Transcription initiation by archaeal RNA polymerase (RNAP) is assisted by transcription factors, TBP, TFB, and TFEα, which show sequence, structural and functional homology to eukaryotic TBP, TFIIB, and TFIIEα. Previous low-resolution cross-linking studies of transcription complexes from the hyperthermophile archaeon Pyrococcus furiosus showed that the transcription bubble DNA makes extensive contacts with TFB and TFEα. Here, contacts between TFB and TFEα surfaces and promoter DNA are mapped with amino acid-to-base precision, using a high-resolution cross-link mapping method. The non-canonical amino acid p-benzoyl phenylalanine (Bpa) was incorporated into TFB1 or TFEα at several positions, transcription complexes were formed and cross-links induced, and the positions of cross-links were mapped by primer extension. In transcription pre-initiation complexes forming a transcription bubble (PICs), the TFB1 B-reader helix and loop conserved sequences interact with the DNA transcribed (T) strand near the upstream side of the transcription bubble, while the B-linker strand conserved sequence interacts with both the transcribed and the non-transcribed (NT) strands. In a 5 nucleotide initially transcribing complex (ITC), cross-links to TFB1 changed for the B-reader but not the B-linker, suggesting scrunching of part of the T strand in the RNAP main channel. The TFEα winged helix cross-linked the NT strand at the upstream edge of the transcription bubble in the PIC. In the ITC, the NT strand changed position dramatically relative to the TFEα wing tip, indicating that the DNA NT strand can bulge out of the main channel as transcription begins and downstream DNA is pulled in. The cross-linking data were compared with structural models of the archaeal PIC and ITC, and indicate that interactions between TFB conserved motifs and DNA are conserved from archaea to humans, while archaeal TFEα contacts to DNA are apparently dissimilar from what is seen in human RNAP II initiation complexes.
MicroRNAs (miRNAs) are small, noncoding RNAs that have emerged as powerful players in the post-transcriptional regulation of gene expression. In the context of viral infections, cellular miRNAs play significant roles in modulating the complex host responses to pathogens and have essential roles in shaping viral replication. Moreover, several virus families encode viral miRNAs that manipulate the host environment and can tap into miRNA-regulated gene networks. Viral antigen expression itself can be directly miRNA regulated, and infection outcomes are substantially shaped by the miRNA activities that influence host anti-viral defense mechanisms. This review discusses common themes for both human and viral miRNAs in viral infection processes, highlighting their dual functions in both suppressing and promoting viral life cycles.
Recombinant proteins with applications in human and veterinary medicine are mainly produced in mammalian expression systems, particularly Chinese hamster ovary (CHO) cells. Although these cells enable proper folding and post-translational modifications, they often exhibit low specific productivity, which represents a critical limitation in large-scale biopharmaceutical manufacturing. Strong viral promoters, including CMV and its enhancer-enriched variant CMV+E, are commonly used to drive transgene expression. However, they are susceptible to epigenetic silencing and progressive loss of activity during prolonged culture, which can compromise yield stability. Endogenous host-cell promoters constitute a promising alternative, as they can maintain transgene expression in alignment with physiological and bioprocess regulatory mechanisms. Here, we assessed the activity of endogenous eukaryotic promoters previously identified in our laboratory from highly expressed CHO-K1 genes. The receptor-binding domain (RBD) of SARS-CoV-2 was selected as a model of a complex secretory glycoprotein domain. Our results demonstrate that the Hspa5 and Vim promoters enable sustained and high-level expression of recombinant RBD protein in CHO-K1 cells, achieving performance comparable to the enhancer-containing viral promoter CMV+E. Notably, Hspa5 and Vim are unoptimized genomic sequences lacking classical enhancers; nevertheless, their intrinsic stability and reduced susceptibility to silencing make them attractive regulatory elements for recombinant protein production. These findings highlight their potential for next-generation promoter engineering and for the development of long-term, high-yield mammalian expression systems for complex therapeutic proteins.
