scholarly journals Conserved Dynamic Mechanism of Allosteric Response to L-arg in Divergent Bacterial Arginine Repressors

Molecules ◽  
2020 ◽  
Vol 25 (9) ◽  
pp. 2247
Author(s):  
Saurabh Kumar Pandey ◽  
Milan Melichercik ◽  
David Řeha ◽  
Rüdiger H. Ettrich ◽  
Jannette Carey
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Large Scale ◽  
Rotational Transition ◽  
Sequence Motifs ◽  
E Coli ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Distinct Sequence ◽  
Dynamics Simulations

Hexameric arginine repressor, ArgR, is the feedback regulator of bacterial L-arginine regulons, and sensor of L-arg that controls transcription of genes for its synthesis and catabolism. Although ArgR function, as well as its secondary, tertiary, and quaternary structures, is essentially the same in E. coli and B. subtilis, the two proteins differ significantly in sequence, including residues implicated in the response to L-arg. Molecular dynamics simulations are used here to evaluate the behavior of intact B. subtilis ArgR with and without L-arg, and are compared with prior MD results for a domain fragment of E. coli ArgR. Relative to its crystal structure, B. subtilis ArgR in absence of L-arg undergoes a large-scale rotational shift of its trimeric subassemblies that is very similar to that observed in the E. coli protein, but the residues driving rotation have distinct secondary and tertiary structural locations, and a key residue that drives rotation in E. coli is missing in B. subtilis. The similarity of trimer rotation despite different driving residues suggests that a rotational shift between trimers is integral to ArgR function. This conclusion is supported by phylogenetic analysis of distant ArgR homologs reported here that indicates at least three major groups characterized by distinct sequence motifs but predicted to undergo a common rotational transition. The dynamic consequences of L-arg binding for transcriptional activation of intact ArgR are evaluated here for the first time in two-microsecond simulations of B. subtilis ArgR. L-arg binding to intact B. subtilis ArgR causes a significant further shift in the angle of rotation between trimers that causes the N-terminal DNA-binding domains lose their interactions with the C-terminal domains, and is likely the first step toward adopting DNA-binding-competent conformations. The results aid interpretation of crystal structures of ArgR and ArgR-DNA complexes.

scholarly journals Transcription Factor Allosteric Regulation Through Substrate Coordination to Zinc

2020 ◽  
Author(s):  
Beatriz Almeida ◽  
Jennifer Kaczmarek ◽  
Pedro Figueiredo ◽  
Kristala LJ Prather ◽  
Alexandra Carvalho
Keyword(s):  
Dna Binding ◽  
Systems Engineering ◽  
Site Directed Mutagenesis ◽  
Structural Isomer ◽  
E Coli ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Zinc Coordination ◽  
Dynamics Simulations ◽  
Positive Character

The development of new synthetic biology circuits for biotechnology and medicine requires deeper mechanistic insight on allosteric transcription factors (aTFs). Here we studied the aTF UxuR, which is a dimer, with each monomer consisting of two structured domains connected by a highly flexible linker region. In order to explore how ligand binding to UxuR affects protein dynamics we performed molecular dynamics simulations in the free protein and the aTF bound to the inducer D-fructuronate or the structural isomer D-glucuronate. We then validated our results by constructing a sensor plasmid for D-fructuronate in <i>E. coli</i> and performed site-directed mutagenesis. Our results show that zinc coordination is necessary for UxuR function, since mutation to alanines prevents expression de-repression by D-fructuronate. Analyzing the different complexes, we found that the disordered linker regions allow the N-terminal domains to display fast and large movements. When the inducer is bound, UxuR is able to sample an open conformation with a more pronounced negative charge at the surface of the N-terminal DNA binding domains. In opposition, in the free and D-glucuronate bond forms the protein samples closed conformations, with a more positive character at the surface of the DNA binding regions. These molecular insights provide a new basis to better harness these systems for biological systems engineering.

scholarly journals Transcription Factor Allosteric Regulation Through Substrate Coordination to Zinc

2020 ◽  
Author(s):  
Beatriz Almeida ◽  
Jennifer Kaczmarek ◽  
Pedro Figueiredo ◽  
Kristala LJ Prather ◽  
Alexandra Carvalho
Keyword(s):  
Dna Binding ◽  
Systems Engineering ◽  
Principal Component ◽  
Site Directed Mutagenesis ◽  
Free Form ◽  
E Coli ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Zinc Coordination ◽  
Dynamics Simulations

