Biophysical characteristics of Tus, the replication arrest protein of Escherichia coli.
Tus, a DNA-binding protein, mediates arrest of DNA replication in Escherichia coli. Tus binds to DNA sequences called Ter sites, located in the terminus region of the chromosome, and forms replication-arrest complexes that block movement of DNA replication forks in a polar fashion. We have analyzed Tus to determine some of its physical parameters and biochemical characteristics. Native Tus had an 8(20,w) of 3.2, a Stokes' radius of 23 A, an axial ratio of 2, and a molar absorption coefficient of 39,700 M-1 cm-1. The data also indicated that Tus existed as a monomeric protein in solution and when complexed with its cognate DNA binding site. Secondary structure estimated from the circular dichroism spectrum suggested that Tus consisted of 40% alpha-helix, 0% beta-sheet, 15% turn, and 45% aperiodic structure. The isoelectric point of native Tus (pH 7.5) was significantly different than that calculated from its amino acid sequence (pH 10.1), possibly because the tertiary structure of Tus perturbs the ionization of several residues. In addition, partial proteolytic digests of free Tus protein did not produce a subfragment of Tus that retained DNA binding activity, but did demonstrate that Tus was resistant to proteolysis when complexed with a Ter site.
Coskun-Ari
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Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania 19104.
Skokotas
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A
Moe
G R
GR
Hill
T M
TM
D001426:Bacterial Proteins / Q000737:chemistry* / Q000648:ultrastructure; D055592:Biophysical Phenomena; D001703:Biophysics; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D007526:Isoelectric Point; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C061910:tus protein, Bacteria; C489236:tus protein, E coli