GalR mutants defective in repressosome formation

Mark Geanacopoulos, George Vasmatzis, Dale E A Lewis, Siddartha Roy, Byungkook Lee, Sankar Adhya

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

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 isolated galR 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 the gal promoter. This observation suggests that the mutations define sites of GaIR-GalR interaction rather than HU-GaIR interaction in the repressosome.

Original languageEnglish (US)
Pages (from-to)1251-1262
Number of pages12
JournalGenes and Development
Volume13
Issue number10
StatePublished - May 15 1999
Externally publishedYes

Fingerprint

Mutation
Proteins
HMGN2 Protein
Superhelical DNA
Secondary Protein Structure
Nucleoproteins
Mutant Proteins
Amino Acid Substitution
Operon
Circular Dichroism
Galactose
Protein Binding
Histones
Spectrum Analysis
Binding Sites
Escherichia coli
DNA
In Vitro Techniques

Keywords

  • DNA looping
  • GalR
  • GalR mutants
  • Protein-protein interactions
  • Repression
  • Repressosome
  • Transcription

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology

Cite this

Geanacopoulos, M., Vasmatzis, G., Lewis, D. E. A., Roy, S., Lee, B., & Adhya, S. (1999). GalR mutants defective in repressosome formation. Genes and Development, 13(10), 1251-1262.

GalR mutants defective in repressosome formation. / Geanacopoulos, Mark; Vasmatzis, George; Lewis, Dale E A; Roy, Siddartha; Lee, Byungkook; Adhya, Sankar.

In: Genes and Development, Vol. 13, No. 10, 15.05.1999, p. 1251-1262.

Research output: Contribution to journalArticle

Geanacopoulos, M, Vasmatzis, G, Lewis, DEA, Roy, S, Lee, B & Adhya, S 1999, 'GalR mutants defective in repressosome formation', Genes and Development, vol. 13, no. 10, pp. 1251-1262.
Geanacopoulos M, Vasmatzis G, Lewis DEA, Roy S, Lee B, Adhya S. GalR mutants defective in repressosome formation. Genes and Development. 1999 May 15;13(10):1251-1262.
Geanacopoulos, Mark ; Vasmatzis, George ; Lewis, Dale E A ; Roy, Siddartha ; Lee, Byungkook ; Adhya, Sankar. / GalR mutants defective in repressosome formation. In: Genes and Development. 1999 ; Vol. 13, No. 10. pp. 1251-1262.
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