Hexamerization of RepA from the Escherichia coli plasmid pKL1.
The Escherichia coli plasmid pKL1 is one of the smallest bacterial plasmids. It encodes a single, autoregulating structural gene, repA, responsible for replication and copy number control. The oligomerization of RepA was previously proposed as the basis of a strategy for pKL1 copy number control. To elucidate the oligomerization properties of RepA in solution, RepA was expressed in E. coli; purified by ion exchange and hydrophobic chromatography; and examined in solution by spectrapolarimetry, light scattering, sedimentation velocity, and equilibrium ultracentrifugation. RepA behaved as a concentration-dependent equilibrium of dimers and hexamers. Conformational parameters of the RepA hexameric complex were determined. These results support the proposed autogenous regulatory model whereby RepA hexamers negatively regulate repA expression thereby affecting the copy number control of pKL1. RepA of pKL1 is the first plasmid replication initiation protein documented to be in dimeric-hexameric forms.
Burian
Ján
J
Microtek International Ltd., 6761 Kirkpatrick Crescent, Saanichton, British Columbia, V8M 1Z8, Canada.
Ausió
Juan
J
Phipps
Barry
B
Moore
Susan
S
Dougan
Douglas
D
Kay
William
W
D001483:Base Sequence; D002850:Chromatography, Gel / Q000379:methods; D004265:DNA Helicases; D004261:DNA Replication / Q000502:physiology; D004268:DNA-Binding Proteins; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics; D008027:Light; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012542:Scattering, Radiation; D015534:Trans-Activators; D014461:Ultracentrifugation / Q000379:methods
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases