Search

Transcription enables the production of RNA from a DNA template. Due to the highly dynamic nature of transcription, live‐cell imaging methods play a crucial role in measuring the kinetics of this process. For instance, transcriptional bursts have been visualized using fluorescent phage‐coat proteins that associate tightly with messenger RNA (mRNA) stem loops formed on nascent transcripts. To convert the signal emanating from a transcription site into meaningful estimates of transcription dynamics, the influence of various parameters on the measured signal must be evaluated. Here, the effect of gene length on the intensity of the transcription site focus was analyzed. Intuitively, a longer gene can support a larger number of transcribing polymerases, thus leading to an increase in the measured signal. However, measurements of transcription induced by hyper‐osmotic stress responsive promoters display independence from gene length. A mathematical model of the stress‐induced transcription process suggests that the formation of gene loops that favor the recycling of polymerase from the terminator to the promoter can explain the observed behavior. One experimentally validated prediction from this model is that the amount of mRNA produced from a short gene should be higher than for a long one as the density of active polymerase on the short gene will be increased by polymerase recycling. Our data suggest that this recycling contributes significantly to the expression output from a gene and that polymerase recycling is modulated by the promoter identity and the cellular state. Take‐away: Quantification of stress‐induced promoter transcription dynamics using a live assays reporter system displays no dependence of signal intensity with gene length.Mathematical modeling predicts that the formation of gene loops leading to the recycling of polymerases can explain the observed behavior.More prevalent polymerase recycling on short genes results in a higher transcriptional output. [ABSTRACT FROM AUTHOR]

Eukaryotic transcription is a complex process involving a vast network of protein and RNA factors that influence gene expression. The main player in transcription is the RNA polymerase that synthesizes the RNA from the DNA template. RNA polymerase II (RNAPII) transcribes all protein coding genes and some noncoding RNAs in eukaryotic cells. The polymerase is aided by interacting partners that shuttle it along the gene for initiation, elongation and termination of transcription. One of the many factors that assist RNAPII in transcription of genes is Ssu72. It is a carboxy-terminal-domain (CTD)-phosphatase that plays pleiotropic roles in the transcription cycle. It is essential for cell viability in Saccharomyces cerevisiae, the organism in which it was discovered. The homologues of Ssu72 have been identified in humans, mice, plants, flies, and fungi thereby suggesting the evolutionarily conserved nature of the protein. Recent studies have implicated the factor beyond the confines of transcription in homeostasis and diseases.

Abstract Background Transcription in mammalian cells is a complex stochastic process involving shuttling of polymerase between genes and phase-separated liquid condensates. It occurs in bursts, which results in vastly different numbers of an mRNA species in isogenic cell populations. Several factors contributing to transcriptional bursting have been identified, usually classified as intrinsic, in other words local to single genes, or extrinsic, relating to the macroscopic state of the cell. However, some possible contributors have not been explored yet. Here, we focus on processes at the 3 ′ and 5 ′ ends of a gene that enable reinitiation of transcription upon termination. Results Using Bayesian methodology, we measure the transcriptional bursting in inducible transgenes, showing that perturbation of polymerase shuttling typically reduces burst size, increases burst frequency, and thus limits transcriptional noise. Analysis based on paired-end tag sequencing (PolII ChIA-PET) suggests that this effect is genome wide. The observed noise patterns are also reproduced by a generative model that captures major characteristics of the polymerase flux between the ends of a gene and a phase-separated compartment. Conclusions Interactions between the 3 ′ and 5 ′ ends of a gene, which facilitate polymerase recycling, are major contributors to transcriptional noise.

