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Reverse transcription with radioactively labeled primers complementary to RNA transcripts from in vitro transcription reactions (A). Lanes A, C, G, and T are sequencing reactions generated from the same primer used for the reverse transcription reactions. Lane 1 was a control transcription reaction without NTPs. The transcripts used as a template for lanes 2 through 5 were generated in the presence of the following activators: none (lane 2), CRP (lane 3), RhaR(lane 4), and RhaR and CRP (lane 5). Panel B shows the positions of the RNAP binding sites (gray boxes labeled −10 and −35) for pSR compared to the putative RNAP binding sites used to generate the CRP A and CRP B transcripts. The RhaR and CRP binding sites are underlined, and the positions of the CRP sites relative to each transcription start site are shown.
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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...
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Citations
... In addition, some AraC/XylS family members directly contact the α-CTD of RNAP for full activation. 21,22 For RhaR, evidence also suggests that important interactions occur with the transcription initiation factor σ 70 Domain 4. 23 We reasoned that domain recombination of the AraC/XylS homologs could be used to build transcription activators that recognize the same DNA binding sites but respond to different effector ligands. For the engineered chimeras to be active, they must have optimal interactions between domains and the two monomers of a dimer: Proper inter-domain interactions are required for effector regulation of the DNA binding domain activities. ...
To create bacterial transcription “circuits” for biotechnology, one approach is to recombine natural transcription factors, promoters, and operators. Additional novel functions can be engineered from existing transcription factors such as the E. coli AraC transcriptional activator, for which binding to DNA is modulated by binding L-arabinose. Here, we engineered chimeric AraC/XylS transcription activators that recognized ara DNA binding sites and responded to varied effector ligands. The first step, identifying domain boundaries in the natural homologs, was challenging because (i) no full-length, dimeric structures were available and (ii) extremely low sequence identities (≤10%) among homologs precluded traditional assemblies of sequence alignments. Thus, to identify domains, we built and aligned structural models of the natural proteins. The designed chimeric activators were assessed for function, which was then further improved by random mutagenesis. Several mutational variants were identified for an XylS•AraC chimera that responded to benzoate; two enhanced activation to near that of wild-type AraC. For an RhaR•AraC chimera, a variant with five additional substitutions enabled transcriptional activation in response to rhamnose. These five changes were dispersed across the protein structure, and combinatorial experiments testing subsets of substitutions showed significant non-additivity. Combined, the structure modeling and epistasis suggest that the common AraC/XylS structural scaffold is highly interconnected, with complex intra-protein and inter-domain communication pathways enabling allosteric regulation. At the same time, the observed epistasis and the low sequence identities of the natural homologs suggest that the structural scaffold and function of transcriptional regulation are nevertheless highly accommodating of amino acid changes.
... CRP-cAMP binds near divergent promoters that control araC and the araBAD operon and promotes expression from both promoters (19). Similarly, the divergent rhaRS and rhaBAD operons are activated by RhaR and RhaS, respectively, and each promoter region has a CRP-cAMP binding site (2,20,21). Maximal expression of arabinose or rhamnose utilization genes, therefore, requires both the specific inducing sugar and elevated cAMP levels (i.e., low glucose levels). ...
... Vfr-P Rha also formed a second lower-mobility complex at the higher concentrations of Vfr (Fig. 4B). This is consistent with the presence of multiple CRP binding sites in the P Rha region (21). ...
