Eukaryotic Transcription Research Articles

Correlation of Cry1Ac mRNA and protein abundance in transgenic Gossypium hirsutum plant.

The online version contains supplementary material available at 10.1007/s13205-021-02828-2.

Real-time single-cell characterization of the eukaryotic transcription cycle reveals correlations between RNA initiation, elongation, and cleavage.

The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel application of Bayesian inference techniques to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters as well as single-cell correlations between these parameters. These measurements, combined with theoretical modeling, suggest a substantial variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.

Peculiarities of Plasmodium falciparum Gene Regulation and Chromatin Structure.

The highly complex life cycle of the human malaria parasite, Plasmodium falciparum, is based on an orchestrated and tightly regulated gene expression program. In general, eukaryotic transcription regulation is determined by a combination of sequence-specific transcription factors binding to regulatory DNA elements and the packaging of DNA into chromatin as an additional layer. The accessibility of regulatory DNA elements is controlled by the nucleosome occupancy and changes of their positions by an active process called nucleosome remodeling. These epigenetic mechanisms are poorly explored in P. falciparum. The parasite genome is characterized by an extraordinarily high AT-content and the distinct architecture of functional elements, and chromatin-related proteins also exhibit high sequence divergence compared to other eukaryotes. Together with the distinct biochemical properties of nucleosomes, these features suggest substantial differences in chromatin-dependent regulation. Here, we highlight the peculiarities of epigenetic mechanisms in P. falciparum, addressing chromatin structure and dynamics with respect to their impact on transcriptional control. We focus on the specialized chromatin remodeling enzymes and discuss their essential function in P. falciparum gene regulation.

Competition for DNA binding between paralogous transcription factors determines their genomic occupancy and regulatory functions.

Most eukaryotic transcription factors (TFs) are part of large protein families, with members of the same family (i.e., paralogous TFs) recognizing similar DNA-binding motifs but performing different regulatory functions. Many TF paralogs are coexpressed in the cell and thus can compete for target sites across the genome. However, this competition is rarely taken into account when studying the in vivo binding patterns of eukaryotic TFs. Here, we show that direct competition for DNA binding between TF paralogs is a major determinant of their genomic binding patterns. Using yeast proteins Cbf1 and Pho4 as our model system, we designed a high-throughput quantitative assay to capture the genomic binding profiles of competing TFs in a cell-free system. Our data show that Cbf1 and Pho4 greatly influence each other's occupancy by competing for their common putative genomic binding sites. The competition is different at different genomic sites, as dictated by the TFs’ expression levels and their divergence in DNA-binding specificity and affinity. Analyses of ChIP-seq data show that the biophysical rules that dictate the competitive TF binding patterns in vitro are also followed in vivo, in the complex cellular environment. Furthermore, the Cbf1-Pho4 competition for genomic sites, as characterized in vitro using our new assay, plays a critical role in the specific activation of their target genes in the cell. Overall, our study highlights the importance of direct TF-TF competition for genomic binding and gene regulation by TF paralogs, and proposes an approach for studying this competition in a quantitative and high-throughput manner.

Inter‐modular interaction between the head and CDK8 modules of Mediator regulates a recruitment of TFIIH to the promoters

Mediator is an essential co-activator, which regulates RNA polymerase II (Pol II) transcription in eukaryotes. Mediator is a large multi-protein complex composed of four distinct sub-complexes termed Head, Middle, Tail, often termed core Mediator (cMed), as well as the CDK8 module (CKM), which is dissociable from core Mediator. While cMed regulates Pol II transcription by involving in formation and pre-initiation complex (PIC) via interaction with Pol II and general transcription factors (GTFs), the role of CKM still remains elusive. In higher eukaryotes, CKM appears to function as both in negative and positive regulator. In yeast Saccharomyces cerevisiae, CKM has been believed to function as a negative regulator. The one mode of inhibition by CKM comes from disruption of activators by triggering ubiquitin-mediated degradation by phosphorylation. However, overall the mode of transcription inhibition by CKM has not been fully understood. It is proposed that CKM inhibits transcription blocking cMed-Pol II interactions. However, genome-wide studies completely disputed this model. In this work, our structure-based functional study has led to a totally unexpected discovery that while Mediator head module facilitates recruitment of TFIIH, CKM appears to inhibit a process. In essence, the inter-modular interaction within the Mediator modules regulates conversion from initiation to elongation step of transcription by modulating a recruitment of a general transcription factor TFIIH.

