scholarly journals Chromatin Opening and Transactivator Potentiation by RAP1 in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (8) ◽  
pp. 5279-5288 ◽  
Author(s):  
Liuning Yu ◽  
Randall H. Morse
Keyword(s):  
Binding Site ◽  
Protein Interactions ◽  
Binding Sites ◽  
Accessory Protein ◽  
Dna Binding Domain ◽  
Transcriptional Activators ◽  
Protein Protein Interactions ◽  
Chromatin Opening

ABSTRACT Transcriptional activators function in vivo via binding sites that may be packaged into chromatin. Here we show that whereas the transcriptional activator GAL4 is strongly able to perturb chromatin structure via a nucleosomal binding site in yeast, GCN4 does so poorly. Correspondingly, GCN4 requires assistance from an accessory protein, RAP1, for activation of the HIS4 promoter, whereas GAL4 does not. The requirement for RAP1 for GCN4-mediated HIS4activation is dictated by the DNA-binding domain of GCN4 and not the activation domain, suggesting that RAP1 assists GCN4 in gaining access to its binding site. Consistent with this, overexpression of GCN4 partially alleviates the requirement for RAP1, whereas HIS4activation via a weak GAL4 binding site requires RAP1. RAP1 is extremely effective at interfering with positioning of a nucleosome containing its binding site, consistent with a role in opening chromatin at the HIS4 promoter. Furthermore, increasing the spacing between binding sites for RAP1 and GCN4 by 5 or 10 bp does not impair HIS4 activation, indicating that cooperative protein-protein interactions are not involved in transcriptional facilitation by RAP1. We conclude that an important role of RAP1 is to assist activator binding by opening chromatin.

Getting into chromatin: how do transcription factors get past the histones?

2003 ◽  
Vol 81 (3) ◽  
pp. 101-112 ◽  
Author(s):  
Randall H Morse
Keyword(s):  
Transcriptional Activation ◽  
Binding Sites ◽  
Dna Binding Proteins ◽  
Transcriptional Activators ◽  
Activation Domains ◽  
Nucleosome Dynamics ◽  
Gain Access ◽  
General Transcription Machinery

Transcriptional activators and the general transcription machinery must gain access to DNA that in eukaryotes may be packaged into nucleosomes. In this review, I discuss this problem from the standpoint of the types of chromatin structures that these DNA-binding proteins may encounter, and the mechanisms by which they may contend with various chromatin structures. The discussion includes consideration of experiments in which chromatin structure is manipulated in vivo to confront activators with nucleosomal binding sites, and the roles of nucleosome dynamics and activation domains in facilitating access to such sites. Finally, the role of activators in facilitating access of the general transcriptional machinery to sites in chromatin is discussed. Key words: nucleosome, chromatin, transcriptional activation, Saccharomyces cerevisiae.

scholarly journals A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

1995 ◽  
Vol 15 (10) ◽  
pp. 5214-5225 ◽  
Author(s):  
A D Catling ◽  
H J Schaeffer ◽  
C W Reuter ◽  
G R Reddy ◽  
M J Weber
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Serum Stimulation ◽  
And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

scholarly journals Hidden partners: Using cross-docking calculations to predict binding sites for proteins with multiple interactions

2018 ◽  
Author(s):  
Nathalie Lagarde ◽  
Alessandra Carbone ◽  
Sophie Sacquin-Mora
Keyword(s):  
Nucleic Acid ◽  
Binding Site ◽  
Protein Interactions ◽  
Binding Sites ◽  
Protein Protein Interactions ◽  
Protein Binding Sites ◽  
Cross Docking ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Docking Calculations