The Pseudomonas aeruginosa virulence factor regulator (Vfr) is a cyclic AMP (cAMP)-responsive transcription factor homologous to the Escherichia coli cAMP receptor protein (CRP). Unlike CRP, which plays a central role in E. coli energy metabolism and catabolite repression, Vfr is primarily involved in the control of P. aeruginosa virulence factor expression. Expression of the Vfr regulon is controlled at the level of vfr transcription, Vfr translation, cAMP synthesis, and cAMP degradation. While investigating mechanisms that regulate Vfr translation, we placed vfr transcription under the control of the rhaBp rhamnose-inducible promoter system (designated PRha) and found that PRha promoter activity was highly dependent upon vfr. Vfr dependence was also observed for the araBp arabinose-inducible promoter (designated PBAD). The observation of Vfr dependence was not entirely unexpected. Both promoters are derived from E. coli, where maximal promoter activity is dependent upon CRP. Like CRP, we found that Vfr directly binds to promoter probes derived from the PRha and PBAD promoters in vitro. Because Vfr-cAMP activity is highly integrated into numerous global regulatory systems, including c-di-GMP signaling, the Gac/Rsm system, MucA/AlgU/AlgZR signaling, and Hfq/sRNAs, the potential exists for significant variability in PRha and PBAD promoter activity in a variety of genetic backgrounds, and use of these promoter systems in P. aeruginosa should be employed with caution.
... Operon rha pada E. coli terdiri dari tiga promotor (Gambar 4) yang teraktivasi saat penambahan ramnosa (Wickstrum et al., 2005) Gambar 4. Representasi skematis ramnosa operon E. coli (Hjelm, 2015). ...
... Aktivasi penuh ketiga operon rha juga membutuhkan cAMP selain penggunaan RhaS ataupun RhaR. CRP berikatan sebagai dimer pada sisi DNA spesifik dan menginduksi suatu lengkungan DNA yang kira-kira 90 derajat dengan kehadiran ligannya, cAMP (Wickstrum et al. 2005). Bagaimanapun juga, bahkan dalam keadaan absennya glukosa, promotor ini masih terbilang baik. ...
ABSTRAK
Escherichia coli (E. coli) merupakan bakteri yang telah lama digunakan secara luas pada industri bioteknologi. Media pertumbuhan murah, pertumbuhannya cepat, dan tingkat ekspresi protein yang tinggi membuat E. coli banyak digunakan sebagai inang untuk ekspresi protein rekombinan. Sistem ekspresi pada E. coli menggunakan sistem operon di mana gen-gen struktural akan ditranskripsikan menjadi mRNA polisistronik yang kemudian akan ditranslasikan membentuk protein. Selain gen struktural, dalam satu operon terdapat pula gen regulator dan promotor. Banyak sistem promotor dari E. coli telah diketahui sebagai sarana untuk ekspresi protein, namun hanya beberapa yang umum digunakan untuk produksi protein rekombinan, diantaranya adalah sistem promotor lac, T7 RNA Polimerase, gal, ara dan rha. Dalam sistem promotor tersebut diperlukan adanya induser untuk meregulasi aktivitas transkripsi dari promotor seperti IPTG, L-arabinosa, galaktosa dan L-rhamnosa. Regulasi dari sistem promotor ini dapat dimanfaatkan dalam ekspresi protein rekombinan seperti Human Epidermal Growth Factor (hEGF) dan Pretrombin-2 (PT2)
Kata kunci : Escherichia coli, sistem promotor, induser, protein rekombinan
... Operon ramnosa E. coli terdiri dari tiga promotor yang teraktivasi saat penambahan ramnosa (Wickstrum et al., 2007;Wickstrum et al., 2005) Namun untuk aplikasi sistem ekspresi ramnosa, ekpresi protein regulator dalam jumlah besar tidak diperlukan karena jumlah yang diekspresikan oleh kromosom cukup untuk mengaktifkan transkripsi walaupun pada plasmid multikopi. Sehingga hanya promotor rhaPBAD yang perlu diklon pada gen untuk dapat diekspresikan (Wagerer et al., 2008). ...
... Aktivasi penuh ketiga operon rha juga membutuhkan cAMP selain penggunaan RhaS ataupun RhaR. CRP berikatan sebagai dimer pada sisi DNA spesifik dan menginduksi suatu lengkungan DNA yang kira-kira 90 derajat dengan kehadiran ligannya, cAMP (Wickstrum et al. 2005). Bagaimanapun juga, bahkan dalam keadaan absennya glukosa, promotor ini masih terbilang baik. ...