Repurposing the CRISPR‐Cas9 System for Targeted Chromatin O ‐linked β‐ N ‐acetylglucosamine Editing

Eukaryotic gene transcription is controlled by many proteins, including the basal transcription machinery, chromatin remodeling complexes, and transcription cofactors. Chromatin and genome-mapping consortia have identified O-linked β-N-acetylglucosamine (O-GlcNAc) as an abundant post-translational modification involved in numerous transcriptional processes, including RNA polymerase function, chromatin dynamics, and cofactor activity. Decoding the precise function of all the intracellular O-GlcNAc-regulated elements identified in these studies remains challenging. Technologies to manipulate O-GlcNAcylation at specific DNA loci for functional analysis without pleiotropic consequences were lacking, but the advent of CRISPR-Cas9-based technologies has enabled the precise perturbation of DNA sequences and delivery of proteins to specific cis-acting DNA regulatory elements to elucidate their role in transcription. We reasoned that a CRISPR-Cas9 system could be developed to study the function of O-GlcNAcylation in transcription, which would circumvent the problems inherent in conventional gene manipulation and pharmacological approaches. We developed a programmable CRISPR-Cas9-based system that allows for O-GlcNAc manipulation of transcriptional complex function at specific cis-regulatory elements. The tools consist of nuclease-null Cas9 (dCas9) fused to O-GlcNAc transferase (OGT), which adds the O-GlcNAc modification, or O-GlcNAcase (OGA), which removes the modification. Previously, we demonstrated that O-GlcNAc plays a role in regulating human □-globin gene transcription. Increasing fetal hemoglobin (HbF) via activation of □-globin chain synthesis is widely accepted as the most effective treatment for SCD and certain types of β-thalassemias. O-GlcNAcylation modulates the formation of a GATA-1-FOG-1-NuRD repressor complex that binds the -566 GATA site of the A□-globin promoter when □-globin gene expression is silent. OGT and OGA interact with GATA-1 and CHD4, a component of the NuRD complex. O-GlcNAcylation of CHD4 stimulates the formation of this repressor complex, whereas removing this PTM results in activation of A□-globin gene expression. As proof of principle to demonstrate the utility of our system, we targeted dCas9-OGT or dCas9-OGA fusion proteins to DNA sequences flanking the -566 GATA site of the A□-globin gene promoter. Increased □-globin gene transcription was measured by RT-qPCR when dCas9-OGA was targeted to this area, whereas □-globin gene expression decreased when dCas9-OGT was targeted to the identical sequences. These data support our published work, confirming that O-GlcNAcylation regulates A□-globin gene transcription. RT-qPCR data coupled with Cut and Run assays demonstrate the robust and highly specific nature of our targeting system, which can be employed as a generally applicable tool to study the role of O-GlcNAcylation in transcriptional regulation.

CTD Kinases’ structural elements enable them to phosphorylate specific residues in the CTD of RNA Polymerase II

The phosphorylation states of RNA polymerase II (Pol II) coordinate the process of eukaryotic transcription by recruitment of transcription regulators. The largest subunit of pol II consists of a highly disordered C-Terminal Domain (CTD). Individual residues of repetitive heptads (Consensus sequence - Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) of the CTD are phosphorylated temporally at different stages of transcription. Intriguingly, despite similar flanking residues, phosphorylation of Ser2 and Ser5 in CTD heptads play dramatically different roles. The uncanny ability of kinases to recognize the correct serine is not well understood. In this paper, we use biochemical assays, mass spectrometry, molecular modeling, and structural analysis to understand the structural elements determining which serine of the CTD heptad is subject to phosphorylation. We identified three motifs in the activation/P+1 loops differentiating the intrinsic specificity of CTD in various CTD kinases. We characterized the enzyme specificity of the CTD kinases, CDK7 as Ser5-specific, Erk2 with dual specificity for Ser2 and Ser5, and Dyrk1a as a Ser2-specific kinase. We also show that the specificities of kinases are malleable and can be modified by incorporating mutations in their activation/P+1 loops that alter the interactions of the three motifs. Our results provide an important clue to the understanding of post-translational modification of RNA polymerase II temporally during active transcription.

Transcriptomics Provides a Genetic Signature of Vineyard Site and Offers Insight into Vintage-Independent Inoculated Fermentation Outcomes.