AbstractProtein-protein interactions control a large range of biological processes and their identification is essential to understand the underlying biological mechanisms. To complement experimental approaches, in silico methods are available to investigate protein-protein interactions. Cross-docking methods, in particular, can be used to predict protein binding sites. However, proteins can interact with numerous partners and can present multiple binding sites on their surface, which may alter the binding site prediction quality. We evaluate the binding site predictions obtained using complete cross-docking simulations of 358 proteins with two different scoring schemes accounting for multiple binding sites. Despite overall good binding site prediction performances, 68 cases were still associated with very low prediction quality, presenting individual area under the specificity-sensitivity ROC curve (AUC) values below the random AUC threshold of 0.5, since cross-docking calculations can lead to the identification of alternate protein binding sites (that are different from the reference experimental sites). For the large majority of these proteins, we show that the predicted alternate binding sites correspond to interaction sites with hidden partners, i.e. partners not included in the original cross-docking dataset. Among those new partners, we find proteins, but also nucleic acid molecules. Finally, for proteins with multiple binding sites on their surface, we investigated the structural determinants associated with the binding sites the most targeted by the docking partners.AbbreviationsANOVA: ANalysis Of Variance; AUC: Area Under the Curve; Best Interface: BI; CAPRI: Critical Assessment of Prediction of Interactions; CC-D: Complete Cross-Docking; DNA: DesoxyriboNucleic Acid; FDR: False Discovery Rate; FRIres(type): Fraction of each Residue type in the Interface; FP: False Positives; GI: Global Interface; HCMD: Help Cure Muscular Dystrophy; JET: Joint Evolutionary Tree; MAXDo: Molecular Association via Cross Docking; NAI: Nucleic Acid Interface; NPV: Negative Predicted Value; PDB: Protein Data Bank; PIP: Protein Interface Propensity; PiQSi: Protein Quaternary Structure investigation; PPIs: Protein-Protein Interactions; PPV: Positive Predicted Value; Prec.: Precision; PrimI: Primary Interface; RNA: RiboNucleic Acid; ROC: Receiver Operating Characteristic; SecI: Secondary Interface; Sen.: Sensitivity; Spe.: Specificity; TN: True Negatives; TP: True Positives; WCG: World Community Grid.

scholarly journals A Conditional Role of U2AF in Splicing of Introns with Unconventional Polypyrimidine Tracts

2007 ◽  
Vol 27 (20) ◽  
pp. 7334-7344 ◽  
Author(s):  
Vinod Sridharan ◽  
Ravinder Singh
Keyword(s):  
Protein Interactions ◽  
Splice Site ◽  
Large Subunit ◽  
Strong Dependence ◽  
Rna Recognition Motif ◽  
Branch Points ◽  
Protein Protein Interactions ◽  
Site Identification

ABSTRACT Recognition of polypyrimidine (Py) tracts typically present between the branch point and the 3′ splice site by the large subunit of the essential splicing factor U2AF is a key early step in pre-mRNA splicing. Diverse intronic sequence arrangements exist, however, including 3′ splice sites lacking recognizable Py tracts, which raises the question of how general the requirement for U2AF is for various intron architectures. Our analysis of fission yeast introns in vivo has unexpectedly revealed that whereas introns lacking Py tracts altogether remain dependent on both subunits of U2AF, introns with long Py tracts, unconventionally positioned upstream of branch points, are unaffected by U2AF inactivation. Nevertheless, mutation of these Py tracts causes strong dependence on the large subunit U2AF59. We also find that Py tract diversity influences the requirement for the conserved C-terminal domain of U2AF59 (RNA recognition motif 3), which has been implicated in protein-protein interactions with other splicing factors. Together, these results suggest that in addition to Py tract binding by U2AF, supplementary mechanisms of U2AF recruitment and 3′ splice site identification exist to accommodate diverse intron architectures, which have gone unappreciated in biochemical studies of model pre-mRNAs.

scholarly journals The yeast SWI-SNF complex facilitates binding of a transcriptional activator to nucleosomal sites in vivo.

1997 ◽  
Vol 17 (8) ◽  
pp. 4811-4819 ◽  
Author(s):  
L G Burns ◽  
C L Peterson
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Protein Assembly ◽  
Transcriptional Activators ◽  
Regulate Gene Expression ◽  
Activation Of Transcription ◽  
Free Region ◽  
And Function

