GalR Represses galP1 by Inhibiting the Rate-determining Open Complex Formation Through RNA Polymerase Contact: A GalR Negative Control Mutant

Roy, Siddhartha; Semsey, Szabolcs; Liu, Mofang; Gussin, Gary N.

doi:10.1016/j.jmb.2004.09.070

Cited by 28 publications

(33 citation statements)

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“…At this point, we speculate that the physical basis for frustration is the highly degenerate nature of protein-DNA interactions in a hydrated, fluid interface. The fact that the identified roughness is compatible with the evolutionary constrains for the E2C-DNA interaction suggests that it may play a functional role (34). Fig.…”

Section: Resultsmentioning

confidence: 61%

“…In this context, it is interesting to note that E2C (31) and other DNA-binding proteins (32) are able to bind specifically to distinct target sequences. We propose that the alternative E2C-DNA complex might have a functional role different from that of the native one, such as modulating the search for the target sequence (33) and/or the coalescence of other regulatory molecules through allosteric effects (34).…”

Section: Resultsmentioning

confidence: 99%

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Experimental snapshots of a protein-DNA binding landscape

Sánchez

Ferreiro

Dellarole

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Protein recognition of DNA sites is a primary event for gene function. Its ultimate mechanistic understanding requires an integrated structural, dynamic, kinetic, and thermodynamic dissection that is currently limited considering the hundreds of structures of protein-DNA complexes available. We describe a protein-DNA-binding pathway in which an initial, diffuse, transition state ensemble with some nonnative contacts is followed by formation of extensive nonnative interactions that drive the system into a kinetic trap. Finally, nonnative contacts are slowly rearranged into native-like interactions with the DNA backbone. Dissimilar protein-DNA interfaces that populate along the DNA-binding route are explained by a temporary degeneracy of protein-DNA interactions, centered on "dual-role" residues. The nonnative species slow down the reaction allowing for extended functionality.energy landscape | human papillomavirus E2 protein | kinetic trap | kinetics | protein-DNA interaction

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Section: Resultsmentioning

confidence: 61%

Section: Resultsmentioning

confidence: 99%

Experimental snapshots of a protein-DNA binding landscape

Sánchez

Ferreiro

Dellarole

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…The promoters are repressed by GalR by two different mechanisms. Either (i) GalR represses P1 by inhibiting open complex formation at the promoter-bound RNA polymerase (33) or (ii) P2 is repressed by a DNA loop that makes the promoter inadequate for transcription initiation (10,11). The presence of D-galactose inactivates GalR to release the repression in each case (25).…”

Section: Resultsmentioning

confidence: 99%

Induction of the Galactose Enzymes in Escherichia coli Is Independent of the C-1-Hydroxyl Optical Configuration of the Inducer d -Galactose

