Transcriptional regulation of the Escherichia coli rhaT gene

Vía, Pilar; Badı́a, Josefa; Baldomà, Laura; Obradors, Nuria; Aguilar, Juan

doi:10.1099/13500872-142-7-1833

Cited by 39 publications

(41 citation statements)

References 13 publications

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“…Neither was found crucial, suggesting that the linkers do not play a direct role in activation, but rather simply connect the two domains.In the presence of the sugar L-rhamnose, RhaS and RhaR activate transcription of Escherichia coli genes, whose products are required for the uptake and catabolism of L-rhamnose (6,7,29,30). RhaS and RhaR comprise a regulatory cascade in which L-rhamnose stimulates RhaR to activate transcription of rhaSR (29) and then RhaS activates transcription of the Lrhamnose catabolic operon rhaBAD (7) and the L-rhamnose transport gene rhaT (30).…”

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“…RhaS and RhaR comprise a regulatory cascade in which L-rhamnose stimulates RhaR to activate transcription of rhaSR (29) and then RhaS activates transcription of the Lrhamnose catabolic operon rhaBAD (7) and the L-rhamnose transport gene rhaT (30). Both RhaS and RhaR are members of the large AraC/XylS family of transcription regulatory proteins, which share sequence similarity in a 100-amino-acid DNA-binding domain (5,10,11,21).…”

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Linker Regions of the RhaS and RhaR Proteins

et al. 2007

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Substitutions within the interdomain linkers of the AraC/XylS family proteins RhaS and RhaR were tested to determine whether side chain identity or linker structure was required for function. Neither was found crucial, suggesting that the linkers do not play a direct role in activation, but rather simply connect the two domains.In the presence of the sugar L-rhamnose, RhaS and RhaR activate transcription of Escherichia coli genes, whose products are required for the uptake and catabolism of L-rhamnose (6,7,29,30). RhaS and RhaR comprise a regulatory cascade in which L-rhamnose stimulates RhaR to activate transcription of rhaSR (29) and then RhaS activates transcription of the Lrhamnose catabolic operon rhaBAD (7) and the L-rhamnose transport gene rhaT (30). Both RhaS and RhaR are members of the large AraC/XylS family of transcription regulatory proteins, which share sequence similarity in a 100-amino-acid DNA-binding domain (5,10,11,21). AraC/XylS family proteins regulate the expression of genes whose functions include carbon metabolism (3,12,25,28), stress responses (4, 15-18, 22, 32), and pathogenesis (9,14,23,24).RhaS and RhaR each consist of two independently functional domains: an N-terminal domain required for dimerization and response to L-rhamnose (A. Kolin, J. R. Wickstrum, and S. M. Egan, unpublished results) and a C-terminal domain involved in DNA binding and transcription activation (1, 2, 6, 31; J. R. Wickstrum and S. M. Egan, unpublished results). Based on alignment with the AraC dimerization domain and its structure (26) and the MarA protein and its structure (22), we can predict the approximate boundaries of the RhaS and RhaR domains. Such analysis, combined with knowledge of AraC (8), leads to the prediction that there is a flexible linker that connects the two domains of RhaS and RhaR. This linker spans approximately residues 166 to 172 in RhaS and 198 to 207 in RhaR (Fig. 1). Genetic and biochemical studies have shown that many different single and multiple substitutions could be made within the AraC linker without impacting activation (8), suggesting that the AraC arabinose response does not depend on the identity of its linker residues. However, our finding that the ligand responses of RhaS and RhaR differ from that of AraC (A. Kolin and S. M. Egan, unpublished results) left open the possibility that the linker might participate in transmission of the L-rhamnose signal.In this study, we used a genetic approach to analyze the linker regions of RhaS and RhaR. Our previous results indicate that L-rhamnose binds to the N-terminal domains of RhaS and RhaR, while transcription activation involves their C-terminal domains (2, 31; Kolin and Egan, unpublished). Therefore, we sought to determine whether the linker regions of RhaS and/or RhaR were involved in transmitting the L-rhamnose status from the N-to the C-terminal domain or whether, similar to AraC, the linker was required only to flexibly connect the two domains.Single alanine substitutions in the linker region of RhaS and RhaR have, at most,...

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Linker Regions of the RhaS and RhaR Proteins

et al. 2007

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“…The other L-rhamnose-responsive activator protein, RhaR, activates transcription of the rhaSR operon (31). Full activation by RhaR requires contact with 70 (RhaR D276 contacts 70 R599), and a rhaSR promoter containing a RhaR binding site, but not a cyclic AMP (cAMP) receptor protein (CRP) binding site, requires the carboxy-terminal domain of the alpha subunit (␣-CTD) of RNAP for full activation (15,34).Full activation of each of the three rha operons also requires the cyclic AMP receptor protein (CRP, also known as the catabolite activator protein or CAP) in addition to either RhaS or RhaR (12,15,32). CRP binds as a dimer to specific DNA sites and induces a DNA bend of approximately 90 degrees in the presence of its ligand, cAMP (21).…”

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“…Full activation of each of the three rha operons also requires the cyclic AMP receptor protein (CRP, also known as the catabolite activator protein or CAP) in addition to either RhaS or RhaR (12,15,32). CRP binds as a dimer to specific DNA sites and induces a DNA bend of approximately 90 degrees in the presence of its ligand, cAMP (21).…”

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Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l -Rhamnose-Responsive rhaSR Promoter in Escherichia coli

