Three tandemly repeated structural genes encoding tRNAf1Met in the metZ operon of Escherichia coli K-12

Gene, 138 (1994) 261-262 0 1994 Elsevier Science B.V. All rights reserved. SSDI 0378-1119 (93) E0616-L

GENE

261

037%1119/94/$07.00

07663

Three tandemly repeated structural genes encoding tRNAEf in the met2 operon of Escherichia coli K-12” (Recombinant plasmid)

Tsuyoshi

DNA; met Y genes; spacer region; formylmethionyl-tRNA;

Kenri, Fumio Iniamoto

and Yasunobu

RecA-dependent recombination; multicopy

Kano

Department of Molecular Genetics, Institute of Molecular and Cellular Biologyfor Pharmaceutical Sciences, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto, Japan Received by A. Nakazawa:

21 July 1993; Revised/Accepted:

16 September/20

September

1993; Received at publishers:

26 October

1993

SUMMARY

The metZ operon of Escherichia coli K-12 consists of three tandemly repeated structural genes encoding tRNAp, separated by two well-conserved spacer sequences. A multicopy plasmid carrying an intact metZ operon is unstable, deleting tRNAp -coding regions by RecA-dependent recombination during cell growth.

Two initiator tRNA species, tRNAy and tRNAp, have been identified in E. coli and shown to be encoded by the genes metZ and metY, mapped at 60.2 min and 69.0 min, respectively (for review, see Gualerzi and Pon, 1990). metY is located in the most proximal region of the nusA operon as one tRNAr-encoding structural gene, whereas metZ was found by nt sequence analysis of the gene cloned in a multicopy vector to consist of two tandemly repeated structural genes encoding tRNAF”‘, forming an operon (Nagase et al., 1988). A cloned gene in a multi-copy plasmid is known to often be inactivated or eliminated during plasmid replication if overexpression of this gene is harmful to the host cells. The tRNAy’ Correspondence to: Dr. Y. Kano, Department of Molecular Genetics, Institute of Molelcular and Cellular Biology for Pharmaceutical Sciences, Kyoto Yamashina-ku,

Pharmaceutical Kyoto 607,

University, 1, Shichono-cho, Misasagi, Japan. Tel. (81-075) 5954720; Fax

(81-075) 595-4799. *On request, the authors will supply detailed the conclusions reached in this Brief Note.

experimental

evidence for

Abbreviations: Ap, ampicillin; bp, base pair(s); kb, kilobase or 1000 bp; LB, Luria-Bertani (medium); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; tRNAy, formylmethionine-specific transfer RNA; wt, wild type; XGal, 5-bromo-4-chloro-3-indolyl-f3-p-galactopyranoside.

operon cloned in a plasmid has been reported to be frequently deleted by recombination between the first and second tRNAy’-coding sequences (Komine et al., 1990). To determine whether metZ operon carried by pMZ17 (Nagase et al., 1988) remains intact or is rearranged, we compared the sizes of restriction fragments of the plasmid and the genomic DNA of E. coli K-12 wt strain YKllOO (Kenri et al., 1991) by Southern blot hybridization and found that DNA of about 0.1 kb had been deleted in the metZ operon carried by pMZ17. To determine the nt sequence of the intact metZ operon, we recloned the 1.3-kb TaqI fragment including metZ (Kenri et al., 1991) from the YKllOO genomic DNA. A JM83 (YanischPerron et al., 1985) derivative harboring a metZ deletion mutation (Kenri et al., 1991) was transformed with plasmids in which about 1.3-kb fragments of genomic DNA digested with TuqI were inserted into the AccI site of pUC19 (2.6 kb). White-colored colonies were selected on LB-agar plates containing 50 ug XGal and 50 ug Ap/ml, and plasmids were extracted from large colonies since a mutant depleted of tRNAF grows more slowly than wt cell (Kenri et al., 1991). One of these 3.9-kb plasmids hybridizable with the 33-mer synthetic oligo (Fig. 1) complementary to the spacer sequence of the metZ gene,

262 t1 -30 -20 -10 10 20 AAGGTTGCAT~AAAACGCGA~CGGAG~~_~~GCGCATCC~CGGACGCG~GGTGGAGCA~ AAGGTTGCATCAAAACGCGAGCGGAGThTAGTGCGCATCCACGGACGCGGGGTGGAGCAG CCTGGTAGCTCGTCGGGCTCATAACCCGAAGGTCGTCGCTTCAAATCCGGCCCCCGCAAC 80 CCTGGTAGCTCGTCGGGCTCATAACCCGAAGGTCGTCGGTTCAAATCCGGCCCCCGCAAC 80 CAATTAAAATTTGATGAAGTAAAGCAGTACGGTGACGCGGGGTGGAGCAGCCTGGTAGCT140 ~----~---_._______--------_____----_----------_-_---____-_-_ 82 CGTCGGGCTCATAACCCGAAGGTCGTCGGTTCAAATCCGGCCCCCGCAACCAATCAAATT203 90 TGATGAAGTAAAAGCAGTACGGTGACGCGGGGTGGAGCAGCCTGGTAGCTCGTCGGGCTC 260 TGATGAAGTAAAAGCAGTACGGTGACGCGGGGTGGAGCAGCCTGGTAGCTCGTCGGGCTC150 ATAACCCGAAGGTCGTCGGTTCAAATCCGGCCCCCGCAACCAATTATTGAACACCCTAAC320 ATAACCCGAAGGTCGTCGGTTCAAATCCGFCCCCCGCAACCAATTATTGAACACCCTAAC210 GGGTGTTTTTTTGTTTCTGGTCTCCCATAAAAAAGCGCCATTCAGCGCCTTTTTATCATC380 GGGTGTTTTTTTGTTTCTGGTCTCCCATAAAAAAGCGCCATTCAGCGCCTTTTTATCATC270

