A mutation outside the two zinc fingers of ADR1 can suppress defects in either finger.

A second-site mutation that restored DNA binding to ADR1 mutants altered at different positions in the two zinc fingers was identified. This mutation (called IS1) was a conservative change of arginine 91 to lysine in a region amino terminal to the two zinc fingers and known from previous experiments to be necessary for DNA binding. IS1 increased binding to the UAS1 sequence two- to sevenfold for various ADR1 mutants and twofold for wild-type ADR1. The change of arginine 91 to glycine decreased binding twofold, suggesting that this arginine is involved in DNA binding in the wild-type protein. The increase in binding by IS1 did not involve protein-protein interactions between the two ADR1 monomers, nor did it require the presence of the sequences flanking UAS1. However, the effect of IS1 was influenced by the sequence of the first finger, suggesting that interactions between the region amino terminal to the fingers and the fingers themselves could exist. A model for the role of the amino-terminal region based on these results and sequence homologies with other DNA-binding motifs is proposed.

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Domain swapping reveals the modular nature of Fos, Jun, and CREB proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4565 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4565-4573 ◽

Cited By ~ 21

Author(s):

L J Ransone ◽

P Wamsley ◽

K L Morley ◽

I M Verma

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Leucine Zipper ◽

Chimeric Protein ◽

Domain Swapping ◽

Amino Terminus ◽

Protein Protein Interactions ◽

Amino Terminal ◽

Nih 3T3 ◽

Basic Region

The products of the Jun and Fos proto-oncogenes form a heterodimer that binds to and activates transcription from 12-O-tetradecanoylphorbol-13-acetate-responsive promoter elements (TGACTCA) and AP-1-binding sites (TGACATCA). These two proteins belong to a family of related transcription factors which contain similar domains required for protein dimerization and DNA binding but display different protein and DNA binding specificities. The basic region, required for DNA binding, is followed by a leucine zipper structure, a domain that mediates protein-protein interactions. To assess the role of these two domains in three related proteins, Fos, Jun, and CREB, we carried out extensive domain-swapping analysis. We found that (i) dimers formed by two Jun leucine zipper-containing proteins were unable to bind DNA as efficiently as a Fos-Jun combination, regardless of the source of the basic region; (ii) the Fos leucine zipper was unable to form either homo- or heterodimers with a chimeric protein containing a Fos leucine zipper; (iii) the Fos basic region was capable of binding to an AP-1 site; (iv) replacement of the Jun amino terminus with that of CREB had little effect on dimerization, whereas replacement with the amino terminus of Fos disrupted both protein-protein and protein-DNA interactions; (v) changes in relative affinities of the Fos and Jun basic regions for the AP-1 element were dependent on the secondary contributions of amino-terminal residues; and (vi) the Fos-Jun chimeric constructs cooperated in transcriptional transactivation of the Jun promoter in NIH 3T3 cells.

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Domain swapping reveals the modular nature of Fos, Jun, and CREB proteins

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4565-4573.1990 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4565-4573

Author(s):

L J Ransone ◽

P Wamsley ◽

K L Morley ◽

I M Verma

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Leucine Zipper ◽

Chimeric Protein ◽

Domain Swapping ◽

Amino Terminus ◽

Protein Protein Interactions ◽

Amino Terminal ◽

Nih 3T3 ◽

Basic Region

The products of the Jun and Fos proto-oncogenes form a heterodimer that binds to and activates transcription from 12-O-tetradecanoylphorbol-13-acetate-responsive promoter elements (TGACTCA) and AP-1-binding sites (TGACATCA). These two proteins belong to a family of related transcription factors which contain similar domains required for protein dimerization and DNA binding but display different protein and DNA binding specificities. The basic region, required for DNA binding, is followed by a leucine zipper structure, a domain that mediates protein-protein interactions. To assess the role of these two domains in three related proteins, Fos, Jun, and CREB, we carried out extensive domain-swapping analysis. We found that (i) dimers formed by two Jun leucine zipper-containing proteins were unable to bind DNA as efficiently as a Fos-Jun combination, regardless of the source of the basic region; (ii) the Fos leucine zipper was unable to form either homo- or heterodimers with a chimeric protein containing a Fos leucine zipper; (iii) the Fos basic region was capable of binding to an AP-1 site; (iv) replacement of the Jun amino terminus with that of CREB had little effect on dimerization, whereas replacement with the amino terminus of Fos disrupted both protein-protein and protein-DNA interactions; (v) changes in relative affinities of the Fos and Jun basic regions for the AP-1 element were dependent on the secondary contributions of amino-terminal residues; and (vi) the Fos-Jun chimeric constructs cooperated in transcriptional transactivation of the Jun promoter in NIH 3T3 cells.