ABSTRACTRibosomal DNA amplicon sequencing of grape musts has demonstrated that microorganisms occur nonrandomly and are associated with the vineyard of origin, suggesting a role for the vineyard, grape, and wine microbiome in shaping wine fermentation outcomes. Here, ribosomal DNA amplicon sequencing from grape musts and RNA sequencing of eukaryotic transcripts from primary fermentations inoculated with the wine yeast Saccharomyces cerevisiae RC212 were used to profile fermentations from 15 vineyards in California and Oregon across two vintages. These data demonstrate that the relative abundance of fungal organisms detected by ribosomal DNA amplicon sequencing correlated with neither transcript abundance from those same organisms within the RNA sequencing data nor gene expression of the inoculated RC212 yeast strain. These data suggest that the majority of the fungi detected in must by ribosomal DNA amplicon sequencing were not active during the primary stage of these inoculated fermentations and were not a major factor in determining RC212 gene expression. However, unique genetic signatures were detected within the ribosomal DNA amplicon and eukaryotic transcriptomic sequencing that were predictive of vineyard site and region. These signatures included S. cerevisiae gene expression patterns linked to nitrogen, sulfur, and thiamine metabolism. These genetic signatures of site offer insight into specific environmental factors to consider with respect to fermentation outcomes and vineyard site and regional wine characteristics.IMPORTANCE The wine industry generates billions of dollars of revenue annually, and economic productivity is in part associated with regional distinctiveness of wine sensory attributes. Microorganisms associated with grapes and wineries are influenced by region of origin, and given that some microorganisms play a role in fermentation, it is thought that microbes may contribute to the regional distinctiveness of wine. In this work, as in previous studies, it is demonstrated that specific bacteria and fungi are associated with individual wine regions and vineyard sites. However, this work further shows that their presence is not associated with detectable fungal gene expression during the primary fermentation or the expression of specific genes by the inoculate Saccharomyces cerevisiae strain RC212. The detected RC212 gene expression signatures associated with region and vineyard site also allowed the identification of flavor-associated metabolic processes and environmental factors that could impact primary fermentation outcomes. These data offer novel insights into the complexities and subtleties of vineyard-specific inoculated wine fermentation and starting points for future investigations into factors that contribute to regional wine distinctiveness.

Integrated requirement of non‐specific and sequence‐specific DNA binding in Myc‐driven transcription

Eukaryotic transcription factors recognize specific DNA sequence motifs, but are also endowed with generic, non-specific DNA-binding activity. How these binding modes are integrated to determine select transcriptional outputs remains unresolved. We addressed this question by site-directed mutagenesis of the Myc transcription factor. Impairment of non-specific DNA backbone contacts caused pervasive loss of genome interactions and gene regulation, associated with increased intra-nuclear mobility of the Myc protein in murine cells. In contrast, a mutant lacking base-specific contacts retained DNA-binding and mobility profiles comparable to those of the wild-type protein, but failed to recognize its consensus binding motif (E-box) and could not activate Myc-target genes. Incidentally, this mutant gained weak affinity for an alternative motif, driving aberrant activation of different genes. Altogether, our data show that non-specific DNA binding is required to engage onto genomic regulatory regions; sequence recognition in turn contributes to transcriptional activation, acting at distinct levels: stabilization and positioning of Myc onto DNA, and-unexpectedly-promotion of its transcriptional activity. Hence, seemingly pervasive genome interaction profiles, as detected by ChIP-seq, actually encompass diverse DNA-binding modalities, driving defined, sequence-dependent transcriptional responses.

Transcription and Cancer in Eukaryotes

Organisms demand pinpoint and correlated gene expression controls for growth, advancement, and operation. This control is also known as transcriptional regulation. It is a complicated process especially in eukaryotes since it is responsible for all biological processes. Transcription control and its concept were brought up about half a century ago1. These concepts gave a basic understanding on DNA binding transcription factors (trans-factors) which inhabit specific DNA sequences at control elements (cis-elements). They regulate transcription apparatus2. Mis-regulation of transcription may cause failure of gene expression that is responsible for cell division, through disproportionate activation of once positively acting transcriptional factors and nuclear oncogenes3. Epigenetic is learning heritable alterations in gene expression that do not occur and involve in DNA sequence. These changes can be established all the time almost randomly, and alternations are passed and inherited through cell division and replication, allowing cells to have different identities while having the same genetic sequence. Due to loss of epigenetic control and failure of keeping proper epigenetic marks, which result in inappropriate activation and will lead to disease state including cancer4. This review focuses on transcriptions, epigenetics, cancers, and potential therapies to regulate and control cancer cells.

