Universität zu Köln

Fragel, Susann M., Montada, Anna, Heermann, Ralf ORCID: 0000-0003-0631-6156, Baumann, Ulrich, Schacherl, Magdalena ORCID: 0000-0002-5478-2509 and Schnetz, Karin ORCID: 0000-0003-4693-6202 (2019). Characterization of the pleiotropic LysR-type transcription regulator LeuO of Escherichia coli. Nucleic Acids Res., 47 (14). S. 7363 - 7380. OXFORD: OXFORD UNIV PRESS. ISSN 1362-4962

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Abstract

LeuO is a pleiotropic LysR-type transcriptional regulator (LTTR) and co-regulator of the abundant nucleoid-associated repressor protein H-NS in Gammaproteobacteria. As other LTTRs, LeuO is a tetramer that is formed by dimerization of the N-terminal DNA-binding domain (DBD) and C-terminal effector-binding domain (EBD). To characterize the Escherichia coli LeuO protein, we screened for LeuO mutants that activate the cas (CRISPR-associated/Cascade) promoter more effectively than wild-type LeuO. This yielded nine mutants carrying amino acid substitutions in the dimerization interface of the regulatory EBD, as shown by solving the EBD's crystal structure. Superimposing of the crystal structures of LeuO-EBD and LeuO-S120D-EBD suggests that the Ser120 to Asp substitution triggers a structural change that is related to effector-induced structural changes of LTTRs. Corresponding functional analyses demonstrated that LeuO-S120D has a higher DNA-binding affinity than wild-type LeuO. Further, a palindromic DNA-binding core-site and a consensus sequence were identified by DNase I footprinting with LeuO-S120D as well as with the dimeric DBD. The data suggest that LeuO-S120D mimics an effector-induced form of LeuO regulating a distinct set of target loci. In general, constitutive mutants and determining the DNA-binding specificity of the DBD-dimer are feasible approaches to characterize LTTRs of unknown function.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Fragel, Susann M. UNSPECIFIED UNSPECIFIED UNSPECIFIED Montada, Anna UNSPECIFIED UNSPECIFIED UNSPECIFIED Heermann, Ralf UNSPECIFIED orcid.org/0000-0003-0631-6156 UNSPECIFIED Baumann, Ulrich UNSPECIFIED UNSPECIFIED UNSPECIFIED Schacherl, Magdalena UNSPECIFIED orcid.org/0000-0002-5478-2509 UNSPECIFIED Schnetz, Karin UNSPECIFIED orcid.org/0000-0003-4693-6202 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-143983
DOI:	10.1093/nar/gkz506
Journal or Publication Title:	Nucleic Acids Res.
Volume:	47
Number:	14
Page Range:	S. 7363 - 7380
Date:	2019
Publisher:	OXFORD UNIV PRESS
Place of Publication:	OXFORD
ISSN:	1362-4962
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language NS-MEDIATED REPRESSION; H-NS; VIBRIO-CHOLERAE; DNA-BINDING; GLOBAL REGULATOR; GENE-EXPRESSION; STRUCTURE REFINEMENT; BGL OPERON; PROTEIN; ACTIVATION Multiple languages Biochemistry & Molecular Biology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/14398