The Saccharomyces cerevisiae SWI-SNF complex is a 2-MDa protein assembly that is required for the function of many transcriptional activators. Here we describe experiments on the role of the SWI-SNF complex in activation of transcription by the yeast activator GAL4. We find that while SWI-SNF activity is not required for the GAL4 activator to bind to and activate transcription from nucleosome-free binding sites, the complex is required for GAL4 to bind to and function at low-affinity, nucleosomal binding sites in vivo. This SWI-SNF dependence can be overcome by (i) replacing the low-affinity sites with higher-affinity, consensus GAL4 binding sequences or (ii) placing the low-affinity sites into a nucleosome-free region. These results define the criteria for the SWI-SNF dependence of gene expression and provide the first in vivo evidence that the SWI-SNF complex can regulate gene expression by modulating the DNA binding of an upstream activator protein.

scholarly journals Identification of protein-protein and ribonucleoprotein complexes containing Hfq

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joël Caillet ◽  
Bruno Baron ◽  
Irina V. Boni ◽  
Célia Caillet-Saguy ◽  
Eliane Hajnsdorf
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Direct Interaction ◽  
Rnase A ◽  
Rnase E ◽  
Protein Protein Interactions ◽  
Control Of Gene Expression ◽  
Ribonucleoprotein Complexes

Abstract Hfq is a RNA-binding protein that plays a pivotal role in the control of gene expression in bacteria by stabilizing sRNAs and facilitating their pairing with multiple target mRNAs. It has already been shown that Hfq, directly or indirectly, interacts with many proteins: RNase E, Rho, poly(A)polymerase, RNA polymerase… In order to detect more Hfq-related protein-protein interactions we have used two approaches, TAP-tag combined with RNase A treatment to access the role of RNA in these complexes, and protein-protein crosslinking, which freezes protein-protein complexes formed in vivo. In addition, we have performed microscale thermophoresis to evaluate the role of RNA in some of the complexes detected and used far-western blotting to confirm some protein-protein interactions. Taken together, the results show unambiguously a direct interaction between Hfq and EF-Tu. However a very large number of the interactions of proteins with Hfq in E. coli involve RNAs. These RNAs together with the interacting protein, may play an active role in the formation of Hfq-containing complexes with previously unforeseen implications for the riboregulatory functions of Hfq.

scholarly journals Norstictic acid is a selective allosteric transcriptional regulator

2021 ◽  
Author(s):  
Julie M Garlick ◽  
Steven M Sturlis ◽  
Paul A Bruno ◽  
Joel A Yates ◽  
Amanda Peiffer ◽  
...  
Keyword(s):  
Binding Site ◽  
Protein Interactions ◽  
Binding Sites ◽  
Triple Negative ◽  
Transcriptional Coactivator ◽  
Protein Protein Interactions ◽  
Ppi Networks ◽  
Binding Surface ◽  
Inhibitor Discovery

Inhibitors of transcriptional protein-protein interactions (PPIs) have high value both as tools and for therapeutic applications. The PPI network mediated by the transcriptional coactivator Med25, for example, regulates stress-response and motility pathways and dysregulation of the PPI networks contributes to oncogenesis and metastasis. The canonical transcription factor binding sites within Med25 are large (~900 square angstroms) and have little topology, and thus do not present an array of attractive small-molecule binding sites for inhibitor discovery. Here we demonstrate that the depsidone natural product norstictic acid functions through an alternative binding site to block Med25-transcriptional activator PPIs in vitro and in cell culture. Norstictic acid targets a binding site comprised of a highly dynamic loop flanking one canonical binding surface and in doing so, it both orthosterically and allosterically alters Med25-driven transcription in a patient-derived model of triple negative breast cancer. These results highlight the potential of Med25 as a therapeutic target as well as the inhibitor discovery opportunities presented by structurally dynamic loops within otherwise challenging proteins.

scholarly journals Selective secretion of alternatively spliced fibronectin variants.