Structure analysis suggests Ess1 isomerizes the carboxy-terminal domain of RNA polymerase II via a bivalent anchoring mechanism

Accurate gene transcription in eukaryotes depends on isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II. Isomerization is part of the “CTD code” that regulates recruitment of proteins required for transcription and co-transcriptional RNA processing. Saccharomyces cerevisiae Ess1 and its human ortholog, Pin1, are prolyl isomerases that engage the long heptad repeat (YSPTSPS)26 of the CTD by an unknown mechanism. Here, we used an integrative structural approach to decipher Ess1 interactions with the CTD. Ess1 has a rigid linker between its WW and catalytic domains that enforces a distance constraint for bivalent interaction with the ends of long CTD substrates (≥4–5 heptad repeats). Our binding results suggest that the Ess1 WW domain anchors the proximal end of the CTD substrate during isomerization, and that linker divergence may underlie evolution of substrate specificity.

Identification of the Elusive Core Promoters Driving Polycistronic Transcription by RNA Polymerase II in Trypanosomes

Protein-coding genes in the trypanosome genome are organized in polycistronic transcription units (PTUs). How RNA Polymerase II (Pol II) initiates PTU transcription has not been resolved and the current model favors initiation driven by chromatin epigenetic marks, rather than core-promoters. Here, we identify sequence-specific promoters by functional characterization of Pol II accumulation peaks identified by ChIP-Seq. Sequences located between oppositely orientated PTUs contained two independent and distinct promoters, each driving unidirectional transcription. Detailed analysis identified a 75-bp promoter necessary and sufficient to drive full reporter expression and containing functional motifs. Analysis of further promoters suggested activity and initiation is regulated and transcription-pattern is either focused or dispersed. In contrast to previous model of unregulated and promoter-independent transcription initiation, sequence-specific promoters determine the initiation of Pol II transcription of protein-coding genes in trypanosomes. These findings resolve the discrepancy between trypanosomes and other eukaryotes in how Pol II initiates protein-coding gene transcription and provide a new framework to events underlying eukaryotic transcription evolution.

YTHDC1 gene polymorphisms and Wilms tumor susceptibility in Chinese children: A five-center case-control study.

Wilms tumor is a common pediatric tumor with abundant genetic drivers. YTHDC1 is an important reader of the N6-methyladenosine modification that widely regulates eukaryotic transcripts. YTHDC1 has been associated with the occurrence and development of some tumors. However, this is the first study on YTHDC1 gene polymorphisms and Wilms tumor susceptibility. In brief, we conducted a five-center case-control study to explore the associations between YTHDC1 polymorphisms (rs2293596 T>C, rs2293595 T>C, and rs3813832 T>C) and Wilms tumor susceptibility in Chinese children. A total of 404 cases and 1198 controls were successfully genotyped using TaqMan real-time PCR. Odds ratios (ORs) and 95% confidence intervals (CIs) were used as the evaluation indicators. We found that children with the 2-3 risk genotypes were more likely to develop Wilms tumor than those with the 0-1 risk genotypes (adjusted OR=1.28, 95% CI=1.01-1.62, P=0.042). However, no other statistically significant results were found in this research study. The combined effect of YTHDC1 polymorphisms significantly increases Wilms tumor susceptibility. Our results need to be verified in different populations after increasing the sample size and controlling for confounding factors.

Structure-Based Approaches to Classify the Functional Impact of ZBTB18 Missense Variants in Health and Disease.

The Cys2His2 type zinc finger is a motif found in many eukaryotic transcription factor proteins that facilitates binding to genomic DNA so as to influence cellular gene expression. One such transcription factor is ZBTB18, characterized as a repressor that orchestrates the development of mammalian tissues including skeletal muscle and brain during embryogenesis. In humans, it has been recognized that disease-associated ZBTB18 missense variants mapping to the coding sequence of the zinc finger domain influence sequence-specific DNA binding, disrupt transcriptional regulation, and impair neural circuit formation in the brain. Furthermore, general population ZBTB18 missense variants that influence DNA binding and transcriptional regulation have also been documented within this domain; however, the molecular traits that explain why some variants cause disease while others do not are poorly understood. Here, we have applied five structure-based approaches to evaluate their ability to discriminate between disease-associated and general population ZBTB18 missense variants. We found that thermodynamic integration and Residue Scanning in the Schrodinger Biologics Suite were the best approaches for distinguishing disease-associated variants from general population variants. Our results demonstrate the effectiveness of structure-based approaches for the functional characterization of missense alleles to DNA binding, zinc finger transcription factor protein-coding genes that underlie human health and disease.

