4928 Nucleic Acids Research, Vol. 20, No. 18 A 1.6 kb region of Bacilus firmnus 0F4 DNA encodes a homolog of Escherichia coli and yeast DNA topoisomerases and may contain a translational readthrough of UGA D.Mack Ivey+, Jianbo Cheng and Terry Ann Krulwich* Department of Biochemistry, Mount Sinai School of Medicine of CUNY, 1 Gustave Levy Place, New York, NY 10029, USA Submitted August 3, 1992 EMBL accession no. Z14112 Several recombinant plasmids from libraries of alkaliphilic Bacillus firmus OF4 have been shown to reverse the sodium sensitivity of an Escherichia coli mutant carrying a deletion in the nhaA Na+/H+ antiporter gene (1, 2, 3). One clone, designated pJB20, was sequenced and shown to contain, in addition to the 1.2 kb region associated with sodium resistance (3), a 1.6 kb region of DNA encoding an apparent homolog of the DNA topoisomerases of E. coli (4, 5) and yeast (6). The region contains two open reading frames; the first ends with a UGA termination codon and is predicted to encode a 30 kDa polypeptide with 26% identity and 54% similarity to the N- terminal 292 residues of the E. coli topoisomerase 3 (topo-3). The second open reading frame begins with an in-frame AUG and is predicted to encode a 27 kDa protein with 27% identitiy and 49% similarity to residues 300-541 of topo-3. The intergenic region contains 5 codons in addition to the termination codon; three codons would specify amino acid residues that are highly conserved among the E. coli and yeast topoisomerases (Figure 1). Translational readthrough of UGA has been reported for both eukaryotes and prokaryotes (7), including Bacillus subtilis (8), where UGA can be decoded as tryptophan at low efficiency. Thus, it is possible that translational readthrough of the B.firmus OF4 topoisomerase open reading frames creates a full-length 60 kDa protein that shares 27% overall sequence identity and 52 % similarity with the E. coli topo-3 and approximately 40 % similarity with the other topoisomerases. We have verified the UGA sequence by PCR-amplifying a 385 bp region of B.firmus OF4 chromosomal DNA encompassing the termination codon. Products from two reactions were cloned and sequenced as described previously (1), and shown to contain the UGA codon. ACKNOWLEDGEMENT This work was supported by grant DCB9018231 from the National Science Foundation. REFERENCES 1. Ivey,D.M., Guffanti,A.A., Bossewitch,J.S., Padan,E. and Krulwich,T.A. (1991) J. Biol. Chem. 266, 23483-23489. 2. Ivey,D.M., Guffanti,A.A., Shen,Z., Kudyan,N. and Krulwich,T.A. (1992) J. Bacteriol. 174, 4878-4884. 3. Ivey,D.M., Guffanti,A.A. and Krulwich,T.A. (1992) In Bakker,E.P. (ed.), Alkali Cation Transport System. CRC Press, Boca Raton. In press. 4. Tse-Dinh,Y.-C. and Wang,J.C. (1986) J. Mol. Biol. 191, 321-331. 5. Digate,R.J. and Marians,K.J. (1989) J. Biol. Chem. 264, 17924-17930. 6. Wallis,J.W., Chrebet,G., Brodsky,G., Rolfe,M. and Rothstein,R. (1989) Cell 58, 409-419. 7. Parker,J. (1989) Microbiol. Rev. 53, 273-298. 8. Lovett,P.S., Ambulos,N.P., Mulbry,W., Noguchi,N. and Rogers,E.J. (1991) J. Bacterial. 173, 1810-1812. Bfi SLGRVQTPLLAEIVKRERLLIBQFTAENFWTVKFNNQGN EWFHEKENRIFTEEQDEQLCELVRN Ec3 SVGJRVQTPVLGLVVRREEEIENFVAK:.PEVKAHIVTPADERFTAIWQPSEACEP., Y,QEEEGkLLHRPLAEHVVNRISG Ecl SAGRVQSVAVRLAVEREREIKAYVPE,F,WEVDA STTTPSGEALAL ... QVTHQNDKPFRPVNKEQTQAAVSLLEK Yst SYGT'CQFPTLGFW..DIU:ERIRNF..VP.,-...YI.QLVVENKDNGGTTTF. . QWDRGHLPDRLSVLTFYETCIETAGNV... UGA AUG Bfi QSSTIMEMKEEMRTYQPPLLY*LSTLMDAGNAFI Ec3 QPAIVT'TSYNDKRESSEAPLPFSLASLQIEAAKRF Ecl ARYSVLERED,KPTTSKPSAPLITSTLQAASTRL Yst ..AQVVDLKSKPTTKYRP''I,.PLTTVELQKNCARYLI MERITES... .DAREL 'RYLPEE.... HFAGR 3TNLSQD .... AV ... PDTFPHAMDLKSLVEK Figure 1. Alignment of the predicted amino acid sequence of the region of the B.firmus OF4 DNA topoisomerase locus that contains a potential readthrough UGA with the corresponding regions of E. coli and yeast topoisomerases. Abbreviations are: Bfi, B.finnus OF4 topoisomerase; Ec3, E. coli topo-3 (5); Ecl , E.coli topo-1 (4); and Yst, Saccharomyces cerevisiae topo-3 (6). Shown are residues 194-344 of E.coli topo-3, 192 -322 of E.coli topo-1, and 230-376 of the yeast topo-3. * To whom correspondence should be addressed +Present address: Department of Biological Sciences, Univeresity of Arkansas, 632 SCEN, Fayetteville, AR 72701, USA .=) 1992 Oxford University Press