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...

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Bibliographic Details
Published in:The Journal of biological chemistry 1994-02, Vol.269 (6), p.4027-4034
Main Authors: FILIZ COSKUN-ARI, F, SKOKOTAS, A, MOE, G. R, HILL, T. M
Format: Article
Language:English
Subjects:
Bacterial Proteins - chemistry
Bacterial Proteins - ultrastructure
Biological and medical sciences
Biophysical Phenomena
Biophysics
Centrifugation, Density Gradient
Chromatography, Gel
Circular Dichroism
DNA Replication
Escherichia coli
Escherichia coli - genetics
Escherichia coli Proteins
Fundamental and applied biological sciences. Psychology
Isoelectric Point
Molecular and cellular biology
Molecular genetics
Peptide Mapping
Protein Structure, Secondary
Replication
Spectrophotometry, Ultraviolet
Ultracentrifugation
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(17)41737-6