The role of general transcription factor TFIIB in transcription extends well beyond its evolutionarily conserved function in initiation. Chromatin localization studies demonstrating binding of TFIIB to both the 5' and 3' ends of genes in a diverse set of eukaryotes strongly suggested a rather unexpected role of the factor in termination. TFIIB indeed plays a role in termination of transcription. TFIIB occupancy of the 3' end is possibly due to its interaction with the termination factors residing there. Interaction of the promoter-bound TFIIB with factors occupying the 3' end of a gene may be the basis of transcription-dependent gene looping. The proximity of the terminator-bound factors with the promoter in a gene loop has the potential to terminate promoter-initiated upstream anti-sense transcription thereby conferring promoter directionality. TFIIB, therefore, is emerging as a factor with pleiotropic roles in the transcription cycle. This could be the reason for preferential targeting of TFIIB by viruses. Further studies are needed to understand the critical role of TFIIB in viral pathogenesis in the context of its newly identified roles in termination, gene looping and promoter directionality. [ABSTRACT FROM AUTHOR]

In eukaryotes, cellular RNAs are produced by three nuclear RNA polymerases (RNAPI, II, and III), which are multisubunit complexes. They share structural and functional features, although they are specialized in the synthesis of specific RNAs. RNAPII transcribes the vast majority of cellular RNAs, including mRNAs and a large number of noncoding RNAs. The structure of RNAPII is highly conserved in all eukaryotes, consisting of 12 subunits (Rpb1-12) organized into five structural modules, among which the Rpb4 and Rpb7 subunits form the stalk. Early studies suggested an accessory role for Rpb4, because is required for specific gene transcription pathways. Far from this initial hypothesis, it is now well established that the Rpb4/7 heterodimer plays much wider roles in gene expression regulation. It participates in nuclear and cytosolic processes ranging from transcription to translation and mRNA degradation in a cyclical process. For this reason, Rpb4/7 is considered a coordinator of gene expression. New functions have been added to the list of stalk functions during transcription, which will be reviewed herein: first, a role in the maintenance of proper RNAPII phosphorylation levels, and second, a role in the establishment of a looped gene architecture in actively transcribed genes. [ABSTRACT FROM AUTHOR]

There is increasing evidence suggesting the contribution of non-coding RNAs (ncRNAs) to the phenotypic and physiological complexity of organisms. A novel ncRNA species has been identified near the transcription boundaries of protein-coding genes in eukaryotes, bacteria, and archaea. This review provides a detailed description of these transcription boundary-associated RNAs (TBARs), including their classification. Based on their genomic distribution, TBARs are divided into two major groups: promoterassociated RNAs (PARs) and terminus-associated RNAs (TARs). Depending on the sequence length, each group is further classified into long RNA species (>200 nt) and small RNA species (<200 nt). According to these rules of TBAR classification, divergent ncRNAs with confusing nomenclatures, such as promoter upstream transcripts (PROMPTs), upstream antisense RNAs (uaRNAs), stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), upstream non-coding transcripts (UNTs), transcription start site-associated RNAs (TSSaRNAs), transcription initiation RNAs (tiRNAs), and transcription termination site-associated RNAs (TTSaRNAs), were assigned to specific classes. Although the biogenesis pathways of PARs and TARs have not yet been clearly elucidated, previous studies indicate that some of the PARs have originated either through divergent transcription or via RNA polymerase pausing. Intriguing findings regarding the functional implications of the TBARs such as the long-range "gene looping" model, which explains their role in the transcriptional regulation of protein-coding genes, are also discussed. Altogether, this review provides a comprehensive overview of the current research status of TBARs, which will promote further investigations in this research area. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) chromatin organization is important for proper gene regulation, yet how the genome is remodeled in response to stress is largely unknown. Here, we use a highly sensitive version of chromosome conformation capture in combination with fluorescence microscopy to investigate Heat Shock Protein (HSP) gene conformation and 3D nuclear organization in budding yeast. In response to acute thermal stress, HSP genes undergo intense intragenic folding interactions that go well beyond 5'-3' gene looping previously described for RNA polymerase II genes. These interactions include looping between upstream activation sequence (UAS) and promoter elements, promoter and terminator regions, and regulatory and coding regions (gene "crumpling"). They are also dynamic, being prominent within 60 s, peaking within 2.5 min, and attenuating within 30 min, and correlate with HSP gene transcriptional activity. With similarly striking kinetics, activated HSP genes, both chromosomally linked and unlinked, coalesce into discrete intranuclear foci. Constitutively transcribed genes also loop and crumple yet fail to coalesce. Notably, a missense mutation in transcription factor TFIIB suppresses gene looping, yet neither crumpling nor HSP gene coalescence is affected. An inactivating promoter mutation, in contrast, obviates all three. Our results provide evidence for widespread, transcription-associated gene crumpling and demonstrate the de novo assembly and disassembly of HSP gene foci. [ABSTRACT FROM AUTHOR]

