Skip Navigation



Nucleic Acids Research Advance Access published online on August 9, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl316
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (999K) Freely available
Right arrow Screen PDF (999K) Freely available
Right arrowOA All Versions of this Article:
34/14/3834    most recent
gkl316v2
gkl316v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Kamionka, A.
Right arrow Articles by Hillen, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kamionka, A.
Right arrow Articles by Hillen, W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.

Article

Induction of single chain tetracycline repressor requires the binding of two inducers

Annette Kamionka, Marius Majewski, Karin Roth, Ralph Bertram, Christine Kraft and Wolfgang Hillen*

Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg Staudtstrasse 5, 91058 Erlangen, Germany

*To whom correspondence should be adressed. Tel: +49 9131 85 28081; Fax: +49 9131 85 28082; Email: whillen{at}biologie.uni-erlangen.de

Received February 15, 2006. Revised March 20, 2006. Accepted April 12, 2006.

In this article we report the in vivo and in vitro characterization of single chain tetracycline repressor (scTetR) variants in Escherichia coli. ScTetR is genetically and proteolytically stable and exhibits the same regulatory properties as dimeric TetR in E.coli. Urea-dependent denaturation of scTetR is independent of the protein concentration and follows the two-state model with a monophasic transition. Contrary to dimeric TetR, scTetR allows the construction of scTetR mutants, in which one subunit contains a defective inducer binding site while the other is functional. We have used this approach to establish that scTetR needs occupation of both inducer binding sites for in vivo and in vitro induction. Single mutations causing loss of induction in dimeric TetR lead to non-inducible scTetR when inserted into one half-side. The construction of scTetR H64K S135L S138I (scTetRi2) in which one half-side is specific for 4-dedimethylamino-anhydrotetracycline (4-ddma-atc) and the other for tetracycline (tc) leads to a protein which is only inducible by the mixture of tc and 4-ddma-atc. Fluorescence titration of scTetRi2 with both inducers revealed distinct occupancy with each of these inducers yielding roughly a 1:1 stoichiometry of each inducer per scTetRi2. The properties of this gain of function mutant clearly demonstrate that scTetR requires the binding of two inducers for induction of transcription.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.