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· 2021 Aug · PMID 34719335
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During transcription, RNA polymerase (RNAP) translocates along the helical template DNA while maintaining high transcriptional fidelity. However, all genomes are dynamically twisted, writhed, and decorated by bound prote...During transcription, RNA polymerase (RNAP) translocates along the helical template DNA while maintaining high transcriptional fidelity. However, all genomes are dynamically twisted, writhed, and decorated by bound proteins and motor enzymes. In prokaryotes, proteins bound to DNA, specifically or not, frequently compact DNA into conformations that may silence genes by obstructing RNAP. Collision of RNAPs with these architectural proteins, may result in RNAP stalling and/or displacement of the protein roadblock. It is important to understand how rapidly transcribing RNAPs operate under different levels of supercoiling or in the presence of roadblocks. Given the broad range of asynchronous dynamics exhibited by transcriptional complexes, single-molecule assays, such as atomic force microscopy, fluorescence detection, optical and magnetic tweezers, etc. are well suited for detecting and quantifying activity with adequate spatial and temporal resolution. Here, we summarize current understanding of the effects of torsion and roadblocks on prokaryotic transcription, with a focus on single-molecule assays that provide real-time detection and readout.
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· 2021 Aug · PMID 34719334
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To exert their functions, RNAs adopt diverse structures, ranging from simple secondary to complex tertiary and quaternary folds. , RNA folding starts with RNA transcription, and a wide variety of processes are coupled to...To exert their functions, RNAs adopt diverse structures, ranging from simple secondary to complex tertiary and quaternary folds. , RNA folding starts with RNA transcription, and a wide variety of processes are coupled to co-transcriptional RNA folding events, including the regulation of fundamental transcription dynamics, gene regulation by mechanisms like attenuation, RNA processing or ribonucleoprotein particle formation. While co-transcriptional RNA folding and associated co-transcriptional processes are by now well accepted as pervasive regulatory principles in all organisms, investigations into the role of the transcription machinery in co-transcriptional folding processes have so far largely focused on effects of the order in which RNA regions are produced and of transcription kinetics. Recent structural and structure-guided functional analyses of bacterial transcription complexes increasingly point to an additional role of RNA polymerase and associated transcription factors in supporting co-transcriptional RNA folding by fostering or preventing strategic contacts to the nascent transcripts. In general, the results support the view that transcription complexes can act as RNA chaperones, a function that has been suggested over 30 years ago. Here, we discuss transcription complexes as RNA chaperones based on recent examples from bacterial transcription.
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· 2021 Aug · PMID 34705601
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Rho is a hexameric bacterial RNA helicase, which became a paradigm of factor-dependent transcription termination. The broadly accepted ("textbook") model posits a series of steps, wherein Rho first binds C-rich () sites...Rho is a hexameric bacterial RNA helicase, which became a paradigm of factor-dependent transcription termination. The broadly accepted ("textbook") model posits a series of steps, wherein Rho first binds C-rich () sites on nascent RNA, uses its ATP-dependent translocase activity to catch up with RNA polymerase (RNAP), and either pulls the transcript from the elongation complex or pushes RNAP forward, thus terminating transcription. However, this appealingly simple mechano-chemical model lacks a biological realism and is increasingly at odds with genetic and biochemical data. Here, we summarize recent structural and biochemical studies that have advanced our understanding of molecular details of RNA recognition, termination signaling, and RNAP inactivation in Rho-dependent transcription termination, rebalancing the view in favor of an alternative "allosteric" mechanism. In the revised model, Rho binds RNAP early in elongation assisted by the cofactors NusA and NusG, forming a pre-termination complex (PTC). The formation of PTC allows Rho to continuously sample nascent transcripts for a termination signal, which subsequently traps the elongation complex in an inactive state prior to its dissociation.
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· 2021 Aug · PMID 34674614
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For survival, bacteria need to continuously evolve and adapt to complex environments, including those that may impact the integrity of the DNA, the repository of genetic information to be passed on to future generations....For survival, bacteria need to continuously evolve and adapt to complex environments, including those that may impact the integrity of the DNA, the repository of genetic information to be passed on to future generations. The multiple factors of DNA repair share the substrate on which they operate with other key cellular machineries, principally those of replication and transcription, implying a high degree of coordination of DNA-based activities. In this review, I focus on progress made in the understanding of the protein factors operating at the crossroads of these three fundamental processes, with emphasis on the protein (Mfd, aka TRCF). Although Mfd research has a rich history that goes back in time for more than half a century, recent reports hint that much remains to be uncovered. I argue that besides being a transcription-repair coupling factor (TRCF), Mfd is also a global regulator of transcription and a pro-mutagenic factor, and that the way it interfaces with transcription, replication and nucleotide excision repair makes it an attractive candidate for the development of strategies to curb molecular evolution, hence, antibiotic resistance.