Gene looping, defined as the physical interaction between the promoter and terminator regions of a RNA polymerase II-transcribed gene, is widespread in yeast and mammalian cells. Gene looping has been shown to play important roles in transcription. Gene-loop formation is dependent on regulatory proteins localized at the 5′ and 3′ ends of genes, such as TFIIB. However, whether other factors contribute to gene looping remains to be elucidated. Here, we investigated the contribution of intrinsic DNA and chromatin structures to gene looping. We found that Saccharomyces cerevisiae looped genes show high DNA bendability around middle and 3/4 regions in open reading frames (ORFs). This bendability pattern is conserved between yeast species, whereas the position of bendability peak varies substantially among species. Looped genes in human cells also show high DNA bendability. Nucleosome positioning around looped ORF middle regions is unstable. We also present evidence indicating that this unstable nucleosome positioning is involved in gene looping. These results suggest a mechanism by which DNA bendability and unstable nucleosome positioning could assist in the formation of gene loops. [ABSTRACT FROM PUBLISHER]

Abstract: More than 70% of breast cancers in women require estrogens for cell proliferation and survival. 17β-estradiol (E2) effect on mammary target cells is almost exclusively mediated by its binding to the estrogen receptor-α (ERα) that joins chromatin where it assembles active transcription complexes. The proliferative and pro-survival action of estrogens is antagonized in most cases by retinoic acid (RA), even though the cognate retinoic acid receptor-α (RARα) cooperates with ERα on promoters of estrogen-responsive genes. We have examined at the molecular level the crosstalk between these nuclear receptors from the point of view of their control of cell growth and show here that RA reverts estrogen-stimulated transcription of the pivotal anti-apoptotic bcl-2 gene by preventing demethylation of dimethyl lysine 9 in histone H3 (HeK9me2). As we previously reported, this is obtained by means of E2-triggered activation of the lysine-specific demethylase 1 (LSD1), an enzyme that manages chromatin plasticity in order to allow specific movements of chromosomal regions within the nucleus. We find that E2 fuels LSD1 by inducing migration of the catalytic subunit of protein kinase A (PKA) into the nucleus, where it targets estrogen-responsive loci. RA rescues LSD1-dependent disappearance of H3K9me2 at bcl-2 regulatory regions upon the prevention of PKA assembly to the same sites. [Copyright &y& Elsevier]

Several ribosomal proteins are down-regulated at the translational, post-transcriptional and transcriptional level in response to abiotic stress, resulting in retardation of growth and productivity in various plants. However, transcriptional mechanisms associated with the stress-mediated down-regulation of such genes have not been well studied. Recently, we reported salt-responsive transcriptional down-regulation of a gene encoding 60S ribosomal protein L32, rpL32_8.1, in rice. In the present work, chromatin remodeling mechanisms associated with down-regulation of this gene under salt stress have been studied. The data obtained from the sodium bisulfite sequencing of a genomic region containing portions of the promoter and 5′-untranslated region of rpL32_8.1 exclude a role for DNA methylation in this down-regulation event. Fine mapping of the first nucleosome after the transcription start site (TSS) revealed its occupancy over the TSS to a greater extent under salt stress than that observed in a controlled environment, possibly causing a hindrance to transcription initiation during its down-regulation. Using chromatin immunoprecipitation, it was observed that histone modifications that are generally associated with highly active genes were markedly reduced under stress conditions at the 5′ end region of this gene under salt stress. By applying a modified chromatin conformation capture (mCCC) technique, promoter–terminator interaction was detected for this gene under control conditions. The efficiency of this interaction was diminished under salt stress. This work indicates that the stress-responsive transcriptional down-regulation of rpL32_8.1 is accompanied by alterations in nucleosome positioning, histone modification and gene looping, but not DNA methylation. [ABSTRACT FROM PUBLISHER]