Lee

Lewis

2008

J Bacteriol

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The two optical forms of aldohexose galactose differing at the C-1 position, ␣-D-galactose and ␤-D-galactose, are widespread in nature. The two anomers also occur in di-and polysaccharides, as well as in glycoconjugates. The anomeric form of D-galactose, when present in complex carbohydrates, e.g., cell wall, glycoproteins, and glycolipids, is specific. Their interconversion occurs as monomers and is effected by the enzyme mutarotase (aldose-1-epimerase). Mutarotase and other D-galactose-metabolizing enzymes are coded by genes that constitute an operon in Escherichia coli. The operon is repressed by the repressor GalR and induced by D-galactose. Since, depending on the carbon source during growth, the cell can make only one of the two anomers of D-galactose, the cell must also convert one anomer to the other for use in specific biosynthetic pathways. Thus, it is imperative that induction of the gal operon, specifically the mutarotase, be achievable by either anomer of D-galactose. Here we report in vivo and in vitro experiments showing that both ␣-D-galactose and ␤-D-galactose are capable of inducing transcription of the gal operon with equal efficiency and kinetics. Whereas all substitutions at the C-1 position in the ␣ configuration inactivate the induction capacity of the sugar, the effect of substitutions in the ␤ configuration varies depending upon the nature of the substitution; methyl and phenyl derivatives induce weakly, but the glucosyl derivative does not.D-Galactose, also known as cerebrose, is a natural aldohexose which is ubiquitous in bacteria, plants, and animals, including human brains (28). It occurs also as part of more complex carbohydrates, e.g., oligo-and polysaccharides, glycoproteins, and glycolipids, and as components of bacterial cell walls. DGalactose has two anomeric forms, ␣-D-galactopyranose and ␤-D-galactopyranose ( Fig. 1) (36). Although the anomers spontaneously interconvert slowly in aqueous solution, an enzyme (mutarotase or aldose-1-epimerase) is responsible for interconverting the two optical varieties in monosaccharide forms in vivo (6,21). Mutarotases are present throughout the prokaryotic and eukaryotic phyla (6,31,34,37). Mutarotase syntheses are also induced in response to specific signals. For example, galactose mutarotase synthesis is induced by retinoic acid in human myeloid cells (30). In the bacterium Escherichia coli, enzymes of D-galactose metabolism (15,20), including the mutarotase (6), are encoded in an operon which is induced by D-galactose. It has been suggested previously that only ␤-Dgalactose is the inducer of the gal operon (7). Since D-galactose moieties in complex carbohydrates are anomerically specific, ␣ or ␤, we hypothesize that the mutarotase enzyme, which interconverts the ␣ and ␤ anomers, must be induced for the interconversion when either one of the two anomers is present in the cell, say, by hydrolysis of D-lactose, which generates ␤-Dgalactose, or of D-melibiose, which generates ␣-D-galactose. In this study, we showed by both in vivo and in vitr...

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“…Up to now, the lac-gal regulon and the gal operon have been reported in Proteobacteria and Firmicutes. The molecular regulation of the gal operon in Proteobacteria such as E. coli is well known (Roy et al, 2004;Semsey et al, 2002Semsey et al, , 2004Semsey et al, , 2006, but the study of Firmicute gal operons is considered to be uncharted territory. Firmicutes are broadly divided into two classes, bacilli and clostridia.…”

Section: Introductionmentioning

confidence: 99%

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

et al. 2009

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On the basis of the Thermoanaerobacter tengcongensis genome, a novel type of gal operon was deduced. The gene expression and biochemical properties of this operon were further characterized. RT-PCR analysis of the intergenic regions suggested that the transcription of the gal operon was continuous. With gene cloning and enzyme activity assays, TTE1929, TTE1928 and TTE1927 were identified to be GalT, GalK and GalE, respectively. Results elicited from polarimetry assays revealed that TTE1925, a hypothetical protein, was a novel mutarotase, termed MR-Tt. TTE1926 was identified as a regulator that could bind to two operators in the operon promoter. The transcriptional start sites were mapped, and this suggested that there are two promoters in this operon. Expression of the gal genes was significantly induced by galactose, whereas only MR-Tt expression was detected in glucose-cultured T. tengcongensis at both the mRNA and the protein level. In addition, the abundance of gal proteins was examined at different temperatures. At temperatures ranging from 60 to 80 6C, the level of MR-Tt protein was relatively stable, but that of the other gal proteins was dramatically decreased. The operator-binding complexes were isolated and identified by electrophoretic mobility shift assay-liquid chromatography (EMSA-LC) MS-MS, which suggested that several regulatory proteins, such as GalR and a sensory histidine kinase, participate in the regulation of the gal operon.

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GalR Represses galP1 by Inhibiting the Rate-determining Open Complex Formation Through RNA Polymerase Contact: A GalR Negative Control Mutant

Cited by 28 publications

References 50 publications

Experimental snapshots of a protein-DNA binding landscape

Experimental snapshots of a protein-DNA binding landscape

Induction of the Galactose Enzymes in Escherichia coli Is Independent of the C-1-Hydroxyl Optical Configuration of the Inducer d -Galactose

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

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