The development of new synthetic biology circuits for biotechnology and medicine requires deeper mechanistic insight on allosteric transcription factors (aTFs). Here we studied the aTF UxuR in the free form and bound to the inducer D-fructuronate or the analog D-glucuronate. We employed molecular dynamics simulations, principal component analysis and electrostatic potential surface calculations. We furthermore constructed a sensor plasmid for Dfructuronate in E. coli and performed site-directed mutagenesis. Our results show that zinc coordination is necessary for UxuR function and that when the inducer is bound, UxuR acquires an open conformation with a more pronounced negative charge at the surface of the N-terminal DNA binding domains. In opposition, in the free and D-glucuronate bond forms the protein acquires closed conformations, with a more positive character at the surface of the DNA binding regions. These processes can be more general than anticipated and harnessed for biological systems engineering.

scholarly journals Transcription Factor Allosteric Regulation Through Substrate Coordination to Zinc

2020 ◽  
Author(s):  
Beatriz Almeida ◽  
Jennifer Kaczmarek ◽  
Pedro Figueiredo ◽  
Kristala LJ Prather ◽  
Alexandra Carvalho
Keyword(s):  
Dna Binding ◽  
Systems Engineering ◽  
Site Directed Mutagenesis ◽  
Structural Isomer ◽  
E Coli ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Zinc Coordination ◽  
Dynamics Simulations ◽  
Positive Character

The development of new synthetic biology circuits for biotechnology and medicine requires deeper mechanistic insight on allosteric transcription factors (aTFs). Here we studied the aTF UxuR, which is a dimer, with each monomer consisting of two structured domains connected by a highly flexible linker region. In order to explore how ligand binding to UxuR affects protein dynamics we performed molecular dynamics simulations in the free protein and the aTF bound to the inducer D-fructuronate or the structural isomer D-glucuronate. We then validated our results by constructing a sensor plasmid for D-fructuronate in <i>E. coli</i> and performed site-directed mutagenesis. Our results show that zinc coordination is necessary for UxuR function, since mutation to alanines prevents expression de-repression by D-fructuronate. Analyzing the different complexes, we found that the disordered linker regions allow the N-terminal domains to display fast and large movements. When the inducer is bound, UxuR is able to sample an open conformation with a more pronounced negative charge at the surface of the N-terminal DNA binding domains. In opposition, in the free and D-glucuronate bond forms the protein samples closed conformations, with a more positive character at the surface of the DNA binding regions. These molecular insights provide a new basis to better harness these systems for biological systems engineering.

Dimeric transcription factor families: it takes two to tango but who decides on partners and the venue?

1992 ◽  
Vol 103 (1) ◽  
pp. 9-14 ◽  
Author(s):  
K.A. Lee
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Experimental Evidence ◽  
Cdna Cloning ◽  
Transcriptional Activation ◽  
Effector Functions ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Dna Elements

Dimeric transcription factors that bind to DNA are often grouped into families on the basis of dimerization and DNA-binding specificities. cDNA cloning studies have established that members of the same family have structurally related dimerisation and DNA-binding domains but diverge in other regions that are important for transcriptional activation. These features lead to the straightforward suggestion that although all members of a family bind to similar DNA elements, individual members exhibit distinct transcriptional effector functions. This simple view is now supported by experimental evidence from those systems that have proved amenable to study. There are however some largely unaddressed questions that concern the mechanisms that allow family members to go about their business without interference from their highly related siblings. Here I will discuss some insights from studies of the bZIP class of transcription factors.

scholarly journals The Activity of Mammalian brm/SNF2α Is Dependent on a High-Mobility-Group Protein I/Y-Like DNA Binding Domain

1999 ◽  
Vol 19 (6) ◽  
pp. 3931-3939 ◽  
Author(s):  
Brigitte Bourachot ◽  
Moshe Yaniv ◽  
Christian Muchardt
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
High Mobility ◽  
High Mobility Group ◽  
Binding Motif ◽  
High Mobility Group Protein ◽  
Interaction Domain ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Group Protein

ABSTRACT The mammalian SWI-SNF complex is a chromatin-remodelling machinery involved in the modulation of gene expression. Its activity relies on two closely related ATPases known as brm/SNF2α and BRG-1/SNF2β. These two proteins can cooperate with nuclear receptors for transcriptional activation. In addition, they are involved in the control of cell proliferation, most probably by facilitating p105Rb repression of E2F transcriptional activity. In the present study, we have examined the ability of various brm/SNF2α deletion mutants to reverse the transformed phenotype ofras-transformed fibroblasts. Deletions within the p105Rb LXCXE binding motif or the conserved bromodomain had only a moderate effect. On the other hand, a 49-amino-acid segment, rich in lysines and arginines and located immediately downstream of the p105Rb interaction domain, appeared to be essential in this assay. This region was also required for cooperation of brm/SNF2α with the glucocorticoid receptor in transfection experiments, but only in the context of a reporter construct integrated in the cellular genome. The region has homology to the AT hooks present in high-mobility-group protein I/Y DNA binding domains and is required for the tethering of brm/SNF2α to chromatin.

scholarly journals Expression of active hormone and DNA-binding domains of the chicken progesterone receptor in E. coli.

