GalR mutants defective in repressosome formation

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 GalR-GalR interaction rather than HU-GalR interaction in the repressosome.[Key Words: Repressosome; transcription; galR mutants; protein-protein interactions; DNA looping; GalR; repression] Received October 13, 1998; revised version accepted April 6, 1999.DNA looping is a general phenomenon in the control of transcription in which two or more DNA-binding proteins recognize DNA sequences flanking a promoter and, by contacting each other, cause a reversible transition to a nucleoprotein complex in which the intervening DNA is looped out and the promoter activity is altered (Adhya 1989; Martin et al. 1990). The relative simplicity of these complexes in prokaryotes compared with their counterparts in eukaryotes has facilitated the investigation by genetic and physicochemical methods of questions critical to the understanding of regulation by DNA looping. Some of these questions are structural in nature: What are the protein-protein and protein-DNA contacts that stabilize these loops? What are the symmetries, if any, of the protein complexes? Other questions are framed in terms of thermodynamics and seek to understand the origin of the negative-free energy of the looped structure.Critical mechanistic questions include how loop formation modulates transcription activity and by what means the stability of the loop is sensitive to environmental conditions.W...

Signal integration in the galactose network of Escherichia coli

“…HU is required for the assembly of the Gal repressosome, a higher-order nucleoprotein complex containing a GalR-mediated DNA loop that represses both galETKM operon promoters (Aki et al, 1996;Geanacopoulos et al, 1999;Semsey et al, 2004) (Fig. 4).…”

“…The galETKM operon and galS has a second operator (OI), whose centre is located 113 and 138 bp downstream from the first operator respectively (Irani et al, 1983;Muller-Hill and Kolkhof, 1994). DNA loop formation by direct interaction of the OE-bound GalR dimer with the OI-bound GalR dimer results in simultaneous repression of both galETKM operon promoters, P1galE and P2galE (Aki et al, 1996;Geanacopoulos et al, 1999;Semsey et al, 2002). As the helical phasing of the galS operator sites could favour DNA looping, the role of OI in the regulation of the PgalS promoter remains a possibility.…”

Genetic Analysis of GalR Tetramerization in DNA Looping during Repressosome Assembly

“…This strategy allowed us to change the GalR surface with a minimal likelihood of disrupting the overall structure of the protein (14). The effect of homology-based site-directed mutagenesis of GalR on repressosome formation was examined in vivo as described under "Experimental Procedures" by the enzyme assay of two gene fusion products that enable the DNA binding activity of a GalR mutant to be measured separately from its ability to form the higher order structure (9). DNA looping was assayed in looping-defective mutants of GalR by measuring the expression of ␤-glucuronidase from a P2ϳgusA fusion whose repression requires repressosome formation (Fig.…”

“…In this approach, a homology model of the dimeric GalR core was constructed based on the structures of two proteins, LacI and PurR, that have high sequence similarity to GalR and are nearly identical in their crystal structures (9,12,13). The particular residues that were substituted into GalR were those that occupied corresponding structural positions in either LacI or PurR when the GalR model was superimposed on the crystal structures.…”

“…The Gal repressosome containing a DNA loop is one such complex whose function is to represses the transcription of the genes for galactose metabolism in Escherichia coli (7)(8)(9). Its assembly requires (i) the binding of two dimeric GalR repressors to two operators flanking the two gal promoters, (ii) the cooperative binding of HU, an abundant component of the nucleoid to a site between the two operators, and (iii) negatively supercoiled DNA.…”