Effect of salt bridge on transcription activation of CRP-dependent lactose operon in Escherichia coli

Tutar, Yusuf; Harman, James G.

doi:10.1016/j.abb.2006.06.019

Cited by 4 publications

(2 citation statements)

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“…The impact of salt bridges formation in the activity of such transcription factor is poorly understood, but the few available studies suggest that they may influence both DNA binding affinities and trans-activation properties of transcription factors. Disruption of the salt bridge by amino acid substitution affected the CRP (cAMP Receptor Protein) protein activity and led to a reduction of the Lac promoter trans-activation, without affecting its DNA binding affinity [42,43]. This reduction is attributed to an alteration of the interaction with the α-subunit of RNA polymerase.…”

Section: Discussionmentioning

confidence: 99%

A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity

et al. 2011

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BackgroundFlavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR). In grapevine (Vitis vinifera L.), several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood.ResultsIn this work, we describe the effects of a single amino acid substitution (R69L) located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5bL, the native protein being referred as VvMYB5bR) was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5bL exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5bR and VvMYB5bL genes in tobacco. Flowers from 35S::VvMYB5bL transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5bR and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation.ConclusionsTaken together, our findings indicate that VvMYB5bL is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s). In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment of protein-protein interactions between MYB and bHLH transcription factors. Mechanisms underlying these interactions are discussed and a model is proposed to explain the transcriptional activity of VvMYB5L observed in the tobacco model.

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Section: Discussionmentioning

confidence: 99%

A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity

et al. 2011

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“…12,13,[29][30][31][32][33][34][35] However, near-UV measurements indicated that the tertiary structure of CRP changes upon cAMP binding. 34,35 Binding of ligand introduces three essential alignments: (i) N-domain C-domain alignment through the C and D helix of each subunit, (ii) subunit-subunit alignment through C-helices of each subunit, (iii) F-helix protrusion from the protein for specific DNA interaction ( Figure 2). [36][37][38][39] Cyclic AMP is not the only factor that induces conformational changes in CRP structure.…”

Section: Conformational States Of Crpmentioning

confidence: 90%

Syn, anti, and finally both conformations of cyclic AMP are involved in the CRP‐dependent transcription initiation mechanism in E. coli lac operon

Tutar¹

2008

Cell Biochemistry & Function

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The cyclic AMP receptor protein (CRP) of Escherichia coli regulates the activity of more than 150 genes. Allosteric changes in CRP structure accompanied by cAMP binding, initiate transcription through protein binding to specific DNA sequences. Initially, researchers proposed a two-site cAMP-binding model for CRP-dependent transcription activation since biophysical methods showed two transitions during titration experiments. Three conformational states were considered; apo-CRP, CRP:(cAMP)(1) and CRP:(cAMP)(2), and CRP:(cAMP)(1) was proposed as the active form in this initial model. X-ray data indicated an anti conformation and in contrast NMR experiments suggested a syn conformation for bound cAMPs. For years this paradigm about ligand conformation has been ambiguous. When CRP was crystallized with four bound cAMP in the last decade, two cAMPs were assigned to syn and the other two to anti conformations. Again three conformational states were suggested; apo-CRP, CRP:(cAMP)(2), and CRP:(cAMP)(4). This new structure changed the view of CRP allosteric activation from a two-site model to a four-site model in the literature and the new model has been supported by biochemical and genetic data so far. According to the accepted model, binding of the first two cAMP molecules displays positive cooperativity, however, binding of the last two cAMP molecules shows negative cooperativity. This resolved the conflict between dynamic and static experimental observations. However, this new model cannot explain the initiation mechanism as previously proposed because functionally active CRP has only one cAMP equivalent. Gene regulation and transcription factors are involved in regulating both prokaryotic and eukaryotic metabolism. Although gene regulation and expression are much more complex in eukaryotes, CRP-mediated transcription initiation is a model of general interest to life sciences and medicine. Therefore, the aim of this review is to summarize recent works and developments on the cAMP-dependent CRP activation mechanism in E. coli.

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cAMP receptor protein (CRP)-mediated resistance/tolerance in bacteria: mechanism and utilization in biotechnology

Geng

Jiang

2015

Appl Microbiol Biotechnol

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Cyclic AMP receptor protein (CRP) is one of the seven global regulators in Escherichia coli, which regulates the expression of over 490 genes. It contains a cAMP binding N-terminal domain and a DNA binding C-terminal domain, connected via a short hinge region. Various stress-tolerant E. coli mutants had been obtained through transcriptional engineering of CRP. This review aims to shed some light on the possible mechanism behind these CRP variants, from the change in CRP structure, transcription profile, and DNA binding affinity. The amino acid substitutions are distributed along the protein-certain mutations have shown higher frequency than others, such as T127N and D138Y. β-Galactosidase reporter gene assay revealed that CRP mutants had lower binding affinity with all three classes of CRP-dependent promoters as compared to native CRP, which probably would change cellular transcription profile. Different CRP mutants would induce different cellular transcription profile in E. coli, but there are common genes differentially expressed in these variants, including upregulated gadAB and downregulated nontransporter genes aspA and tnaA, and transporter/poringenes malE, mglB, cstA, and lamB. We believe that transcriptional engineering of CRP can provide an alternative strain engineering method for E. coli and its detailed mechanism may need further investigations.

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Effect of salt bridge on transcription activation of CRP-dependent lactose operon in Escherichia coli

Cited by 4 publications

References 39 publications

A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity

A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity

Syn, anti, and finally both conformations of cyclic AMP are involved in the CRP‐dependent transcription initiation mechanism in E. coli lac operon

cAMP receptor protein (CRP)-mediated resistance/tolerance in bacteria: mechanism and utilization in biotechnology

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