Comprehensive identification of lysine 2-hydroxyisobutyrylated proteins in Ustilaginoidea virens reveals the involvement of lysine 2-hydroxyisobutyrylation in fungal virulence.

Lysine 2-hydroxyisobutyrylation (Khib ) is a newly identified post-translational modification (PTM) that plays important roles in transcription and cell proliferation in eukaryotes. However, its function remains unknown in phytopathogenic fungi. Here, we performed a comprehensive assessment of Khib in the rice false smut fungus Ustilaginoidea virens, using Tandem Mass Tag (TMT)-based quantitative proteomics approach. A total of 3 426 Khib sites were identified in 977 proteins, suggesting that Khib is a common and complex PTM in U. virens. Our data demonstrated that the 2-hydroxyisobutyrylated proteins are involved in diverse biological processes. Network analysis of the modified proteins revealed a highly interconnected protein network that included many well-studied virulence factors. We confirmed that the Zn-binding reduced potassium dependency3-type histone deacetylase (UvRpd3) is a major enzyme that removes 2-hydroxyisobutyrylation and acetylation in U. virens. Notably, mutations of Khib sites in the mitogen-activated protein kinase (MAPK) UvSlt2 significantly reduced fungal virulence and decreased the enzymatic activity of UvSlt2. Molecular dynamics simulations demonstrated that 2-hydroxyisobutyrylation in UvSlt2 increased the hydrophobic solvent-accessible surface area and thereby affected binding between the UvSlt2 enzyme and its substrates. Our findings thus establish Khib as a major post-translational modification in U. virens and point to an important role for Khib in the virulence of this phytopathogenic fungus.

Watching U4 snRNA Release During Spliceosome Activation with Mango RNA APTamers

Introns from eukaryotic pre-mRNA transcripts are removed by the spliceosome. Splicing is accomplished only after the spliceosomal RNA active site is formed from snRNAs, pre-mRNA, and several proteins. Base pairing between U4 and U6 snRNAs is disrupted during activation, allowing for a conformational change in the U6 snRNA necessary for catalysis at later stages of splicing. The process by which the U4/U6 duplex base pairing contacts are destabilized during activation is unknown but results in irreversible release of U4 with its associated proteins.

Single-Molecule Fret Analysis of Key Protein Conformational Changes During Promoter Escape by RNA Polymerase

Bacterial RNA Polymerases (RNAPs) bind to transcription initiation factors called σ factors to start promoter-specific transcription. Within the σ factor lies a highly conserved structural module, the “σ-finger”, which forms a loop that protrudes towards the RNAP active-center and that interacts with the template strand DNA. The close proximity of the σ-finger to the “heart” of transcription is implicated in the pre-organisation of the template strand DNA, in the synthesis of the first short RNAs, and in the pausing of transcription upon synthesis of a 6-mer RNA. However, the σ-finger also blocks entry of the nascent RNA to the RNAP exit channel and must be displaced during initial transcription as the first step in promoter escape. Despite recent structural studies, σ-finger conformational changes during late transcription initiation and promoter escape are still unknown. Here we report a novel single-molecule FRET ruler that monitors the conformational dynamics of the E.coli σ70-finger from the early stages of transcription initiation to promoter escape. Our results using the σ-finger FRET ruler on transcription complexes formed on a derivative of the lac promoter show that the σ-finger adopts three conformations, which interconvert with rates 0.01-1.5s−1. Intriguingly, we find the σ-finger first displaced in complexes trapped in synthesis of abortive transcripts up to a 10-mer RNA. This observation is likely driven by collision of the σ-finger with the 5’-end of the growing nascent RNA. Based on our results, we propose a new model describing the relation of the conformational changes in the σ-finger to the dynamics of transcription initiation and promoter escape. Archaeal and eukaryotic transcription initiation complexes also contain structural modules that block the RNAP exit channel, so it is therefore likely that this model applies to all kingdoms of life.

