Nucleotide sequence of the hemB gene of Escherichia coli K12

Abstract

The hemB gene of Escherichia coli K12, coding for porphobilinogen synthase (PBG-S; syn., 5-aminolevulinic acid dehydratase, ALA-D), was cloned following insertion of an EcoRI fragment of plasmid F'13 into the mobilizable vector pCR1. The hybrid plasmid carrying the hemB gene was able to complement a hemB mutant of E. coli K12: not only was the PBG-S activity of the mutant restored after the acquisition of the hemB gene, but it was about ten times higher than that of the wild type. Subcloning of the original EcoRI fragment (14.6 kb) enabled us to locate the hemB gene on an NruI-HpaI fragment of about 1.1 kb. The hemB promoter was located toward the NruI end of the fragment, as shown by the use of the pKO promoter-probe series of vectors. Sequencing of the hemB gene indicated the presence of an open reading frame (ORF) of 1051 nucleotides, which should correspond to the HemB protein. Primer extension experiments enabled us to identify the 5' end of the hemB mRNA, and to deduce the -10 and -35 regions of the hemB promoter. Protein synthesis performed by an in vitro coupled transcription-translation system, showed the presence of a protein of about 35 kDa. This is in agreement with the molecular weight of the HemB protein (35.6 kDa), as deduced from the nucleotide sequence of the gene. Comparison of the amino acid sequences of E. coli and human PBG-S allowed the detection of several regions of strong homology between the two proteins. Two of these regions correspond, as expected, to the putative zinc-binding and catalytic sites of the human PBG-S.