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Functional analysis of the competence transcription factor ComK of Bacillus subtilis by characterization of truncation variants

Microbiology ◽

10.1099/mic.0.28357-0 ◽

2006 ◽

Vol 152 (2) ◽

pp. 473-483 ◽

Cited By ~ 11

Author(s):

Kim A. Susanna ◽

Fabrizia Fusetti ◽

Andy-Mark W. H. Thunnissen ◽

Leendert W. Hamoen ◽

Oscar P. Kuipers

Keyword(s):

Transcription Factor ◽

Bacillus Subtilis ◽

Dna Binding ◽

Protein Interactions ◽

Transcription Activation ◽

Wild Type ◽

Protein Protein Interactions ◽

Protein Dna Interactions ◽

Lower Protein

The competence transcription factor ComK is the master regulator of competence development in Bacillus subtilis. In the regulatory pathway, ComK is involved in different interactions: (i) protein–DNA interactions to stimulate transcription of ComK-dependent genes and (ii) protein–protein interactions, divided into interactions with other proteins and interactions between ComK proteins involving oligomerization. The fact that ComK displays different types of interactions suggests the presence of specific, distinct domains in the protein. This paper describes a search for functional domains, by constructing ComK truncation variants, which were tested for DNA binding, oligomerization and transcription activation. Truncations at the C-terminal end of ComK demonstrated the requirement of this part for transcription activation, but not for DNA binding. The C-terminal region is probably involved in oligomerization of ComK-dimers into tetramers. Surprisingly, a ComK truncation variant lacking 9 aa from the N-terminal end (ΔN9ComK) showed higher transcription activation than wild-type ComK, when expressed in Lactococcus lactis. However, in B. subtilis, transcription activation by ΔN9ComK was twofold lower than that by wild-type ComK, resulting from a five- to sixfold lower protein level of ComKΔN9. Thus, relatively, ΔN9ComK is more active in transcription activation than wild-type ComK. These results suggest that the presence of this N-terminal extension on ComK is a trade-off between high transcription activation and a thus far unidentified role in regulation of ComK.

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Neural network modeling of differential binding between wild-type and mutant CTCF reveals putative binding preferences for zinc fingers 1-2

10.1101/2021.09.23.461552 ◽

2021 ◽

Author(s):

Irene Miriam Kaplow ◽

Abhimanyu Banerjee ◽

Chuan-Sheng Foo

Keyword(s):

Neural Network ◽

Dna Binding ◽

Zinc Fingers ◽

Neural Network Modeling ◽

Binding Motif ◽

Wild Type ◽

Core Motif ◽

Binding Motifs ◽

Binding Domains

Background: Many transcription factors (TFs), such as multi zinc-finger (ZF) TFs, have multiple DNA binding domains (DBDs) with multiple components, and deciphering the DNA binding motifs of individual components is a major challenge. One example of such a TF is CCCTC-binding factor (CTCF), a TF with eleven ZFs that plays a variety of roles in transcriptional regulation, most notably anchoring DNA loops. Previous studies found that CTCF zinc fingers (ZFs) 3-7 bind CTCF's core motif and ZFs 9-11 bind a specific upstream motif, but the motifs of ZFs 1-2 have yet to be identified. Results: We developed a new approach to identifying the binding motifs of individual DBDs of a TF through analyzing chromatin immunoprecipitation sequencing (ChIP-seq) experiments in which a single DBD is mutated: we train a deep convolutional neural network to predict whether wild-type TF binding sites are preserved in the mutant TF dataset and interpret the model. We applied this approach to mouse CTCF ChIP-seq data and, in addition to identifying the known binding preferences of CTCF ZFs 3-11, we identified a GAG binding motif for ZF1 and a weak ATT binding motif for ZF2. We analyzed other CTCF datasets to provide additional evidence that ZFs 1-2 interact with the motifs we identified, and we found that the presence of the motif for ZF1 is associated with Ctcf peak strength. Conclusions: Our approach can be applied to any TF for which in vivo binding data from both the wild-type and mutated versions of the TF are available, and our findings provide an unprecedently comprehensive understanding of the binding preferences of CTCF's DBDs.

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Novel trans-Acting Bacillus subtilis glnA Mutations That Derepress glnRA Expression

Journal of Bacteriology ◽

10.1128/jb.01734-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2485-2492 ◽

Cited By ~ 14

Author(s):

Susan H. Fisher ◽

Lewis V. Wray

Keyword(s):

