Unusual Oligomerization Required for Activity of NtrC, a Bacterial Enhancer-Binding Protein

Wyman, Claire; Rombel, Irene; North, Anne K.; Bustamante, Carlos; Kustu, Sydney

doi:10.1126/science.275.5306.1658

Cited by 233 publications

(251 citation statements)

References 24 publications

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“…This is also compatible with the data available for NtrC. In this case, scanning force microscopy suggests that transcriptional activation of glnAp2 may depend on the formation of an NtrC oligomer larger than a tetramer (Rippe et al, 1997 ;Wyman et al, 1997). Furthermore, analytical ultracentrifugation studies indicate that NtrC phosphorylation causes the formation of an octameric complex within the UAS of glnAp2 (Rippe et al, 1998) with an ability to bind simultaneously two target DNA sequences.…”

Section: Conclusion : Oligomerization Of Xylr During Activation Of Pusupporting

confidence: 84%

Visualization of DNA–protein intermediates during activation of the Pu promoter of the TOL plasmid of Pseudomonas putida

Garmendía

2000

Microbiology

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The ATP-dependent multimerization process undergone by the σ 54 -dependent activator XylR of the TOL plasmid pWW0 of Pseudomonas putida when bound to the upstream activating sequences (UAS) of the cognate Pu promoter was examined by transmission electron microscopy (TEM). To this end, supercoiled DNA templates were combined with increasing concentrations of the constitutive XylR variant XylR∆A, with or without ATP or its non-hydrolysable analogue ATPγS, and the resulting complexes were visualized by TEM. The different types of DNA-protein association were analysed and a statistical study of the frequency of the various forms was made. ATP appeared to establish an equilibrium between different molecular associations, as well as major changes in the physical shape of the DNA-protein complexes. The formation of higher nucleoprotein structures frequently bearing DNA bends became manifest. Such complexes often engaged otherwise separated UAScontaining plasmids, indicating that the ATP-driven multimer included XylR molecules recruited in trans. Whilst ATP caused the different types of XylR-DNA complex to occur at quite balanced frequencies, ATPγS appeared to displace the distribution predominantly towards the higher order forms. These data are compatible with the notion that each time ATP is hydrolysed the transcriptional activation complex is disassembled.

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Section: Conclusion : Oligomerization Of Xylr During Activation Of Pusupporting

confidence: 84%

Visualization of DNA–protein intermediates during activation of the Pu promoter of the TOL plasmid of Pseudomonas putida

Garmendía

2000

Microbiology

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“…One explanation is that these amino acid substitutions could affect the ability of the protein to form oligomers. S. typhimurium NtrC must assemble into a large oligomer to activate transcription in a process that involves both protein-DNA and protein-protein interactions (Porter et al, 1993;Wyman et al, 1997). We assume that DctD (⌬1-142) must similarly assemble into an oligomer to activate transcription.…”

Section: Discussionmentioning

confidence: 99%

A conserved region in the σ⁵⁴‐dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation

Wang

Lee

Brewer

et al. 1997

Molecular Microbiology

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SummaryRhizobium melioti DctD activates transcription from the dctA promoter by catalysing the isomerization of closed complexes between 54 -RNA polymerase holoenzyme and the promoter to open complexes. DctD must make productive contact with 54 -holoenzyme and hydrolyse ATP to catalyse this isomerization. To define further the activation process, we sought to isolate mutants of DctD that had reduced affinities for 54 -holoenzyme. Mutagenesis was confined to the well-conserved C3 region of the protein, which is required for coupling ATP hydrolysis to open complex formation in 54 -dependent activators. Mutant forms of DctD that failed to activate transcription and had substitutions in the C-terminal half of the C3 region were efficiently cross-linked to 54 and the ␤-subunit of RNA polymerase, suggesting that they bound normally to 54 -holoenzyme. In contrast, some mutant forms of DctD with amino acid substitutions in the N-terminal half of the C3 region had reduced affinities for 54 and the ␤-subunit in the cross-linking assay. These data suggest that the N-terminal half of the C3 region of DctD contains a site that may contact 54-holoenzyme during open complex formation.

