The University of Southampton
University of Southampton Institutional Repository

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.

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.
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.
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 (KD) 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 KD 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 KD by 200-300-fold, whereas the A173T substitution increased KD 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.
11989-11999
Neylon, Cameron
697f067b-db25-4c41-9618-28f4b74f73aa
Brown, Susan E.
e802b7a1-70e7-4a8b-a108-1a8e93b09061
Kralicek, Andrew V.
5f8de1fd-0d46-4b6c-95d4-c1e8ca5e55f1
Miles, Caroline S.
772c7c83-6cd0-4e39-b86c-c2898d7ec125
Love, Christopher A.
85ba5e10-1ba7-48af-883c-b84d65d88705
Dixon, Nicholas E.
29679e00-7adc-4c6d-bf48-bc68d756a6fa
Neylon, Cameron
697f067b-db25-4c41-9618-28f4b74f73aa
Brown, Susan E.
e802b7a1-70e7-4a8b-a108-1a8e93b09061
Kralicek, Andrew V.
5f8de1fd-0d46-4b6c-95d4-c1e8ca5e55f1
Miles, Caroline S.
772c7c83-6cd0-4e39-b86c-c2898d7ec125
Love, Christopher A.
85ba5e10-1ba7-48af-883c-b84d65d88705
Dixon, Nicholas E.
29679e00-7adc-4c6d-bf48-bc68d756a6fa

Neylon, Cameron, Brown, Susan E., Kralicek, Andrew V., Miles, Caroline S., Love, Christopher A. and Dixon, Nicholas E. (2000) 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. Biochemistry, 39, 11989-11999. (doi:10.1021/bi001174w).

Record type: Article

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 (KD) 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 KD 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 KD by 200-300-fold, whereas the A173T substitution increased KD 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.

Text
0012-BiochemTus.pdf - Other
Download (172kB)

More information

Published date: October 2000

Identifiers

Local EPrints ID: 26769
URI: http://eprints.soton.ac.uk/id/eprint/26769
PURE UUID: c855230a-644e-4051-88d3-dfabb7a68fb8

Catalogue record

Date deposited: 11 Apr 2006
Last modified: 25 Nov 2019 19:26

Export record

Altmetrics

Contributors

Author: Cameron Neylon
Author: Susan E. Brown
Author: Andrew V. Kralicek
Author: Caroline S. Miles
Author: Christopher A. Love
Author: Nicholas E. Dixon

University divisions

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×