Interaction of the Escherichia coli Replication Terminator Protein (Tus) with DNA: A Model Derived from DNA-Binding Studies of Mutant Proteins by Surface Plasmon Resonance

Date

2000

Authors

Neylon, David Cameron
Brown, Susan Elizabeth
Kralicek, Andrew V
Miles, Caroline
Love, Christopher
Dixon, Nicholas

Journal Title

Journal ISSN

Volume Title

Publisher

American Chemical Society

Abstract

The Escherichia coli replication terminator protein (Tus) binds tightly and specifically to termination sites such as TerB in order to halt DNA replication. To better understand the process of Tus-TerB interaction, an assay based on surface plasmon resonance was developed to allow the determination of the equilibrium dissociation constant of the complex (K(D)) and association and dissocation rate constants for the interaction between Tus and various DNA sequences, including TerB, single-stranded DNA, and two nonspecific sequences that had no relationship to TerB. The effects of factors such as the KCl concentration, the orientation and length of the DNA, and the presence of a single-stranded tail on the binding were also examined. The K(D) measured for the binding of wild type and His6-Tus to TerB was 0.5 nM in 250 mM KCl. Four variants of Tus containing single-residue mutations were assayed for binding to TerB and the nonspecific sequences. Three of these substitutions (K89A, R198A, and Q250A) increased K(D) by 200-300-fold, whereas the A173T substitution increased K(D) by 4000-fold. Only the R198A substitution had a significant effect on binding to the nonspecific sequences. The kinetic and thermodynamic data suggest a model for Tus binding to TerB which involves an ordered series of events that include structural changes in the protein.

Description

Keywords

Keywords: amino acid; bacterial protein; mutant protein; potassium chloride; replication terminator protein; single stranded DNA; Tus protein; unclassified drug; amino acid substitution; article; association; concentration response; dissociation constant; DNA bindi

Citation

Source

Biochemistry

Type

Journal article

Book Title

Entity type

Access Statement

License Rights

DOI

10.1021/bi001174w

Restricted until

2037-12-31