Dalam Buku ini dibahas mengenai pengertian, prinsip dan penerapan beberapa teknik biologi molekul, meliputi isolasi templat DNA (deoxcyrebonucleic acid), perbanyakan fragmen DNA dengan PCR (polymerase chain reaction), pemotongan DNA dengan enzim restriksi, kloning atau TDR (teknologi DNA rekombinan), penentuan urutan DNA dengan DNA sequencer, dan overekspresi protein rekombinan pada Escherichia coli baik secara intra- maupun ekstraselular, serta optimasi kodon dan gen sintesis sebagai alternatif isolasi DNA dan PCR.
pemanfaatan teknik biologi molekul ini diarahkan pada non rekayasa genetik maupun rekayasa genetik. pada non rekayasa genetik, teknik biologi molekul diterapkan di bidang kesehatan untuk didiagnosis tingkat molekul penyakit infeksi atau kelainan metabolisme, dan di bidang forensik dan antropologi berdasarkan analisis homologi dan hhubungan kekerabatan. Melalui rekayasa genetik dapat memperbaiki galur suatu organisme sehingga dapat memproduksi produk metabolisme lebih banyak serta dapat menghasilkan galur baru yang dapat membuat produk metabolisme baru. Organisme pada tingkat prokariotik (misalnya Escherichia coli) dapat dijadikan sumber sebagai sel inang untuk memproduksi peptida atau protein rekombinan.
... Operon ramnosa E. coli terdiri dari tiga promotor yang teraktivasi saat penambahan ramnosa (Wickstrum et al., 2005) (Wagerer et al., 2008). ...
... Aktivasi penuh ketiga operon rha juga membutuhkan cAMP selain penggunaan RhaS ataupun RhaR. CRP berikatan sebagai dimer pada sisi DNA spesifik dan menginduksi suatu lengkungan DNA yang kira-kira 90 derajat dengan kehadiran ligannya, cAMP (Wickstrum et al. 2005). ...
Dalam buku ini dibahas mengenai sistem ekspresi ekstraselular yang telah dikembangkan di dalam Escherichia coli (E. coli) meliputi peran sinyal peptidase pada sekresi protein, mekanisme degradasi dan pelipatan protein dalam membran periplasma, sistem sekresi protein ekstraselular Jalur Twin-Arginine Translocation (Tat) dan kemampuannya dalam memfasilitasi sekresi protein, ekspresi protein pada Escherichia coli menggunakan sistem promotor Rhapbad, sekresi ekstraselular menggunakan phospolipase c secara ko-ekspresi.
... In the presence of rhamnose, RhaR acts as inducer of rhaS, which itself induces transcription of rhaBAD and rhaT (Egan and Schleif 1993;Vía et al. 1996). Cyclic AMP receptor protein (CRP) functions as coactivator for the transcription of all three operons rhaBAD, rhaT and rhaS, which render the system susceptible to catabolite repression (Holcroft and Egan 2000a;Holcroft and Egan 2000b;Wickstrum et al. 2005). RhaS itself is capable of activating rhaSR transcription, but due to a lower CRP contribution, it results in a lower transcription than by activation with RhaR. ...
Tuning of transcription is a promising strategy to overcome challenges associated with a non-suitable expression rate like outgrowth of segregants, inclusion body formation, metabolic burden and inefficient translocation. By adjusting the expression rate—even on line—to purposeful levels higher product titres and more cost-efficient production processes can be achieved by enabling culture long-term stability and constant product quality. Some tunable systems are registered for patents or already commercially available. Within this contribution, we discuss the induction mechanisms of various Escherichia coli inherent promoter systems with respect to their tunability and review studies using these systems for expression tuning. According to the current level of knowledge, some promoter systems were successfully used for expression tuning, and in some cases, analytical evidence on single-cell level is still pending. However, only a few studies using tunable strains apply a suitable process control strategy. So far, expression tuning has only gathered little attention, but we anticipate that expression tuning harbours great potential for enabling and optimizing the production of a broad spectrum of products in E. coli.
