Functional characterization of roles of GalR and GalS as regulators of the gal regulon

Abstract

An isorepressor of the gal regulon in Escherichia coli, GalS, has been purified to homogeneity. In vitro DNase I protection experiments indicated that among operators of the gal regulon, GalS binds most strongly to the external operator of the mgl operon, which encodes the high-affinity ␤-methylgalactoside galactose transport system, and with less affinity to the operators controlling expression of the gal operon, which codes for enzymes of galactose metabolism. GalS has even less affinity for the external operator of galP, which codes for galactose permease, the major low-affinity galactose transporter in the cell. This order of affinities is the reverse of that of GalR, which binds most strongly to the operator of galP and most weakly to that of mgl. Our results also show that GalS, like its homolog, GalR, is a dimeric protein which in binding to the bipartite operators of the gal operon selectively represses its P1 promoter. Consistent with the fact that GalR is the exclusive regulator of the low-affinity galactose transporter, galactose permease, and that the major role of GalS is in regulating expression of the high-affinity galactose transporter encoded by the mgl operon, we found that the DNA binding of GalS is 15-fold more sensitive than that of GalR to galactose.The genes and operons comprising the gal regulon in Escherichia coli enable the cell to use galactose both as a source of energy and as a source of intermediates necessary for biosynthetic glycosylation reactions (1). The polycistronic gal operon encodes enzymes participating in the Leloir pathway for galactose metabolism, while the mgl operon and the galP gene encode the high-affinity ␤-methylgalactoside transport system and the galactose permease, respectively, the two major galactose uptake systems in E. coli (18). The expression of the enzymes involved with the Leloir pathway and galactose transport activities is regulated by two highly homologous, galactose-sensitive repressors, GalR and GalS, encoded by the genes galR and galS, respectively. These repressors are members of the GalR-LacI family of transcriptional regulators in E. coli (17). The precise role played by each repressor in regulating expression of the genes of the regulon is unclear. GalR, the first of these proteins to be identified and which has been purified, functions as the major repressor of the gal operon and is sufficient for repression of its genes in the absence of galactose (1). GalS was identified because in strains in which galR was deleted, the gal operon could be further induced by galactose, a phenomenon called ultrainduction (9). Subsequent genetic studies indicated that GalS functions to control its own expression and that of the ␤-methylgalactoside transport system, the galactose transporter in E. coli (16). In order to obtain a better understanding of the roles of GalR and GalS in coordinating both galactose metabolism and uptake, we have purified GalS and studied the interaction of both of these repressors with the regulatory sequences in the regulon. Our re...

Signal integration in the galactose network of Escherichia coli

“…However, transcription from the PgalP, PmglB and PgalS promoters was observed only in the presence of CRP. These in vitro results confirm the previously reported in vivo observations that expression of these three genes is highly dependent upon cAMP and CRP (Weickert and Adhya, 1993b;Geanacopoulos and Adhya, 1997). To investigate the differences in elements that are known to influence the intrinsic activity of promoters, we compared DNA sequences of the PgalP, PmglB and PgalS with that of P1galE and P2galE (Fig.…”

“…GalS was more efficient in mglBAC repression, while it did not completely inhibit P1galE promoter activity. The incomplete repression of P1galE by GalS may be because GalS dimers bound to the two galETKM operators do not interact with each other to bring about tight repression by forming a DNA loop in the way GalR does in the galETKM operon (Geanacopoulos and Adhya, 1997). Therefore, unlike GalR, GalS represses P1galE transcription only by contact inhibition, not by DNA looping.…”

“…About 40% of the CRP-activated PmglB promoter activity was observed even in the presence of higher concentrations (50 nM) of GalR. Both the relatively weak affinity of GalR to the mgl operator (Geanacopoulos and Adhya, 1997) and the different helical phasing of the GalR and CRP binding sites at the mglBAC regulatory region ( Fig. 1) can account for the incomplete repression of mglBAC transcription.…”

How gene order is influenced by the biophysics of transcription regulation

“…However, the difference in DNA binding constants between GalS and the GalS-galactose complex is relatively small, on the order of 2 orders of magnitude. Experimental data suggest (45) that even in the presence of galactose, when GalS reaches a sufficiently large concentration, the repressor again shuts down the transcription of transporter genes encoded in mglBAC operon. It can be speculated that at the low expression levels typical for local regulators such as GalS and LacI, this is possible only in cases where a high local concentration of GalS is reached, making the release of the newly synthesized GalS protein near the 3Ј end of galS gene and the 5Ј end of mglBAC operon a key factor.…”

GalR mutants defective in repressosome formation

“…Transcription reactions were performed as described previously (Geanacopoulos and Adhya 1997). The reaction mixture (50 µl) contained 20 mM Tris acetate (pH 7.8), 10 mM magnesium acetate, 100 mM potassium glutamate, 2 nM DNA template, 1.0 mM ATP, 0.1 mM GTP, 0.1 mM CTP, 0.01 mM UTP, and 10-20 µCi of [␣-32 P]UTP (3000 Ci/mmole).…”