Bacterial nucleomodulins: A coevolutionary adaptation to the eukaryotic command center

Through long-term interactions with their hosts, bacterial pathogens have evolved unique arsenals of effector proteins that interact with specific host targets and reprogram the host cell into a permissive niche for pathogen proliferation. The targeting of effector proteins into the host cell nucleus for modulation of nuclear processes is an emerging theme among bacterial pathogens. These unique pathogen effector proteins have been termed in recent years as “nucleomodulins.” The first nucleomodulins were discovered in the phytopathogens Agrobacterium and Xanthomonas, where their nucleomodulins functioned as eukaryotic transcription factors or integrated themselves into host cell DNA to promote tumor induction, respectively. Numerous nucleomodulins were recently identified in mammalian pathogens. Bacterial nucleomodulins are an emerging family of pathogen effector proteins that evolved to target specific components of the host cell command center through various mechanisms. These mechanisms include: chromatin dynamics, histone modification, DNA methylation, RNA splicing, DNA replication, cell cycle, and cell signaling pathways. Nucleomodulins may induce short- or long-term epigenetic modifications of the host cell. In this extensive review, we discuss the current knowledge of nucleomodulins from plant and mammalian pathogens. While many nucleomodulins are already identified, continued research is instrumental in understanding their mechanisms of action and the role they play during the progression of pathogenesis. The continued study of nucleomodulins will enhance our knowledge of their effects on nuclear chromatin dynamics, protein homeostasis, transcriptional landscapes, and the overall host cell epigenome.

The landscape of alternative splicing reveals novel events associated with tumorigenesis and the immune microenvironment in gastric cancer.

Alternative splicing (AS), contributing to vast protein diversity from a rather limited number of genes in eukaryotic transcripts, has emerged as an important signature for tumor initiation and progression. However, a systematic understanding of its functional impact and relevance to gastric cancer (GC) tumorigenesis is lacking. Differentially expressed AS (DEAS) was verified among GC-associated AS events based on RNA-seq profiles from the TCGA database. Functional enrichment analysis, unsupervised clustering analysis and prognostic models were used to infer the potential roles of DEAS events and their molecular, clinical and immune features. In total, 12,225 AS events were detected from 5,199 genes, among which 314 AS events were identified as DEAS events in GC. The parental genes of the DEAS events were significantly enriched in the regulation of GC-related processes. The splicing correlation network suggested a significant relationship between DEAS events and splicing factors (SFs). Three clusters of DEAS events were identified to be different in prognosis, cancer-specific signatures and immune features between distinct clusters. Univariate and multivariate analyses regarded 3 DEAS events as independent prognostic indicators. Profiling of the AS landscape in GC elucidated the functional roles of the splicing network in GC and might serve as a novel prognostic indicator and therapeutic target.

Conformational Plasticity and DNA-Binding Specificity of the Eukaryotic Transcription Factor Pax5.

The eukaryotic transcription factor Pax5 has a DNA-binding Paired domain composed of two independent helical bundle subdomains joined by a flexible linker. Previously, we showed distinct biophysical properties of the N-terminal (NTD) and C-terminal (CTD) subdomains, with implications for how these two regions cooperate to distinguish nonspecific and cognate DNA sites [Perez-Borrajero, C., et al. (2016) J. Mol. Biol. 428, 2372-2391]. In this study, we combined experimental methods and molecular dynamics (MD) simulations to dissect the mechanisms underlying the functional differences between the Pax5 subdomains. Both subdomains showed a similar dependence of DNA-binding affinity on ionic strength. However, due to a greater contribution of non-ionic interactions, the NTD bound its cognate DNA half-site with an affinity approximately 10-fold higher than that of the CTD with its half-site. These interactions involve base-mediated contacts as evidenced by nuclear magnetic resonance spectroscopy-monitored chemical shift perturbations. Isothermal titration calorimetry revealed that favorable enthalpic and compensating unfavorable entropic changes were substantially larger for DNA binding by the NTD than by the CTD. Complementary MD simulations indicated that the DNA recognition helix H3 of the NTD is particularly flexible in the absence of DNA and undergoes the largest changes in conformational dynamics upon binding. Overall, these data suggest that the differences observed for the subdomains of Pax5 are due to the coupling of DNA binding with dampening of motions in the NTD required for specific base contacts. Thus, the conformational plasticity of the Pax5 Paired domain underpins the differing roles of its subdomains in association with nonspecific versus cognate DNA sites.