GalR mutants defective in repressosome formation

Geanacopoulos, Mark ; Vasmatzis, George ; Lewis, Dale E. A. ; Roy, Siddartha ; Lee, Byungkook ; Adhya, Sankar (1999) GalR mutants defective in repressosome formation Genes & Development, 13 . pp. 1251-1262. ISSN 0890-9369

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Transcription repression of the galactose operon of Escherichia coli requires (1) the binding of the GalR repressor to tandem operators flanking the promoters, (2) the binding of histone-like protein, HU, to a site between the GalR-binding sites, and (3) negatively supercoiled DNA. Under these conditions, protein-protein interactions mediate the formation of a nucleoprotein complex in the form of a DNA loop, which we have termed a repressosome. To analyze the structure of the repressosome, we have screened and isolatedgalR mutants in which single amino acid substitutions in GalR lead to defects in loop formation while the protein's operator-binding activity is retained. The mutant proteins were purified and their properties confirmed in vitro. We verified that in the case of the two stronger mutations, the proteins had secondary structures that were identical to that of wild-type GalR as reflected by circular dichroism spectroscopy. Homology-based modeling of GalR by use of the crystal structures of PurR and LacI has enabled us to place the three sites of mutation in a structural context. They occur in the carboxy-terminal subdomain of the GalR core, are surface exposed, and, therefore, may be involved in protein-protein interactions. On the basis of our model of GalR and its structural alignment with LacI and PurR, we have identified additional residues, the substitution of which leads to a specific defect in repression by looping. The effects of the mutations are the same in the presence of HMG-17, a eukaryotic protein unrelated to HU, which can also mediate GalR-dependent repression of thegal promoter. This observation suggests that the mutations define sites of GalR-GalR interaction rather than HU-GalR interaction in the repressosome.

Item Type:Article
Source:Copyright of this article belongs to Cold Spring Harbor Laboratory Press.
Keywords:Repressosome; Transcription; galR Mutants; Protein-protein Interactions; DNA Looping; GalR; Repression
ID Code:43183
Deposited On:10 Jun 2011 07:36
Last Modified:18 May 2016 00:15

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