Proper transcription termination coupled to pre-mRNA cleavage and polyadenylation is an important step in gene expression as it generates the 3'UTR of mRNAs and defines transcription units. Defective termination (transcriptional readthrough) can suppress transcription of downstream genes by transcriptional interference but can also promote the production of chimeric transcripts, which contain the exons of upstream genes spliced to exons of downstream genes. This review will summarize current findings of chimeric splicing between exons of adjacent genes generated by transcriptional readthrough and the conditions that promote these events. Chimeric splicing can be facilitated by inactivation of factors that promote transcription termination or of specific splicing factors, particularly those involved in 3'-splice site recognition. This effect is due to the tight coupling between recognition of the terminal intron 3'-splice site and of the downstream polyadenylation sites, and its impact on transcriptional termination. Production of chimeric transcripts is also increased in cellular conditions that perturb transcription termination, such as cellular stress, treatment with the chemotherapeutic agent Imatinib or viral infections. However, not all conditions that promote transcriptional readthrough result in chimeric splicing, suggesting that the production of chimeric mRNAs requires a "Goldilocks" state of alteration of the transcriptional machinery that allows for transcriptional readthrough without a general reduction of splicing efficiency.
The TATA-binding protein (TBP) is an essential component of the eukaryotic transcriptional machinery, yet little is known about the structure, evolution, and functional divergence of TBP paralogs in parasitic flatworms. Here, we performed a comprehensive comparative analysis of TBP1 and TBP2 across cestode species of medical and veterinary relevance. Using genomic annotation, multiple sequence alignment, homology modeling, phylogenetics, electrostatic potential mapping, and protein - DNA docking complemented with molecular dynamics (MD) simulations, we reveal that both paralogs maintain a highly conserved genomic organization and the canonical TBP α/β saddle architecture. TBP1 exhibits moderate variability in sequence and electrostatic in peripheral regions, whereas TBP2 is more conserved, suggesting distinct evolutionary constraints. Despite paralog divergence, all cestode TBPs retain the key aromatic and basic residues required for minor-groove recognition of the TATA box and interaction with general transcription factors. Docking and MD simulations confirm a conserved pattern of TATA-binding across species, with TBP2 displaying exceptionally uniform interaction networks. Together, these findings provide genomic and structural data for TBPs in cestodes, suggesting the existence of paralog-specific regulatory roles and offering new insights into transcriptional control in parasitic flatworms.
Transposable elements (TEs) threaten genomic integrity, yet their pervasive presence indicates the limitations of existing silencing mechanisms. A recent paper in Nature (Zhao et al. 2025) discovered the SOS splicing system, which provides an RNA-level defense that excises DNA transposons from mRNAs, thereby restoring gene sequence. This spliceosome-independent pathway, mediated by AKAP17A, CAAP1, and RTCB, recognizes dsRNA hairpins formed by inverted terminal repeats (ITRs) and religates the resulting RNA fragments. From an evolutionary perspective, SOS splicing exemplifies a post-transcriptional error-correction mechanism that mitigates the deleterious consequence of TE insertions, paralleling the Constructive Neutral Evolution (CNE) framework. In contrast, ADAR-mediated A-to-I RNA editing suppresses the MDA5-triggered innate immune responses to TE-derived dsRNAs, effectively tolerating rather than eliminating TEs. There may be partial overlap between ADAR and SOS substrates. ADAR editing may delay but not prevent SOS splicing, while SOS excision removes ADAR substrates. The lethality of ADAR loss underscores its role as the mechanism mitigating purifying selection on TEs and thus may contribute to their genomic tolerance and proliferation. Collectively, while ADAR masks the harm of TEs, SOS splicing actively repairs the resulting damage, together illustrating a delicate evolutionary balance between TE tolerance and transcriptomic rescue.
Transcription termination is a highly regulated step which sets boundaries between genes and maintains genome integrity. Defects in transcription termination will cause unexpected expression of downstream genes and traffic-jam of RNA polymerases with protein machineries. Termination occurs mainly in two types of mechanisms regarding whether it depends on molecular motor action, i.e. factor-dependent termination, or is induced solely by nucleic acid signals, i.e. intrinsic termination. In recent years, great efforts have been devoted to, and significant advances have been achieved in understanding the mechanisms of transcription termination. This review focuses on the topic of factor-dependent termination and intrinsic termination and highlights the recent progress in the structural and functional studies of RNA polymerases that are critical for transcription termination.