... With respect to the transcriptional regulation of the genes involved in rhamnose catabolism, the regulatory system of E. coli is most finely elucidated (19)(20)(21)(22)(23). It was reported that, in the E. coli genome, genes encoding the rhamnose transporter (RhaT), the rhamnose catabolic enzymes (RhaA, RhaB, and RhaD), and the rhamnose-responsive transcriptional activators (RhaS and RhaR) are assembled to form the rhaSR and rhaBAD operons and the rhaT gene, which are located adjacent to one another. ...
... In response to rhamnose, RhaR activates the rhaSR operon, and the resulting RhaS associated with rhamnose, in turn, activates the rhaBAD (possibly rhaBADM) operon and the rhaT gene (20)(21)(22). The cyclic AMP receptor protein (CRP) functions as a coactivator by binding simultaneously with RhaS or RhaR to each of the regulatory regions (20,22,23). This involvement of CRP means that the expression of the genes for rhamnose catabolism is under the carbon catabolite repression. ...
Unlabelled:
The Bacillus subtilis rhaEWRBMA (formerly yuxG-yulBCDE) operon consists of four genes encoding enzymes for l-rhamnose catabolism and the rhaR gene encoding a DeoR-type transcriptional regulator. DNase I footprinting analysis showed that the RhaR protein specifically binds to the regulatory region upstream of the rhaEW gene, in which two imperfect direct repeats are included. Gel retardation analysis revealed that the direct repeat farther upstream is essential for the high-affinity binding of RhaR and that the DNA binding of RhaR was effectively inhibited by L-rhamnulose-1-phosphate, an intermediate of L-rhamnose catabolism. Moreover, it was demonstrated that the CcpA/P-Ser-HPr complex, primarily governing the carbon catabolite control in B. subtilis, binds to the catabolite-responsive element, which overlaps the RhaR binding site. In vivo analysis of the rhaEW promoter-lacZ fusion in the background of ccpA deletion showed that the L-rhamnose-responsive induction of the rhaEW promoter was negated by the disruption of rhaA or rhaB but not rhaEW or rhaM, whereas rhaR disruption resulted in constitutive rhaEW promoter activity. These in vitro and in vivo results clearly indicate that RhaR represses the operon by binding to the operator site, which is detached by L-rhamnulose-1-phosphate formed from L-rhamnose through a sequence of isomerization by RhaA and phosphorylation by RhaB, leading to the derepression of the operon. In addition, the lacZ reporter analysis using the strains with or without the ccpA deletion under the background of rhaR disruption supported the involvement of CcpA in the carbon catabolite repression of the operon.
Importance:
Since L-rhamnose is a component of various plant-derived compounds, it is a potential carbon source for plant-associating bacteria. Moreover, it is suggested that L-rhamnose catabolism plays a significant role in some bacteria-plant interactions, e.g., invasion of plant pathogens and nodulation of rhizobia. Despite the physiological importance of L-rhamnose catabolism for various bacterial species, the transcriptional regulation of the relevant genes has been poorly understood, except for the regulatory system of Escherichia coli. In this study, we show that, in Bacillus subtilis, one of the plant growth-promoting rhizobacteria, the rhaEWRBMA operon for L-rhamnose catabolism is controlled by RhaR and CcpA. This regulatory system can be another standard model for better understanding the regulatory mechanisms of L-rhamnose catabolism in other bacterial species.
... At the bgl operon, which is controlled by HNS, StpA, CRP, and LeuO, BglJ-RcsB and CRP presumably act synergistically to overcome repression by HNS ( Figure 5). Synergistic activation of a transcriptional regulator together with CRP has been shown at several loci including the ara locus by AraC and CRP, malE by MalT and CRP, rha by RhaS/ RhaR and CRP and asc by two CRP dimers, among others (37,(44)(45)(46)(47). However, at the bgl locus BglJ-RcsB shifts transcription initiation from a class I CRP dependent promoter to a promoter with the center of the CRPbinding site mapping at position À41.5, which is typical of class II CRP-dependent promoters (Figures 2 and 5). ...