2005

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The Escherichia coli rhaSR operon encodes two AraC family transcription activator proteins, RhaS and RhaR, which regulate expression of the L-rhamnose catabolic regulon in response to L-rhamnose availability. RhaR positively regulates rhaSR in response to L-rhamnose, and RhaR activation can be enhanced by the cyclic AMP (cAMP) receptor protein (CRP) protein. CRP is a well-studied global transcription regulator that binds to DNA as a dimer and activates transcription in the presence of cAMP. We investigated the mechanism of CRP activation at rhaSR both alone and in combination with RhaR in vivo and in vitro. Base pair substitutions at potential CRP binding sites in the rhaSR-rhaBAD intergenic region demonstrate that CRP site 3, centered at position ؊111.5 relative to the rhaSR transcription start site, is required for the majority of the CRP-dependent activation of rhaSR. DNase I footprinting confirms that CRP binds to site 3; CRP binding to the other potential CRP sites at rhaSR was not detected. We show that, at least in vitro, CRP is capable of both RhaR-dependent and RhaR-independent activation of rhaSR from a total of three transcription start sites. In vitro transcription assays indicate that the carboxy-terminal domain of the alpha subunit (␣-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the ␣-CTD is required for CRP activation of rhaSR. Although CRP requires the presence of RhaR for efficient in vivo activation of rhaSR, DNase I footprinting assays indicated that cooperative binding between RhaR and CRP does not make a significant contribution to the mechanism of CRP activation at rhaSR. It therefore appears that CRP activates transcription from rhaSR as it would at simple class I promoters, albeit from a relatively distant position.The L-rhamnose regulon of Escherichia coli consists of the rhaT, rhaSR, and rhaBAD operons. The rhaSR operon encodes the L-rhamnose-responsive transcription activators RhaS and RhaR, both of which are members of the AraC/XylS family of transcription regulators (13), while the rhaBAD and rhaT operons encode the L-rhamnose catabolic enzymes and the L-rhamnose transport protein, respectively (2, 23). RhaS activates transcription of the rhaBAD and rhaT operons in the presence of L-rhamnose by binding to DNA sites that overlap the Ϫ35 hexamers by four base pairs and extend upstream to Ϫ81 at rhaBAD and Ϫ82 at rhaT (11, 32). Part of the mechanism of RhaS activation involves contact with the 70 subunit of RNA polymerase (RNAP), specifically, RhaS residues D241 and D250 make contact with 70 residues R599 and K593, respectively (7, 34). The other L-rhamnose-responsive activator protein, RhaR, activates transcription of the rhaSR operon (31). Full activation by RhaR requires contact with 70 (RhaR D276 contacts 70 R599), and a rhaSR promoter containing a RhaR binding site, but not a cyclic AMP (cAMP) receptor protein (CRP) binding site, requires the carboxy-terminal domain of the alpha subunit (␣-CTD) of RNAP for full activation (15,34).F...

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“…The sole identified function of RhaR is to activate transcription of rhaSR and thereby increase RhaS protein concentration in the presence of L-rhamnose, while RhaS directly activates transcription of the rhaBAD and rhaT operons (11,12,(27)(28)(29)(30). Cyclic AMP receptor protein (CRP) is required for full expression of all three of the rha operons, but it functions as a coactivator that substantially activates transcription only when RhaS or RhaR also binds to the promoter regions (11,15,31). CRP activation at the rhaSR operon was shown to require the RNA polymerase (RNAP) ␣-subunit C-terminal domain (␣-CTD) (33).…”

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The AraC/XylS Family Activator RhaS Negatively Autoregulates rhaSR Expression by Preventing Cyclic AMP Receptor Protein Activation

et al. 2010

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The Escherichia coli RhaR protein activates expression of the rhaSR operon in the presence of its effector, L-rhamnose. The resulting RhaS protein (plus L-rhamnose) activates expression of the L-rhamnose catabolic and transport operons, rhaBAD and rhaT, respectively. Here, we further investigated our previous finding that rhaS deletion resulted in a threefold increase in rhaSR promoter activity, suggesting RhaS negative autoregulation of rhaSR. We found that RhaS autoregulation required the cyclic AMP receptor protein (CRP) binding site at rhaSR and that RhaS was able to bind to the RhaR binding site at rhaSR. In contrast to the expected repression, we found that in the absence of both RhaR and the CRP binding site at the rhaSR promoter, RhaS activated expression to a level comparable with RhaR activation of the same promoter. However, when the promoter included the RhaR and CRP binding sites, the level of activation by RhaS and CRP was much lower than that by RhaR and CRP, suggesting that CRP could not fully coactivate with RhaS. Taken together, our results indicate that RhaS negative autoregulation involves RhaS competition with RhaR for binding to the RhaR binding site at rhaSR. Although RhaS and RhaR activate rhaSR transcription to similar levels, CRP cannot effectively coactivate with RhaS. Therefore, once RhaS reaches a relatively high protein concentration, presumably sufficient to saturate the RhaS-activated promoters, there will be a decrease in rhaSR transcription. We propose a model in which differential DNA bending by RhaS and RhaR may be the basis for the difference in CRP coactivation.

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Transcriptional regulation of the Escherichia coli rhaT gene

Cited by 39 publications

References 13 publications

Linker Regions of the RhaS and RhaR Proteins

Linker Regions of the RhaS and RhaR Proteins

Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l -Rhamnose-Responsive rhaSR Promoter in Escherichia coli

The AraC/XylS Family Activator RhaS Negatively Autoregulates rhaSR Expression by Preventing Cyclic AMP Receptor Protein Activation

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