Eukaryotic transcription of mRNAs and some non-coding RNAs is governed by RNA Polymerase II (RNA pol II). The full progression of RNA pol II across a gene - from promoter clearance through transcript elongation and termination - is dependent upon the post-translational modifications (PTMs) of its carboxy-terminal domain (CTD) and the dynamic recruitment of numerous trans-acting factors. Rtr1 in yeast and its human orthologue, RPAP2, have emerged as multifunctional regulators of RNA pol II. Despite evidence supporting their role as Ser5-specific CTD phosphatases, structural and biochemical studies have raised doubts about whether they are bona fide phosphatases or instead function as cofactors that influence the activity of established CTD phosphatases. Furthermore, both proteins have been implicated in processes ranging from RNA pol II biogenesis and nuclear transport to transcriptional elongation and termination. Notably, Rtr1's influence also extends to post-transcriptional events like mRNA stability. In this review, we describe the main functions attributed to Rtr1 and RPAP2, and discuss the role of the human homologue in various diseases.
Human Immunodeficiency Virus 1 (HIV-1) is the causative agent for acquired immunodeficiency syndrome (AIDS). Antiretroviral therapy has turned HIV-1 from a lethal disease to a chronic condition but is not curative due to the persistence of a small reservoir of latently infected cells. The molecular mechanisms driving HIV-1 latency have been extensively studied, thus far largely focusing on transcriptional regulation. Here, we summarize well established and newly discovered mechanisms of HIV-1 latency, as well as how studies of the HIV-1 promoter have informed the broader transcription field. As a strategy toward HIV-1 cure, latency reversal agents (LRAs) have been developed to pharmacologically target blocks in HIV-1 transcription to achieve reactivation of viral gene expression. However, clinical studies indicate that LRAs have largely failed to sufficiently activate the reservoir such that viral protein is produced, and there was no reduction in the size of the viral reservoir. Indeed it has become clear that co- and post-transcriptional mechanisms are also at play to regulate HIV-1 gene expression and may also serve as attractive targetable blocks. We also outline recent developments in technologies allowing the ex vivo characterization of the HIV-1 reservoir in people living with HIV (PWH). These novel technologies enable us to interrogate the different molecular compartments such as integrated intact and defective proviral HIV-1 DNA, unspliced and spliced RNA, and protein levels that provide unprecedented new insight into latency mechanisms. Lastly, the potential of different transcription-targeting cure strategies is discussed in light of the contributions of co- and posttranscriptional blocks and the advent of Long Acting (LA)-ART.
Proper regulation of transcription involves not only quantitative control of RNA dosage but also ensuring the correct biochemical properties of transcripts. In all eukaryotes, the epigenetic landscape and the dynamic composition of the RNA Polymerase II complex (PolII) interact to control the transcription of translatable mRNA. Decades of research have described dogmatic rules for model organisms, such as the distribution of individual chromatin marks along the transcription unit or the hierarchical phosphorylation pattern in the C-terminal domain (CTD) of the largest PolII subunit RPB1. Besides this canonical mRNA transcription, there are exceptions; on the one hand, not all genes in a species follow the dogma, and on the other hand, there are species that show general divergence from the models, both in the epigenomic landscape and in the genetically encoded PolII. In the recent literature, protists in particular have shifted their attention as they show considerable differences in chromatin structure and PolII complex composition. Here, we aim to enlighten the transcription machinery of the unicellular ciliate Paramecium as an exciting model to study a divergent transcriptional machinery for vegetative mRNA and developmental ncRNA transcription.