nzetZ may be important

for accurate

tRNAp-coding

sequence,

combination

between

genes for tRNAf’. demly

repeated

plasmid,

PM213 pMZl7

Fig. 1. The nt sequences of merZ of pMZ1.i (upper) and pMZ17 (lower). The underlined sequences indicate structural genes encoding tRNAp. Dashed line represents the region deleted in tnrtZ of pMZI7. Double underlined are the consensus sequences of m&Z promoter 1988). The double-wavy line shows the complementary synthetic oligo used for Southern are numbered from left to right, + (Nagase

blot hybridization

etal. sequence of

(Nagase

analysis. The nt start point

I being the transcriptional

et al., 1988).

possibly carrying the intact merZ operon, was named pMZ13. The nt sequence of a 458-bp portion of the 1.3-kb TclqI fragment in pMZl3 is shown in Fig. 1. Three structural genes encoding tRNAz”’ are found to be tandemly repeated with spacer regions between them. The nt sequences of the three structural genes are identical. Two spacer regions are of the same nt length (33 bp) but show some heterogeneity in their nt sequences. The residue at nt 85 is T in the first spacer region instead of C at nt 195 in the second spacer region. Four A’s and three A’s are present at nt 86 to 89 and nt 101 to 103, respectively, in the first spacer, whereas three A’s and four A’s are present at nt 196 to 198 and nt 210 to 213, respectively, in the second spacer. Thus, the ancestral gene encoding met2 may have contained two tandemly repeated sequences encoding tRNA~ and a spacer region which may have been duplicated during a recent brief period of evolution. Alternatively, the nt sequence preserved in the present

of the

transformants

relative YKllOO strain

A mixture amounts

second

the instability sequences

of tan-

cloned

with pMZ 13 were cultured of 3.9-kb

of about

and

mutation

were

plasmids

slowly and only 3.9-kb plasmids

in

from

transformants with

in a over-

I to 2 was recovered

In contrast,

recA-56

3.7-kb

re-

structural

of 50 ltg Ap/ml, and plasmids

transformants. harboring

by homologous

and

To investigate tRNAF’-coding

night in the presence extracted.

possibly

the first

them. The 3.9-kb plasmids CCCTTCTCGCCACGTCGCCCCATCGGCAAAATACGCTT 418 CCCTTCTCGCCACGTCGCCCCATCGGCAAAATACGCTT 308

processing

precursor tRNA. It is suggested from nt analysis that the metZ of pMZ17 has lost the first spacer and the second

of a

pMZl3

grew

could be detected

from

gave 1.3-kb T~ql fragments,

and the 3.7-kb plasmids gave l.l-kb TaqI fragments which were found to consist of three structural genes and one structural gene encoding tRNAF’, respectively (data not shown). We detected overexpressed levels of both immature and mature tRNAy molecules in YK 1100 transformants. These results suggested that the mctZ operon cloned in a multicopy plasmid is somewhat harmful to cehs and so deletion mutants of tRNAk?-coding regions are presumably selected during cell growth by RecArecombination between tRNAEt-coding dependent regions.

REFERENCES Gualerzi,

CO.

and Pon. CL.:

Initiation

of mRNA

translation

in pro-

karyotes. Biochemistry 29 (1990) 5881-5889. Kenri, T., Kohno, K., Goshima, N., Imamoto, F. and Kano, Y.: Construction and characterization of an Escherichirr coli mutant with a deletion of the merZ gene encoding tRNAF. Gene 103 (1991) 31.-36. Komine, Y., Adachi, T.. Inokuchi, H. and Ozeki, H.: Genomic organization and physical mapping of the transfer RNA genes in Escherkhia co/i K-12. J. Mol. Biol. 212 (1990) 5799598. Nagase. T’., Ishii, S. and Imamoto, F.: Differential trol of the two tRNAy genes of Escherichiu

transcriptional co/i K-12.

con-

Gene

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i 1988) 49957. Yanisch-Perron, C.. Vieira, J. and Messing, J.: Improved M I3 phage cloning vectors and host strains: nucleotide sequences of the M 13mplX and pUC19

vectors.

Gene 33 (1985) 103 119.