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· 2021 Aug · PMID 34570660
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Coordination between the molecular machineries that synthesize and decode prokaryotic mRNAs is an important layer of gene expression control known as transcription-translation coupling. While it has long been known that...Coordination between the molecular machineries that synthesize and decode prokaryotic mRNAs is an important layer of gene expression control known as transcription-translation coupling. While it has long been known that translation can regulate transcription and vice-versa, recent structural and biochemical work has shed light on the underlying mechanistic basis. Complexes of RNA polymerase linked to a trailing ribosome (expressomes) have been structurally characterized in a variety of states at near-atomic resolution, and also directly visualized in cells. These data are complemented by recent biochemical and biophysical analyses of transcription-translation systems and the individual components within them. Here, we review our improved understanding of the molecular basis of transcription-translation coupling. These insights are discussed in relation to our evolving understanding of the role of coupling in cells.
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· 2021 Aug · PMID 34499567
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Genome architecture has proven to be critical in determining gene regulation across almost all domains of life. While many of the key components and mechanisms of eukaryotic genome organization have been described, the i...Genome architecture has proven to be critical in determining gene regulation across almost all domains of life. While many of the key components and mechanisms of eukaryotic genome organization have been described, the interplay between bacterial DNA organization and gene regulation is only now being fully appreciated. An increasing pool of evidence has demonstrated that the bacterial chromosome can reasonably be thought of as chromatin, and that bacterial chromosomes contain transcriptionally silent and transcriptionally active regions analogous to heterochromatin and euchromatin, respectively. The roles played by histones in eukaryotic systems appear to be shared across a range of nucleoid-associated proteins (NAPs) in bacteria, which function to compact, structure, and regulate large portions of bacterial chromosomes. The broad range of extant NAPs, and the extent to which they differ from species to species, has raised additional challenges in identifying and characterizing their roles in all but a handful of model bacteria. Here we review the regulatory roles played by NAPs in several well-studied bacteria and use the resulting state of knowledge to provide a working definition for NAPs, based on their function, binding pattern, and expression levels. We present a screening procedure which can be applied to any species for which transcriptomic data are available. Finally, we note that NAPs tend to play two major regulatory roles - xenogeneic silencers and developmental regulators - and that many unrecognized potential NAPs exist in each bacterial species examined.
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· 2021 Aug · PMID 34486930
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Bacteria in most natural environments spend substantial periods of time limited for essential nutrients and not actively dividing. While transcriptional activity under these conditions is substantially reduced compared t...Bacteria in most natural environments spend substantial periods of time limited for essential nutrients and not actively dividing. While transcriptional activity under these conditions is substantially reduced compared to that occurring during active growth, observations from diverse organisms and experimental approaches have shown that new transcription still occurs and is important for survival. Much of our understanding of transcription regulation has come from measuring transcripts in exponentially growing cells, or from experiments focused on transcription from highly active promoters by the housekeeping RNA polymerase holoenzyme. The fact that transcription during growth arrest occurs at low levels and is highly heterogeneous has posed challenges for its study. However, new methods of measuring low levels of gene expression activity, even in single cells, offer exciting opportunities for directly investigating transcriptional activity and its regulation during growth arrest. Furthermore, much of the rich structural and biochemical data from decades of work on the bacterial transcriptional machinery is also relevant to growth arrest. In this review, the physiological changes likely affecting transcription during growth arrest are first considered. Next, possible adaptations to help facilitate ongoing transcription during growth arrest are discussed. Finally, new insights from several recently published datasets investigating mRNA transcripts in single bacterial cells at various growth phases will be explored. Keywords: Growth arrest, stationary phase, RNA polymerase, nucleoid condensation, population heterogeneity.
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· 2021 Aug · PMID 34403307
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The low G + C Gram-positive bacteria represent some of the most medically and industrially important microorganisms. They are relied on for the production of food and dietary supplements, enzymes and antibiotics, as well...The low G + C Gram-positive bacteria represent some of the most medically and industrially important microorganisms. They are relied on for the production of food and dietary supplements, enzymes and antibiotics, as well as being responsible for the majority of nosocomial infections and serving as a reservoir for antibiotic resistance. Control of gene expression in this group is more highly studied than in any bacteria other than the Gram-negative model Escherichia coli, yet until recently no structural information on RNA polymerase (RNAP) from this group was available. This review will summarize recent reports on the high-resolution structure of RNAP from the model low G + C representative Bacillus subtilis, including the role of auxiliary subunits and , and outline approaches for the development of antimicrobials to target RNAP from this group.