1989 ◽  
Vol 109 (6) ◽  
pp. 3445-3453 ◽  
Author(s):  
J E Schwarzbauer ◽  
C S Spencer ◽  
C L Wilson
Keyword(s):  
Protein Interactions ◽  
Time Course ◽  
Deletion Mapping ◽  
Protein Protein Interactions ◽  
Dimer Formation ◽  
Alternatively Spliced ◽  
Selective Retention ◽  
V Region

We demonstrate that the alternatively spliced variable (V) region of fibronectin (FN) is required for secretion of FN dimers during biosynthesis. Alternative splicing of the V segment of the rat FN transcript generates three subunit variants (V120, V95, V0) that differ by the inclusion or omission of an additional 120 or 95 amino acids. We are exploring the functions of this segment by expressing variant cDNAs in normal and transformed fibroblasts. Like FN itself, the cDNA-encoded polypeptides (deminectins [DNs]) containing the V120 or V95 segment are efficiently secreted as disulfide-bonded homodimers. However, few homodimers of DNs lacking this region, V0 DNs, are secreted. V0 homodimers do form inside the cell, as demonstrated by biosynthetic analyses of dimer formation and secretion using pulse-chase and time course experiments, but these dimers seldom reach the cell surface and are probably degraded intracellularly. Coexpression of V0 and V120 subunits results in intracellular formation of three types of dimers, V0-V0, V0-V120, and V120-V120, but only the V120-containing dimers are secreted. This selective retention of V0 homodimers indicates that the V region is required for formation and secretion of native FN dimers. In an analogous in vivo situation, we show that plasma FN also lacks V0-V0 dimers and consists of V0-V+ and V+-V+ combinations. Dissection of V region sequences by deletion mapping localizes the major site involved in DN dimer secretion to an 18-amino acid segment within V95. In addition, high levels of dimer secretion can be restored by insertion of V into a heterologous site 10 kD COOH terminal to its normal location. We discuss the potential role of intracellular protein-protein interactions in FN dimer formation.

scholarly journals Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA

2009 ◽  
Vol 192 (1) ◽  
pp. 326-335 ◽  
Author(s):  
Brian Callahan ◽  
Kiet Nguyen ◽  
Alissa Collins ◽  
Kayla Valdes ◽  
Michael Caplow ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Helical Structure ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Structures And Properties ◽  
Virulence Protein ◽  
Single Polypeptide ◽  
Specific Association

ABSTRACT Mycobacterium tuberculosis EsxA and EsxB proteins are founding members of the WXG100 (WXG) protein family, characterized by their small size (∼100 amino acids) and conserved WXG amino acid motif. M. tuberculosis contains 11 tandem pairs of WXG genes; each gene pair is thought to be coexpressed to form a heterodimer. The precise role of these proteins in the biology of M. tuberculosis is unknown, but several of the heterodimers are secreted, which is important for virulence. However, WXG proteins are not simply virulence factors, since nonpathogenic mycobacteria also express and secrete these proteins. Here we show that three WXG heterodimers have structures and properties similar to those of the M. tuberculosis EsxBA (MtbEsxBA) heterodimer, regardless of their host species and apparent biological function. Biophysical studies indicate that the WXG proteins from M. tuberculosis (EsxG and EsxH), Mycobacterium smegmatis (EsxA and EsxB), and Corynebacterium diphtheriae (EsxA and EsxB) are heterodimers and fold into a predominately α-helical structure. An in vivo protein-protein interaction assay was modified to identify proteins that interact specifically with the native WXG100 heterodimer. MtbEsxA and MtbEsxB were fused into a single polypeptide, MtbEsxBA, to create a biomimetic bait for the native heterodimer. The MtbEsxBA bait showed specific association with several esx-1-encoded proteins and EspA, a virulence protein secreted by ESX-1. The MtbEsxBA fusion peptide was also utilized to identify residues in both EsxA and EsxB that are important for establishing protein interactions with Rv3871 and EspA. Together, the results are consistent with a model in which WXG proteins perform similar biological roles in virulent and nonvirulent species.

scholarly journals PDZ Motifs in PTP-BL and RIL Bind to Internal Protein Segments in the LIM Domain Protein RIL

1998 ◽  
Vol 9 (3) ◽  
pp. 671-683 ◽  
Author(s):  
Edwin Cuppen ◽  
Herlinde Gerrits ◽  
Barry Pepers ◽  
Bé Wieringa ◽  
Wiljan Hendriks
Keyword(s):  
Protein Interactions ◽  
Tyrosine Phosphatase ◽  
Specific Interaction ◽  
Protein Protein Interactions ◽  
Lim Domain ◽  
Binding Interface ◽  
Mouse Tissues

The specificity of protein–protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells.

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