The bacterial Rcs phosphorelay signals perturbations of the bacterial cell envelope to its response regulator RcsB, which
regulates transcription of multiple loci related to motility, biofilm formation and various stress responses. RcsB is unique,
as its set of target loci is modulated by interaction with auxiliary regulators including BglJ. The BglJ–RcsB heteromer is
known to activate the HNS repressed leuO and bgl loci independent of RcsB phosphorylation. Here, we show that BglJ–RcsB activates the promoters of 10 additional loci (chiA, molR, sfsB, yecT, yqhG, ygiZ, yidL, ykiA, ynbA and ynjI). Furthermore, we mapped the BglJ–RcsB binding site at seven loci and propose a consensus sequence motif. The data suggest
that activation by BglJ–RcsB is DNA phasing dependent at some loci, a feature reminiscent of canonical transcriptional activators,
while at other loci BglJ–RcsB activation may be indirect by inhibition of HNS-mediated repression. In addition, we show that
BglJ–RcsB activates transcription of bgl synergistically with CRP where it shifts the transcription start by 20 bp from a position typical for class I CRP-dependent
promoters to a position typical for class II CRP-dependent promoters. Thus, BglJ–RcsB is a pleiotropic transcriptional activator
that coordinates regulation with global regulators including CRP, LeuO and HNS.
... In this context, several reports provide evidence that two transcription factors work cooperatively in response to the same signal, as in Vibrio vulnificus, where the nan operon is negatively regulated by CRP and NanR in the presence of N-acetylmannosamine 6-phosphate (Kim et al., 2011). Also, in E. coli CRP requires the presence of RhaR to efficiently activate rhaSR in vivo in response to Lrhamnose (Wickstrum et al., 2005). Similarly, studies in Haemophilus influenzae suggest that CRP and SiaR regulate their respective operators by simultaneously binding to an intergenic region between nan and siaPT, where SiaR functions as both a repressor and activator, using glucosamine-6-phosphate as a co-activator, and interacts with CRP to regulate these divergent promoters (Johnston et al., 2010). ...
OmpW is a minor porin that has been involved in osmoregulation and the uptake of ceftriaxone. Evidence obtained in our laboratory indicates that in S. Typhimurium 14028s its expression is increased by SoxS, upon exposure to paraquat, and is required for the resistance to the toxic compound. SoxS belongs to the AraC family of transcriptional regulators, of which MarA and Rob are also members. Due to the high structural similarity among these proteins, genes under their control have been denominated the mar/sox/rob regulon and present a DNA binding consensus sequence denominated marsox box. In this work, we evaluated the role of the transcription factors MarA, SoxS and Rob of S. Typhimurium in regulating ompW expression in response to menadione. To evaluate the changes in ompW expression, we determined the transcript and protein levels of OmpW in the different genetic backgrounds with or without treatment with the toxic compound. ompW expression was up-regulated in response to menadione in the wild type and Δrob strains. In the ΔmarA and ΔsoxS backgrounds, the positive regulation was abolished. Using transcriptional fusions and EMSAs with the wild type and mutant versions of the promoter region we demonstrated that two of the predicted sites were functional. In a double marA soxS mutant strain, ompW transcript levels were lowered after exposure to menadione, and only in trans complementation with both genes was able to restore the positive regulation observed in the wild type strain. Additionally, using EMSAs we demonstrate that MarA increases the affinity of SoxS for the promoter region of ompW, supporting a mechanism of cooperative regulation. In conclusion, we demonstrate that in response to menadione, ompW expression is cooperatively regulated by both MarA and SoxS through a direct interaction with the promoter region.
