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The cAMP-CRP/CytR nucleoprotein complex in Escherichia coli: two pairs of closely linked binding sites for the cAMP-CRP activator complex are involved in combinatorial regulation of the cdd promoter.

ABSTRACT: Transcription initiation at CytR regulated promoters in Escherichia coli is controlled by a combinatorial regulatory system in which the cAMP receptor protein (CRP) functions as both an activator and a co-repressor. By combining genetic studies and footprinting analyses, we demonstrate that regulated expression of the CytR controlled cdd promoter requires three CRP-binding sites: a high affinity site (CRP-1) and two overlapping low affinity sites (CRP-2 and CRP-3) centred at positions -41, -91 and -93, respectively. In the absence of CytR, cAMP-CRP interacts at one set of sites (CRP-1 and CRP-2) and both of these binding sites are required for full promoter activation. In the presence of CytR, however, the two regulators bind cooperatively to cddP forming a nucleoprotein complex in which cAMP-CRP binds to CRP-1 and CRP-3 and CytR occupies the sequence between these sites. Thus, association of the two regulators involves a repositioning of the cAMP-CRP complex. Moreover, mutant cdd promoters in which CRP-2 and CRP-3 have been deleted are partially regulated by CytR, and cAMP-CRP and CytR still bind cooperatively to these promoters. These findings provide clues to an understanding of how cAMP-CRP and CytR interact at a structurally diverse set of promoters.

SUBMITTER: Holst B

PROVIDER: S-EPMC556823 | biostudies-other | 1992 Oct

REPOSITORIES: biostudies-other

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The cAMP-CRP/CytR nucleoprotein complex in Escherichia coli: two pairs of closely linked binding sites for the cAMP-CRP activator complex are involved in combinatorial regulation of the cdd promoter.

Holst B B Søgaard-Andersen L L Pedersen H H Valentin-Hansen P P

The EMBO journal 19921001 10

Transcription initiation at CytR regulated promoters in Escherichia coli is controlled by a combinatorial regulatory system in which the cAMP receptor protein (CRP) functions as both an activator and a co-repressor. By combining genetic studies and footprinting analyses, we demonstrate that regulated expression of the CytR controlled cdd promoter requires three CRP-binding sites: a high affinity site (CRP-1) and two overlapping low affinity sites (CRP-2 and CRP-3) centred at positions -41, -91 a ...[more]

PMID: 1327747

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Project description:BackgroundCyclic AMP receptor protein (CRP), also known as catabolite gene activator protein (CAP), is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally studied in only two cyanobacterial strains: Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; therefore, a systematic genome-scale study of the potential CRP target genes and binding sites in cyanobacterial genomes is urgently needed.ResultsWe have predicted and analyzed the CRP binding sites and regulons in 12 sequenced cyanobacterial genomes using a highly effective cis-regulatory binding site scanning algorithm. Our results show that cyanobacterial CRP binding sites are very similar to those in E. coli; however, the regulons are very different from that of E. coli. Furthermore, CRP regulons in different cyanobacterial species/ecotypes are also highly diversified, ranging from photosynthesis, carbon fixation and nitrogen assimilation, to chemotaxis and signal transduction. In addition, our prediction indicates that crp genes in modern cyanobacteria are likely inherited from a common ancestral gene in their last common ancestor, and have adapted various cellular functions in different environments, while some cyanobacteria lost their crp genes as well as CRP binding sites during the course of evolution.ConclusionThe CRP regulons in cyanobacteria are highly diversified, probably as a result of divergent evolution to adapt to various ecological niches. Cyanobacterial CRPs may function as lineage-specific regulators participating in various cellular processes, and are important in some lineages. However, they are dispensable in some other lineages. The loss of CRPs in these species leads to the rapid loss of their binding sites in the genomes.

Project description:Biofilm formation by Klebsiella pneumoniae on indwelling medical devices increases the risk of infection. Both type 1 and type 3 fimbriae are important factors in biofilm formation by K. pneumoniae. We found that a putative enzyme II (EII) complex of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), etcA (EIIA)-etcB (EIIB)-etcC (EIIC), regulated biofilm and type 3 fimbriae formation by K. pneumoniae STU1. In this study, the regulatory mechanism of etcABC in K. pneumoniae type 3 fimbriae formation was investigated. We found via quantitative RT-PCR that overexpression of etcABC enhanced the transcription level of the mrk operon, which is involved in type 3 fimbriae synthesis, and reduced the transcription level of the fim operon, which is involved in type 1 fimbriae synthesis. To gain further insight into the role of etcABC in type 3 fimbriae synthesis, we analyzed the region upstream of the mrk operon and found the potential cyclic 3'5'-adenosine monophosphate (cAMP) receptor protein (CRP) binding site. After crp was deleted in K. pneumoniae STU1 and two clinical isolates, these three crp mutant strains could not express MrkA, the major subunit of the fimbrial shaft, indicating that CRP positively regulated type 3 fimbriae synthesis. Moreover, a crp mutant overexpressing etcABC could not express MrkA, indicating that the regulation of type 3 fimbriae by etcABC was dependent on CRP. In addition, deletion of cyaA, which encodes the adenylyl cyclase that synthesizes cAMP, and deletion of crr, which encodes the glucose-specific EIIA, led to a reduction in lac operon regulation and therefore bacterial lactose uptake in K. pneumoniae. Exogenous cAMP but not etcABC overexpression compensated for the role of cyaA in bacterial lactose uptake. However, either etcABC overexpression or exogenous cAMP compensated for the role of crr in bacterial lac operon regulation that would eventually restore lactose uptake. We also found via ELISA and the luxCDABE reporter system that overexpression of etcABC increased intracellular cAMP levels and the transcription level of crp, respectively, in K. pneumoniae. In conclusion, overexpression of etcABC positively regulated cAMP production and cAMP-CRP activity to activate the mrk operon, resulting in increased type 3 fimbriae synthesis in K. pneumoniae.

Project description:Protein-protein interactions play an essential role in cellular processes. Certain proteins form stable complexes with their partner proteins, whereas others function by forming transient complexes. The conventional protein-protein interaction model describes an interaction between two proteins under the assumption that a protein binds to its partner protein through a single binding site. In this study, we improved the conventional interaction model by developing a Multiple-Site (MS) model in which a protein binds to its partner protein through closely located multiple binding sites on a surface of the partner protein by transiently docking at each binding site with individual binding free energies. To test this model, we used the protein-protein interaction mediated by Src homology 3 (SH3) domains. SH3 domains recognize their partners via a weak, transient interaction and are therefore promiscuous in nature. Because the MS model requires large amounts of data compared with the conventional interaction model, we used experimental data from the positionally addressable syntheses of peptides on cellulose membranes (SPOT-synthesis) technique. From the analysis of the experimental data, individual binding free energies for each binding site of peptides were extracted. A comparison of the individual binding free energies from the analysis with those from atomistic force fields gave a correlation coefficient of 0.66. Furthermore, application of the MS model to 10 SH3 domains lowers the prediction error by up to 9% compared with the conventional interaction model. This improvement in prediction originates from a more realistic description of complex formation than the conventional interaction model. The results suggested that, in many cases, SH3 domains increased the protein complex population through multiple binding sites of their partner proteins. Our study indicates that the consideration of general complex formation is important for the accurate description of protein complex formation, and especially for those of weak or transient protein complexes.

Dataset Information

The cAMP-CRP/CytR nucleoprotein complex in Escherichia coli: two pairs of closely linked binding sites for the cAMP-CRP activator complex are involved in combinatorial regulation of the cdd promoter.

Publications

The cAMP-CRP/CytR nucleoprotein complex in Escherichia coli: two pairs of closely linked binding sites for the cAMP-CRP activator complex are involved in combinatorial regulation of the cdd promoter.

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