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“…It can also activate transcription in trans (12). NtrC is an activator that binds to the enhancer, and, when phosphorylated by NtrB protein kinase, forms higher order homo-oligomers and is capable of activating the transcription of the glnAp2 gene (13)(14)(15)(16). Phosphorylation of NtrC also activates its ATPase activity, which is required to stimulate conversion from the closed initiation complex (RP c ) to the open initiation complex (RP o ) (13)(14)(15)(16).…”

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confidence: 99%

DNA supercoiling allows enhancer action over a large distance

Liu

Бондаренко

Ninfa

et al. 2001

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

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Enhancers are regulatory DNA elements that can activate their genomic targets over a large distance. The mechanism of enhancer action over large distance is unknown. Activation of the glnAp2 promoter by NtrC-dependent enhancer in Escherichia coli was analyzed by using a purified system supporting multiple-round transcription in vitro. The data suggest that DNA supercoiling is an essential requirement for enhancer action over a large distance (2,500 bp) but not over a short distance (110 bp). DNA supercoiling facilitates functional enhancer-promoter communication over a large distance, probably by bringing the enhancer and promoter into close proximity.T ranscriptional enhancers are relatively short (30 -200 bp) DNA sequences usually composed of several binding sites for activator protein(s). The landmark of enhancers is their ability to communicate with promoters and activate genes over a large distance (up to 60 kb; see ref. 1 for a review). Prokaryotic and eukaryotic enhancers share several key properties such as ability to activate transcription over a large distance, tight coupling of DNA melting with ATP hydrolysis and high stability of the initiation complexes (see refs. 2 and 3 for recent reviews). In contrast to eukaryotic enhancers, prokaryotic enhancers can activate transcription over a large distance in vitro. This ability allows detailed analysis of the mechanism of enhancer action impossible in the case of eukaryotic enhancers.The mechanism of enhancer action over a large distance is unknown. It is most likely that proteins bound at the enhancer and promoter directly interact such that intervening DNA forms a loop (see refs. 4 and 5 for reviews). The main problem for communication over a large distance is low concentration of the DNA regions in the vicinity of each other (see ref. 4 for a review). Measurements of local concentration of linear DNA ends in the vicinity of each other suggest that it is relatively high (10 Ϫ7 M) only when the distance between DNA ends is 100-900 bp (6). When distance between the DNA ends is increased to 3 kb, their local concentration is decreased by an order of magnitude (6). Thus, it is remarkable that DNA regions positioned far away from each other (such as an enhancer and a promoter) can communicate efficiently.Several models have been proposed to explain the mechanism of enhancer action over a large distance. One class of models suggests that initial communication of an enhancer with a promoter leads to formation of a stable DNA-protein complex in the vicinity of the promoter. This stable complex may facilitate subsequent rounds of transcription serving as a ''memory'' of initial enhancer-promoter interaction (see ref. 1 for a review). Alternatively, the average distance between promoter and enhancer could be considerably decreased if the intervening DNA is supercoiled (sc) or bent (see ref. 4 for a review). It has also been shown that sequence-dependent superhelical DNA inserts can facilitate enhancer action (7).The mechanism of action of the NtrC-dependent, 54...

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Unusual Oligomerization Required for Activity of NtrC, a Bacterial Enhancer-Binding Protein

Cited by 233 publications

References 24 publications

Visualization of DNA–protein intermediates during activation of the Pu promoter of the TOL plasmid of Pseudomonas putida

Visualization of DNA–protein intermediates during activation of the Pu promoter of the TOL plasmid of Pseudomonas putida

A conserved region in the σ⁵⁴‐dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation

DNA supercoiling allows enhancer action over a large distance

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Unusual Oligomerization Required for Activity of NtrC, a Bacterial Enhancer-Binding Protein

Cited by 233 publications

References 24 publications

Visualization of DNA–protein intermediates during activation of the Pu promoter of the TOL plasmid of Pseudomonas putida

Visualization of DNA–protein intermediates during activation of the Pu promoter of the TOL plasmid of Pseudomonas putida

A conserved region in the σ54‐dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation

DNA supercoiling allows enhancer action over a large distance

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A conserved region in the σ⁵⁴‐dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation