DNA sequence of the E. coli gyrB gene: application of a new sequencing strategy

We have determined the sequence of the IS. coll gyrB gene, using a new sequencing approach in which transposition from a mini-Mu plasmid into the DNA provides random start points for dideoxynucleotide sequence analysis. The gyrB sequence corresponds to a protein 804 amino acids long; a previously isolated protein fragment with partial enzymatic activity has been identified as the C-termlnal half-molecule. A plausible terminator of gyrB transcription is located just beyond the structural gene. DNA gyrase from E^. coli is made up of two subunlts, A and B, with molec- ular weights of about 100,000 and 90,000, respectively. The corresponding structural genes have been identified, originally as loci of mutations to drug-resistance: one class of mutations in gyrA is responsible for resistance to quinolone compounds (e.g., nalidixic acid, oxolinic acid, and norfloxacin), while a class of mutations in gyrB confers resistance to the coumarin anti- biotics novobiocin, coumerraycin A^, and clorobiocin. The use of these inhibitors has helped to show that the A subunit is responsible for the DNA breakage-rejoining activity of DNA gyrase, and that the B subunit carries out ATP hydrolysis. The DNA-supercolling and DNA-relaxing activities require the presence of both subunits; the B subunit is apparently needed not only for the energy coupling used in the supercoiling reaction but even for the less demanding relaxation reaction. The Isolation of a fragment of the Gyr B protein which was able to confer a DNA-relaxing activity on the Gyr A protein supported the Idea that there are distinct functional domains of the B subunit; this complex was no longer active in DNA supercoiling or ATP hydrolysis. (For reviews incorporating the information cited above, see (1, 2)). As a first step In more detailed studies of the functional organization of the gyrB gene, we have now determined the DNA sequence of the gene and Its