Bacillus Subtilis ◽

Transcription Factors ◽

Dna Binding ◽

Glutamine Synthetase ◽

Protein Interactions ◽

Feedback Inhibition ◽

Wild Type ◽

Protein Protein Interactions ◽

Enzymatic Assays

ABSTRACT Bacillus subtilis contains two nitrogen transcription factors, GlnR and TnrA. The activities of GlnR and TnrA are regulated by direct protein-protein interactions with the feedback-inhibited form of glutamine synthetase (GS). To look for other factors involved in regulating GlnR activity, we isolated mutants with constitutive glnRA expression (GlnC). The twenty-seven GlnC mutants isolated in this mutant screen all contained mutations tightly linked to the glnRA operon which encodes GlnR (glnR) and GS (glnA). Four GlnC mutants contained mutations in the glnR gene that most likely impair the ability of GlnR to bind DNA. Three other GlnC mutants contained novel glnA mutations (S55F, V173I, and L174F). GlnR regulation was completely relieved in the three glnA mutants, while only modest defects in TnrA regulation were observed. In vitro enzymatic assays showed that the purified S55F mutant enzyme was catalytically defective while the V173I and L174F enzymes were highly resistant to feedback inhibition. The V173I and L174F GS proteins were found to require higher glutamine concentrations than the wild-type GS to regulate the DNA-binding activities of GlnR and TnrA in vitro. These results are consistent with a model where feedback-inhibited GS is the only cellular factor involved in regulating the activity of GlnR in B. subtilis.

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Zinc fingers: DNA binding and protein-protein interactions

Biological Research ◽

10.4067/s0716-97602000000100009 ◽

2000 ◽

Vol 33 (1) ◽

Cited By ~ 49

Author(s):

OSCAR LEON ◽

MONICA ROTH

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Zinc Fingers ◽

Protein Protein Interactions

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Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism

Nature Communications ◽

10.1038/s41467-021-22253-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Christopher R. Horne ◽

Hariprasad Venugopal ◽

Santosh Panjikar ◽

David M. Wood ◽

Amy Henrickson ◽

...

Keyword(s):

Sialic Acid ◽

Dna Binding ◽

Protein Interactions ◽

Acid Metabolism ◽

Environmental Changes ◽

Dna Interaction ◽

Protein Protein Interactions ◽

Nutrient Source ◽

Cryo Electron Microscopy ◽

Close Proximity

AbstractBacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.

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Transactivation Functions of the N-Terminal Domains of Nuclear Hormone Receptors: Protein Folding and Coactivator Interactions

Molecular Endocrinology ◽

10.1210/me.2002-0258 ◽

2003 ◽

Vol 17 (1) ◽

pp. 1-10 ◽

Cited By ~ 125

Author(s):

Raj Kumar ◽

E. Brad Thompson

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Hormone Receptors ◽

Nuclear Hormone Receptor ◽

Binding Domain ◽

Dna Binding Domain ◽

Protein Protein Interactions ◽

Carboxy Terminal ◽

And Function ◽

Terminal Domains

Abstract The N-terminal domains (NTDs) of many members of the nuclear hormone receptor (NHR) family contain potent transcription-activating functions (AFs). Knowledge of the mechanisms of action of the NTD AFs has lagged, compared with that concerning other important domains of the NHRs. In part, this is because the NTD AFs appear to be unfolded when expressed as recombinant proteins. Recent studies have begun to shed light on the structure and function of the NTD AFs. Recombinant NTD AFs can be made to fold by application of certain osmolytes or when expressed in conjunction with a DNA-binding domain by binding that DNA-binding domain to a DNA response element. The sequence of the DNA binding site may affect the functional state of the AFs domain. If properly folded, NTD AFs can bind certain cofactors and primary transcription factors. Through these, and/or by direct interactions, the NTD AFs may interact with the AF2 domain in the ligand binding, carboxy-terminal portion of the NHRs. We propose models for the folding of the NTD AFs and their protein-protein interactions.

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Interactions between Integrase and Excisionase in the Phage Lambda Excisive Nucleoprotein Complex

Journal of Bacteriology ◽

10.1128/jb.184.18.5200-5203.2002 ◽

2002 ◽

Vol 184 (18) ◽

pp. 5200-5203 ◽

Cited By ~ 34

Author(s):

Eun Hee Cho ◽

Richard I. Gumport ◽

Jeffrey F. Gardner

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions ◽

Mobility Shift ◽

Host Chromosome ◽

Amino Terminal ◽

Nucleoprotein Complex ◽

Carboxyl Terminal ◽

Α Helix ◽

Gel Mobility Shift ◽

Amino Terminal Domain

ABSTRACT Bacteriophage lambda site-specific recombination comprises two overall reactions, integration into and excision from the host chromosome. Lambda integrase (Int) carries out both reactions. During excision, excisionase (Xis) helps Int to bind DNA and introduces a bend in the DNA that facilitates formation of the proper excisive nucleoprotein complex. The carboxyl-terminal α-helix of Xis is thought to interact with Int through direct protein-protein interactions. In this study, we used gel mobility shift assays to show that the amino-terminal domain of Int maintained cooperative interactions with Xis. This finding indicates that the amino-terminal arm-type DNA binding domain of Int interacts with Xis.

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