NUCLEOTIDE-SEQUENCE OF THE ESCHERICHIA-COLI THYMIDINE KINASE GENE PROVIDES EVIDENCE FOR CONSERVATION OF FUNCTIONAL DOMAINS AND QUATERNARY STRUCTURE

Using lambda bacteriophage clones from the Kohara Escherichia coli library spanning minutes 25.5 to 28.5 on the E. coli chromosome (strain W3110), two overlapping DNA fragments were identified which were able to confer thymidine kinase (TK) enzyme activity to a TK- strain of E. coli (KY895). This genetic complementation assay was used in concert with subcloning procedures to identify the minimal region (a 900bp EcoRI-SalI fragment and a small portion of the adjoining downstream fragment was determined. Computer analysis of the derived sequence indicated the presence of a rightward-reading open reading frame of 615bp which was capable of encoding a 205-amino-acid polypeptide with a predicted M(r) of 23458 daltons. The in vivo transcriptional activity of this locus was confirmed by Northern blot hybridization analysis of RNA isolated from E. coli JM101 or KY895 which detected a 650-nucleotide RNA transcribed from this region. This places the tdk gene at approximately minute 27.35 on the E. coli W3110 chromosome, about 15 kb downstream from the narG locus and approximately 25 kb upstream of the trp operon. Although the predicted M(r) of the E. coli TK protein was 23.5 kDa, gel-filtration analyses suggested that, like eukaryotic thymidine kinases, the active form of this enzyme is a multimeric complex. Furthermore, comparisons between the predicted amino acid sequences of the TK proteins encoded by E. coli, bacteriophage T4, and a mammalian poxvirus (vaccinia virus) revealed several regions of amino acid identity, one of which appears to be the ATP-binding site, thereby suggesting that the essential functional domains of this enzyme have been well-conserved throughout evolution.