Investigation of the domain structure of the Escherichia coli DNA gyrase A protein.
thesisposted on 19.11.2015 by Richard Jonathan. Reece
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Treatment of Escherichia coli DNA gyrase A protein with trypsin generates two large, stable fragments of molecular masses 64 kDa and 33 kDa which are derived respectively from the N-and C-terminus of GyrA. The trypsin-cleaved A protein (A'), can support DNA supercoiling, relaxation and other reactions of gyrase. The isolated 64 kDa fragment will also catalyse DNA supercoiling but the 33 kDa fragment shows no enzymic activity. An amber mutation, introduced into gyrA near the point which corresponds to the tryptic cleavage site, yields GyrA(1-573) which shares the same properties as the 64 kDa tryptic fragment. Using genetic engineering, large numbers of 3'-gyrA deletion mutants have been produced; those encoding a protein smaller than 58 kDa (GyrA(1-523)) did not obviously overproduce truncated GyrA. GyrA(1-523) shows similar enzymic properties to GyrA(1-573) but cannot perform DNA supercoiling. Deletion of fifty C-terminal residues from GyrA(1-573) has the effect of disrupting part of the protein essential for supercoiling. I propose that the N-terminal 64 kDa represents the DNA breakage/reunion domain of the A protein, while the 33 kDa fragment contributes to gyrase-DNA complex stability. Certain N-terminal deletion mutants of the GyrA protein were also constructed. Removal of the N-terminal 6 amino acids had no effect on the properties compared to GyrA. Removal of the N-terminal 69 amino acids yields a protein with no supercoiling or cleavage ability. The start of the N-terminal breakage-reunion domain is probably located within this 63 amino acid region. The domains of GyrA were investigated by microcalorimetry. GyrA yields two unfolding transitions. GyrA(1-573) and GyrA(1-523) both yield a single unfolding transition corresponding to one of the GyrA transitions. Therefore GyrA contains two structural domains that can be assigned to the functional moieties described above. GyrA(1-573) has been crystallized, and four crystal forms identified. A diffraction pattern to 7 A has been obtained.