1989 ◽  
Vol 8 (1) ◽  
pp. 83-90 ◽  
Author(s):  
J. Eul ◽  
M. E. Meyer ◽  
L. Tora ◽  
M. T. Bocquel ◽  
C. Quirin-Stricker ◽  
...  
Keyword(s):  
Dna Binding ◽  
Progesterone Receptor ◽  
E Coli ◽  
Dna Binding Domains ◽  
Binding Domains

scholarly journals Domains Required for Transcriptional Activation Show Conservation in the Mga Family of Virulence Gene Regulators

2006 ◽  
Vol 188 (3) ◽  
pp. 863-873 ◽  
Author(s):  
Cheryl M. Vahling ◽  
Kevin S. McIver
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Group A Streptococcus ◽  
Gene Activation ◽  
Random Mutagenesis ◽  
Virulence Gene ◽  
Common Mechanism ◽  
Dna Binding Domains ◽  
Amino Terminal ◽  
Binding Domains

ABSTRACT Mga, or the multigene regulator of the group A streptococcus (GAS) (Streptococcus pyogenes), is a transcriptional regulator of virulence genes important for colonization and immune evasion. All serotypes of the GAS possess one of two divergent mga alleles (mga-1 or mga-2), and orthologues of Mga have also been identified in other pathogenic streptococci. To date, the only functional motifs established within Mga are two amino-terminal DNA-binding domains (HTH-3 and HTH-4). To uncover novel domains, a random mutagenesis screen using an M6 Mga (mga-1) was undertaken to find mutations leading to a defect in transcriptional activation of the Mga-regulated emm gene. In addition to mutations in the established DNA-binding domains, the screen also revealed mutations in a region conserved among several Mga orthologues. Alanine scanning helped resolve the boundaries of this conserved Mga domain (CMD-1) spanning from residues 10 to 15 of the protein, with the two flanking amino acid residues likely involved in protein stability. Transcriptional reporter analyses demonstrated the importance of CMD-1 for activation of Pemm and autoactivation of Pmga in the serotype M6 Mga. Mutational analyses showed that both CMD-1 and HTH-4 are also necessary for activation of the promoter target Pmrp in a divergent serotype M4 Mga (mga-2), suggesting a conserved functionality. However, in contrast to M6, the M4 Mga mutants did not show a defect in autoregulation. Mutation of similar conserved residues in the Mga-like regulator DmgB from S. dysgalactiae subsp. dysgalactiae showed that CMD-1 and HTH-4 are critical for transcriptional activation in this orthologue, implying that a common mechanism of virulence gene activation may exist for members of the Mga family of regulators.

scholarly journals Charged residues in the H-NS linker drive DNA binding and gene silencing in single cells

2017 ◽  
Vol 114 (47) ◽  
pp. 12560-12565 ◽  
Author(s):  
Yunfeng Gao ◽  
Yong Hwee Foo ◽  
Ricksen S. Winardhi ◽  
Qingnan Tang ◽  
Jie Yan ◽  
...  
Keyword(s):  
Dna Binding ◽  
Gene Silencing ◽  
Single Cells ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Superresolution Microscopy ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Associated Proteins ◽  
Dynamics Simulations

Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains. In the present work we demonstrate that linker amino acids promote engagement with DNA. In the absence of linker contacts, H-NS binding is significantly reduced, although no change in chromosome compaction is observed. H-NS is not localized to two distinct foci; rather, it is scattered all around the nucleoid. The linker makes DNA contacts that are required for gene silencing, while chromosome compaction does not appear to be an important H-NS function.

scholarly journals Fungal Mediator Tail Subunits Contain Classical Transcriptional Activation Domains

2015 ◽  
Vol 35 (8) ◽  
pp. 1363-1375 ◽  
Author(s):  
Zhongle Liu ◽  
Lawrence C. Myers
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Weak Interactions ◽  
Primary Role ◽  
Eukaryotic Transcription ◽  
Content Type ◽  
Activation Domains ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Transcriptional Activation Domains

Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits ofSaccharomyces cerevisiae,Candida albicans, andCandida dubliniensisMediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains ofS. cerevisiaeMed2 and Med3, as well asC. dubliniensisTlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen.

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