TATA-box binding protein (TBP) is a core subunit of the transcription factor TFIID and plays a pivotal role in recognizing the TATA-box in protein-coding genes, facilitating the assembly of the transcription preinitiation complex. In Taenia solium, only one TBP isoform (TsTBP1) has been previously reported. Here, we identify and characterize a second isoform, TsTBP2, using a combination of molecular biology and bioinformatics approaches. TsTBP2 shares 42% primary sequence identity with TsTBP1 and exhibits distinct expression patterns between cysticerci and adult stages. To investigate the molecular determinants of DNA recognition, selectivity, and binding affinity, we performed molecular docking and molecular dynamics simulations for both TsTBPs with various TATA-box sequences. Our results reveal that TsTBP1 exhibits higher affinity for T. solium TATA-box sequences compared to the consensus AdML TATA-box (TATAAAAG), largely due to the specific interaction of critical phenylalanine residues with the DNA minor groove, which induces DNA bending and stabilizes the TBP-DNA complex. Furthermore, analysis of the Buckle parameter indicates that these Phe residues are the principal contributors to DNA distortion. To our knowledge, this study represents the first analysis of TBP selectivity and affinity in cestodes, providing insights into the molecular mechanisms underlying transcriptional regulation in T. solium.
Epstein-Barr Virus (EBV) establishes life-long latent infection in >90% of adults and is a causal agent for diverse cancers and autoimmune diseases. EBV has a complex life cycle in multiple different tissue types that involve dynamic variations in viral gene expression. These gene expression changes account for the success of the virus in long-term persistence and evading host immune control, as well as its potential for driving cancer evolution and autoimmune disease. Here, we review some of the salient features of EBV gene regulation highlighting the many variations of viral transcription. We review recent advances in our understanding of the factors that bind and regulate EBV gene expression. Based on this diversity of viral transcription patterns, we propose that EBV genome consists of gene modules regulated by local promoter-proximal transcription factor combinations that are further regulated by distal regulatory interactions among the various modules that interact through architectural factors, such as CTCF and cohesion. These modules are likely to represent chromatin architectural domains, and can also interact with host chromosome domains that further regulate viral and host gene expression. We propose that this gene regulatory hierarchy provides EBV with necessary plasticity for viral persistence, as well as a strong potentiator for cancer and autoimmune disease.
The nuclear receptor (NR) superfamily of ligand-activated receptors plays a key role in maintaining cellular homeostasis and in pathophysiology. NRs can be subdivided into functional activities structural similarity and the existence of endogenous ligands. Most NRs are classified as those that are adopted orphan or orphan receptors which have only possible ligands or no identified endogenous ligands, respectively. In this review, the activities of the complete orphan receptor sub-family of transcription factors have been reviewed with a focus on the effects of possible endogenous (biochemicals), natural product-derived and synthetic ligands. Despite their lack of a bona-fide ligand, the orphan receptors bind structurally diverse compounds that exhibit tissue-specific agonist, antagonist and inverse agonist activities with potential for future development as clinical therapeutics for the treatment of multiple diseases.
The regulation of transcription is a major control point in the flow of information from the genome to the phenome. Central to this regulation are transcription factors (TFs), which bind specific DNA motifs in gene regulatory regions. In both metazoans and plants, 5-7% of all genes encode TFs. Although individual TFs can recognize and regulate thousands of target genes, an important question remains: how many TFs are required to precisely control the expression of a single gene? In this review, we compare the regulation of gene expression in plants and metazoans, outline key methodologies for identifying genes recognized or regulated by TFs, and explore what is currently known about the number of TFs needed to define the expression of any given plant gene. As the volume of high-throughput sequencing data continues to grow exponentially, it becomes increasingly clear that transcriptional regulatory networks exhibit remarkable complexity, characterized by many targets influenced by each TF; and that many TFs, often several dozens, contribute to the regulation of individual genes.
The WD repeat domain 77 (WDR77) protein plays a critical role in prostate development and dysregulation of WDR77 expression is associated with prostate tumorigenesis. This study investigated the regulatory effects of GATA3 and E2F6 on WDR77 gene expression. A negative correlation between GATA3/E2F6 and WDR77 expression at both mRNA and protein levels was observed during prostate development and prostate tumorigenesis. Prostate cancer cells lost expression of GATA3 and E2F6 and re-expression of GATA3 and E2F6 resulted in a dose-dependent reduction in WDR77 expression and cell growth. Exogenous expression of WDR77 relieved the growth inhibition by GATA3. GATA3 and E2F6 directly interact with the promoter of the WDR77 gene in vitro and in vivo and repress WDR77 promoter activity. These results provide valuable insights into the molecular mechanisms governing WDR77 expression during prostate development and prostate tumorigenesis.