Abstract: The retention of transcription proteins at an actively transcribed gene contributes to maintenance of the active transcriptional state and increases the rate of subsequent transcription cycles relative to the initial cycle. This process, called transcription reinitiation, generates the abundant RNAs in living cells. The persistence of stable preinitiation intermediates on activated genes representing at least a subset of basal transcription components has long been recognized as a shared feature of RNA polymerase (Pol) I, II and III-dependent transcription in eukaryotes. Studies of the Pol III transcription machinery and its target genes in eukaryotic genomes over the last fifteen years, has uncovered multiple details on transcription reinitiation. In addition to the basal transcription factors that recruit the polymerase, Pol III itself can be retained on the same gene through multiple transcription cycles by a facilitated recycling pathway. The molecular bases for facilitated recycling are progressively being revealed with advances in structural and functional studies. At the same time, progress in our understanding of Pol III transcriptional regulation in response to different environmental cues points to the specific mechanism of Pol III reinitiation as a key target of signaling pathway regulation of cell growth. This article is part of a Special Issue entitled: Transcription by Odd Pols. [Copyright &y& Elsevier]

Abstract: The cycle of eukaryotic transcription, from initiation to elongation and termination is regulated at multiple steps. Coordinated action of regulatory factors keeps in check the transcriptional competence of RNA polymerase II (RNAPII) at different stages. Productive transcription requires the escape of the paused RNAPII from the promoter and transition to rapid elongation of the transcript. Numerous studies have identified diverse mechanisms of initiating transcription by overriding inhibitory signals at the gene promoter. The general theme that has emerged is that the balance between positive and negative regulatory factors determines the overall rate of transcription. Recently transcription termination has emerged as an important area of transcriptional regulation that is coupled with the efficient recycling of RNAPII. The factors associated with transcription termination can also mediate gene looping and thereby determine the efficiency of re-initiation. This review highlights these regulatory steps, the key modulators involved in transcription dynamics, and the emerging tools to analyze them. [Copyright &y& Elsevier]

The transcription cycle of RNAPII is comprised of three consecutive steps; initiation, elongation and termination. It has been assumed that the initiation and termination steps occur in spatial isolation, essentially as independent events. A growing body of evidence, however, has challenged this dogma. First, factors involved in initiation and termination exhibit both a genetic and a physical interaction during transcription. Second, the initiation and termination factors have been found to occupy both ends of a transcribing gene. Third, physical interaction of initiation and termination factors occupying distal ends of a gene sometime results in the entire terminator region of a genes looping back and contact its cognate promoter, thereby forming a looped gene architecture during transcription. A logical interpretation of these findings is that the initiation and termination steps of transcription do not occur in isolation. There is extensive communication of factors occupying promoter and terminator ends of a gene during transcription cycle. This review entails a discussion of the promoter-terminator crosstalk and its implication in the context of transcription. • Promoter and terminator crosstalk during transcription. • Crosstalk involves physical or functional interaction of initiation and termination factors. • Physical interaction of initiation factors at the promoter with the termination factors at the 3′ end of a gene may result in formation of a looped gene architecture. • Gene looping has been implicated in termination, reinitiation, promoter directionality and transcription memory. • Not all promoter-terminator crosstalk, however, results in gene loop formation. [ABSTRACT FROM AUTHOR]

