, account for the low levels of the enzyme produced by B. stearothermophilus cells. It is interesting to note that the E. coli trpS gene has a potentially hairpin-loop-generating-sequence overlapping with a promoter (Hall and Yanofsky, 1981). Hence these sequences may be functionally similar and may be involved in binding regulatory proteins. No striking homology to the consensus E. coli trp operator has been observed for both the E. coli and B. stearothemophilus trpS genes. Downstream of the TAA stop codon is a G + C-rich region (X2) of dyad symmetry (AG = -33.8 kcal) followed by a stretch of T residues, a sequence characteristic of E. coli Rho-independent transcription terminators. The stem loop is preceded by a stretch of A residues and hence this sequence may function as a transcription terminator in both orientations. Upstream from this sequence is a smaller hairpin loop (TI) with a calculated AG of -9.2 kcal. These

enzyme

TABLE II

Comparison of the GC content of the B. stearothennophilus t&3 gene with other thermophihc Bacillus genes, the E. coli trpS gene and an average of 64 E. coli genes Gene

%G + C total

%G+Catthird base of codon

Reference

This report Ihara et al. (1985) Takagi et al. (1985)

B. stearothermophilus trpS

54

69

Bacillus spp. a-amylase

50

57

B. stearothermophilus

58

12

52

60

neutral protease B. stearothermophilus

lactate dehydrogenase B. srearothermophilus tyrS

54

70

E. coli trpS

52

60

average of 64

53

55

E. coli genes

D.A.B.,A.R. Clarke, W.N. Chia, D. Wigley,A.F.S., J.J. Holbrook, T.A. and N.P.M., manuscript in preparation Winter et al. (1983) Hall et al. (1982) Grosjean and Fiers (1982)

44

structures may therefore act together to terminate transcription of the t+S gene. (e) Comeluslons The cloning and nt sequence determination of the B. stearothennophilus trpS gene has enabled us to

confii (and resolve ambiguities in) the primary structure of the WTS enzyme. These data are necessary for detailed X-ray crystallographic analysis of the enzyme. In addition, to facilitate isolation of large quantities of the WTS enzyme for both crystallographic and biochemical studies, we are constructing high expression vectors. Finally, we are now in a position to carry out aa replacement experiments by site-directed mutagenesis to analyse in detail the mode of action of the WTS enzyme.

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