... CRP activates transcription from over 150 different promoters in E. coli (Kolb et al., 1993; Zheng et al., 2004) and this CRP regulon includes several operons mainly involved in catabolism of carbon sources other than glucose (Kolb et al., 1993). As observed for the MhpR regulator, CRP also regulates expression of other transcription factors in E. coli such as MelR (Webster et al., 1988), MalT (Chapon & Kolb, 1983), RhaS (Wickstrum et al., 2005), MaoB (Yamashita et al., 1996), HcaR (Turlin et al., 2001), GalS (Weickert & Adhya, 1993), RpoH (Kallipolitis & Valentin-Hansen, 1998), BlgG (Gulati & Mahadevan, 2000), PrpR (Lee et al., 2005) and Fis (Nasser et al., 2001). Taking our results together with previous data (Torres et al., 2003) we have demonstrated that the glucose effect on mhp expression is exerted at the level of both catabolic and regulatory promoters. ...
... Taking our results together with previous data (Torres et al., 2003) we have demonstrated that the glucose effect on mhp expression is exerted at the level of both catabolic and regulatory promoters. This simultaneous CRP control is not unique to the mhp pathway, since it has been also described for the genes for propionate catabolism in E. coli and Salmonella enterica (Lee et al., 2005) and for some regulons from E. coli such as those for L-rhamnose (Holcroft & Egan, 2000; Wickstrum et al., 2005), melibiose (Webster et al., 1988; Belyaeva et al., 2000) and maltose (Chapon & Kolb, 1983; Richet & Sogaard-Andersen, 1994; Richet, 2000). While the expression of the mhpR, melR and malT regulators depends only on the presence of CRP (Webster et al., 1988; Chapon & Kolb, 1983), the expression of rhaSR and prpR depends on the presence of CRP and the transcription activators RhaR and PrpR, respectively (Tobin & Schleif, 1990a, b; Wickstrum et al., 2005; Lee et al., 2005). ...
... This simultaneous CRP control is not unique to the mhp pathway, since it has been also described for the genes for propionate catabolism in E. coli and Salmonella enterica (Lee et al., 2005) and for some regulons from E. coli such as those for L-rhamnose (Holcroft & Egan, 2000; Wickstrum et al., 2005), melibiose (Webster et al., 1988; Belyaeva et al., 2000) and maltose (Chapon & Kolb, 1983; Richet & Sogaard-Andersen, 1994; Richet, 2000). While the expression of the mhpR, melR and malT regulators depends only on the presence of CRP (Webster et al., 1988; Chapon & Kolb, 1983), the expression of rhaSR and prpR depends on the presence of CRP and the transcription activators RhaR and PrpR, respectively (Tobin & Schleif, 1990a, b; Wickstrum et al., 2005; Lee et al., 2005). The mechanism of CRP activation of these promoters is different, the Pr promoter of mhpR and the promoter of melR being class II CRP-dependent promoters (Webster et al., 1988; Samarasinghe et al., 2008) whereas the promoter of malT, and the P SR and P prpR promoters behave as class I CRP-dependent promoters (Chapon & Kolb, 1983; Wickstrum et al., 2005; Lee et al., 2005).Busby & Ebright, 1999) is compared to the Pr promoter sequence from nt " 51 to " 30 and the known cAMP–CRP binding sites at pgalP1 and pmelR, which are located from " 52 to " 31 with respect to its transcription start point. ...
The expression of the mhp genes involved in the degradation of the aromatic compound 3-(3-hydroxyphenyl)propionic acid (3HPP) in Escherichia coli is dependent on the MhpR transcriptional activator at the Pa promoter. This catabolic promoter is also subject to catabolic repression in the presence of glucose mediated by the cAMP-CRP complex. The Pr promoter drives the MhpR-independent expression of the regulatory gene. In vivo and in vitro experiments have shown that transcription from the Pr promoter is downregulated by the addition of glucose and this catabolic repression is also mediated by the cAMP-CRP complex. The activation role of the cAMP-CRP regulatory system was further investigated by DNase I footprinting assays, which showed that the cAMP-CRP complex binds to the Pr promoter sequence, protecting a region centred at position -40.5, which allowed the classification of Pr as a class II CRP-dependent promoter. Open complex formation at the Pr promoter is observed only when RNA polymerase and cAMP-CRP are present. Finally, by in vitro transcription assays we have demonstrated the absolute requirement of the cAMP-CRP complex for the activation of the Pr promoter.