The transcription cycle of RNAPII is comprised of three consecutive steps; initiation, elongation and termination. It has been assumed that the initiation and termination steps occur in spatial isolation, essentially as independent events. A growing body of evidence, however, has challenged this dogma. First, factors involved in initiation and termination exhibit both a genetic and a physical interaction during transcription. Second, the initiation and termination factors have been found to occupy both ends of a transcribing gene. Third, physical interaction of initiation and termination factors occupying distal ends of a gene sometime results in the entire terminator region of a genes looping back and contact its cognate promoter, thereby forming a looped gene architecture during transcription. A logical interpretation of these findings is that the initiation and termination steps of transcription do not occur in isolation. There is extensive communication of factors occupying promoter and terminator ends of a gene during transcription cycle. This review entails a discussion of the promoter-terminator crosstalk and its implication in the context of transcription., (Copyright © 2020 Elsevier B.V. All rights reserved.)

There is increasing evidence suggesting the contribution of non-coding RNAs (ncRNAs) to the phenotypic and physiological complexity of organisms. A novel ncRNA species has been identified near the transcription boundaries of protein-coding genes in eukaryotes, bacteria, and archaea. This review provides a detailed description of these transcription boundary-associated RNAs (TBARs), including their classification. Based on their genomic distribution, TBARs are divided into two major groups: promoter-associated RNAs (PARs) and terminus-associated RNAs (TARs). Depending on the sequence length, each group is further classified into long RNA species (>200 nt) and small RNA species (<200 nt). According to these rules of TBAR classification, divergent ncRNAs with confusing nomenclatures, such as promoter upstream transcripts (PROMPTs), upstream antisense RNAs (uaRNAs), stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), upstream non-coding transcripts (UNTs), transcription start site-associated RNAs (TSSaRNAs), transcription initiation RNAs (tiRNAs), and transcription termination site-associated RNAs (TTSaRNAs), were assigned to specific classes. Although the biogenesis pathways of PARs and TARs have not yet been clearly elucidated, previous studies indicate that some of the PARs have originated either through divergent transcription or via RNA polymerase pausing. Intriguing findings regarding the functional implications of the TBARs such as the long-range "gene looping" model, which explains their role in the transcriptional regulation of protein-coding genes, are also discussed. Altogether, this review provides a comprehensive overview of the current research status of TBARs, which will promote further investigations in this research area.

Functional cross-talk between the promoter and terminator of a gene has long been noted. Promoters and terminators are juxtaposed to form gene loops in several organisms, and gene looping is thought to be involved in transcriptional regulation. The general transcription factor IIB (TFIIB) and the C-terminal domain phosphatase Ssu72, essential factors of the transcription preinitiation complex and the mRNA processing and polyadenylation complex, respectively, are important for gene loop formation. TFIIB and Ssu72 interact both genetically and physically, but the molecular basis of this interaction is not known. Here we present a crystal structure of the core domain of TFIIB in two new conformations that differ in the relative distance and orientation of the two cyclin-like domains. The observed extraordinary conformational plasticity may underlie the binding of TFIIB to multiple transcription factors and promoter DNAs that occurs in distinct stages of transcription, including initiation, reinitiation, and gene looping. We mapped the binding interface of the TFIIB-Ssu72 complex using a series of systematic, structure-guided in vitro binding and site-specific photocross-linking assays. Our results indicate that Ssu72 competes with acidic activators for TFIIB binding and that Ssu72 disrupts an intramolecular TFIIB complex known to impede transcription initiation. We also show that the TFIIB-binding site on Ssu72 overlaps with the binding site of symplekin, a component of the mRNA processing and polyadenylation complex. We propose a hand-off model in which Ssu72 mediates a conformational transition in TFIIB, accounting for the role of Ssu72 in transcription reinitiation, gene looping, and promoter-terminator cross-talk., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)