Since the Modern Synthesis, interest has grown in resolving the "black box" between genotype and phenotype. Contained within this black box are highly plastic RNA and proteins with global effects on chromosome integrity and gene expression that serve as evolutionary capacitors - elements that enable the accumulation and buffering of genetic variation in normal conditions and reveal hidden genetic variation when induced by environmental stress. Discussion of evolutionary capacitors has primarily focused on eukaryotic translation factors and chaperones, such as Hsp90 and PSI+ prion. However, due to the coupling of transcription and translation in prokaryotes, transcription factors can be equally impactful in the modulation of gene expression and phenotypes. In this review, we discuss the prokaryotic transcription terminator Rho and how mutagenesis and plasticity of Rho influence epistasis, evolvability, and adaptation to stress in bacteria. We discuss the effects of variation in Rho generated by nature, laboratory mutagenesis, and experimental evolution; and how this variation is constrained or encouraged by Rho's extensive network of protein interactors. Exploring Rho's role as an evolutionary capacitor, along with identifying additional elements that can serve this function, can significantly advance our understanding of how organisms adapt to thrive in diverse environments.
The lncRNAs have deepened our understanding of crop domestication and improvement. These regulators influence key traits like yield, germination, and stress response. Future research should identify functional lncRNAs, explore their interactions, and use CRISPR for targeted improvements. Understanding their roles in polyploid crops may enhance resilience and productivity.
The transcription factor p53 is the most frequently impaired tumor suppressor in human cancers. In response to various stress stimuli, p53 activates transcription of genes that mediate its tumor-suppressive functions. Distinctive characteristics of p53 outlined here enable a well-defined program of genes involved in cell cycle arrest, apoptosis, senescence, differentiation, metabolism, autophagy, DNA repair, anti-viral response, and anti-metastatic functions, as well as facilitating autoregulation within the p53 network. This versatile, anti-cancer network governed chiefly by a single protein represents an immense opportunity for targeted cancer treatment, since about half of human tumors retain unmutated p53. During the last two decades, numerous compounds have been developed to block the interaction of p53 with the main negative regulator MDM2. However, small molecule inhibitors of MDM2 only induce a therapeutically desirable apoptotic response in a limited number of cancer types. Moreover, clinical trials of the MDM2 inhibitors as monotherapies have not met expectations and have revealed hematological toxicity as a characteristic adverse effect across this drug class. Currently, combination treatments are the leading strategy for enhancing efficacy and reducing adverse effects of MDM2 inhibitors. This review summarizes efforts to identify and test therapeutics that work synergistically with MDM2 inhibitors. Two main types of drugs have emerged among compounds used in the following combination treatments: first, modulators of the p53-regulated transcriptome (including chromatin modifiers), translatome, and proteome, and second, drugs targeting the downstream pathways such as apoptosis, cell cycle arrest, DNA repair, metabolic stress response, immune response, ferroptosis, and growth factor signaling. Here, we review the current literature in this field, while also highlighting overarching principles that could guide target selection in future combination treatments.
Protein engineering has emerged as a powerful approach toward the development of novel therapeutics targeting the MYC/MAX/E-box network, an active driver of >70% of cancers. The MYC/MAX heterodimer regulates numerous genes in our cells by binding the Enhancer box (E-box) DNA site and activating the transcription of downstream genes. Traditional small molecules that inhibit MYC face significant limitations that include toxic effects, drug delivery challenges, and resistance. Recent advances in protein engineering offer promising alternatives by creating protein-based drugs that directly disrupt the MYC/MAX dimerization interface and/or MYC/MAX's binding to specific DNA targets. Designed DNA binding proteins like Omomyc, DuoMyc, ME47, MEF, and Mad inhibit MYC activity through specific dimerization, sequestration, and DNA-binding mechanisms. Compared to small molecules, these engineered proteins can offer superior specificity and efficacy and provide a potential pathway for overcoming the limitations of traditional cancer therapies. The success of these protein therapeutics highlights the importance of protein engineering in developing cancer treatments.
