Nucleotide sequence of the Escherichia coli thymidine kinase gene provides evidence for conservation of functional domains and quaternary structure

Abstract

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 900 bp EcoRI-SalI fragment) which contained the E. coli thymidine kinase gene (tdk). The nucleotide sequence of the 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 615 bp which was capable of encoding a 205-amino-acid polypeptide with a predicted Mr 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 Mr 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.(ABSTRACT TRUNCATED AT 250 WORDS)