The N-terminal methyltransferase NRMT1 is an important regulator of protein/DNA interactions and plays a role in many cellular processes, including mitosis, cell cycle progression, chromatin organization, DNA damage repa...The N-terminal methyltransferase NRMT1 is an important regulator of protein/DNA interactions and plays a role in many cellular processes, including mitosis, cell cycle progression, chromatin organization, DNA damage repair, and transcriptional regulation. Accordingly, loss of NRMT1 results in both developmental pathologies and oncogenic phenotypes. Though NRMT1 plays such important and diverse roles in the cell, little is known about its own regulation. To better understand the mechanisms governing NRMT1 expression, we first identified its predominant transcriptional start site and minimal promoter region with predicted transcription factor motifs. We then used a combination of luciferase and binding assays to confirm CREB1 as the major regulator of NRMT1 transcription. We tested which conditions known to activate CREB1 also activated NRMT1 transcription, and found CREB1-mediated NRMT1 expression was increased during recovery from serum starvation and muscle cell differentiation. To determine how NRMT1 expression affects myoblast differentiation, we used CRISPR/Cas9 technology to knock out NRMT1 expression in immortalized C2C12 mouse myoblasts. C2C12 cells depleted of NRMT1 lacked expression and were unable to proceed down the muscle differentiation pathway. Instead, they took on characteristics of C2C12 cells that have transdifferentiated into osteoblasts, including increased alkaline phosphatase and type I collagen expression and decreased proliferation. These data implicate NRMT1 as an important downstream target of CREB1 during muscle cell differentiation.
Recent studies have identified multiple polyadenylation sites in nearly all mammalian genes. Although these are interpreted as evidence for alternative polyadenylation, our knowledge of the underlying mechanisms is still...Recent studies have identified multiple polyadenylation sites in nearly all mammalian genes. Although these are interpreted as evidence for alternative polyadenylation, our knowledge of the underlying mechanisms is still limited. Most studies only consider the immediate surroundings of gene ends, even though experiments have uncovered the involvement of external factors such as splicing. Whereas splicing manipulation was impracticable until recently, we now used mutants in the () gene to study their impact on 3' end processing. We observe multiple rounds of readthrough and gene fusions, suggesting that no arbitration between polyadenylation sites occurs. Instead, a window of opportunity seems to control end processing. Through the identification of T-rich sequence motifs, our data indicate that splicing and transcriptional pausing interact to regulate alternative polyadenylation. We propose that 3' splice site activation comprises a variable timer, which determines how long transcription proceeds before polyadenylation signals are recognized. Thus, the role of core polyadenylation signals could be more passive than commonly believed. Our results provide new insights into the mechanisms of alternative polyadenylation and expand the catalog of related aberrations. APA: alternative polyadenylation; bp: basepair; MEF: mouse embryonic fibroblasts; PA: polyadenylation; PAS: polyadenylation site; Pol II: (RNA) polymerase II ; RT-PCR:reverse-transcriptase PCR; SF:splicing factor; SFPQ:splicing factor rich in proline and glutamine; SS:splice site; TRSM:Thymidine rich sequence motif; UTR:untranslated terminal region.
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· 2021 Feb · PMID 34036896
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Noncoding RNAs are proclaimed to be expressed in various cancer types and one such type is found to be pancreatic ductal adenocarcinoma (PDAC). The long noncoding RNAs (LncRNAs) affect the migration, invasion, and growth...Noncoding RNAs are proclaimed to be expressed in various cancer types and one such type is found to be pancreatic ductal adenocarcinoma (PDAC). The long noncoding RNAs (LncRNAs) affect the migration, invasion, and growth of tumor cells by playing important roles in the process of epigenesis, post-transcription, and transcriptional regulation along with the maintenance of apoptosis and cell cycle. It is quite subtle whether the alterations in lncRNAs would impact PDAC progression and development. This review throws a spotlight on the lncRNAs associated with tumor functions: MALAT-1, HOTAIR, HOXA13, H19, LINC01559, LINC00460, SNHG14, SNHG16, DLX6-AS1, MSC-AS1, ABHD11-AS1, DUXAP8, DANCR, XIST, DLEU2, etc. are upregulated lncRNAs whereas GAS5, HMlincRNA717, MIAT, LINC01111, lncRNA KCNK15-AS1, etc. are downregulated lncRNAs inhibiting the invasion and progression of PDAC. These data provided helps in the assessment of lncRNAs in the development, metastasis, and occurrence of PDAC and also play a vital role in the evolution of biomarkers and therapeutic agents for the treatment of PDAC.
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· 2021 Feb · PMID 34000965
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Diverse models have been advanced for the evolution of the genetic code. Here, models for tRNA, aminoacyl-tRNA synthetase (aaRS) and genetic code evolution were combined with an understanding of EF-Tu suppression of tRNA...Diverse models have been advanced for the evolution of the genetic code. Here, models for tRNA, aminoacyl-tRNA synthetase (aaRS) and genetic code evolution were combined with an understanding of EF-Tu suppression of tRNA 3 anticodon position wobbling. The result is a highly detailed scheme that describes the placements of all amino acids in the standard genetic code. The model describes evolution of 6-, 4-, 3-, 2- and 1-codon sectors. Innovation in column 3 of the code is explained. Wobbling and code degeneracy are explained. Separate distribution of serine sectors between columns 2 and 4 of the code is described. We conclude that very little chaos contributed to evolution of the genetic code and that the pattern of evolution of aaRS enzymes describes a history of the evolution of the code. A model is proposed to describe the biological selection for the earliest evolution of the code and for protocell evolution.
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· 2021 Feb · PMID 33622180
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The innate immune system has numerous signal transduction pathways that lead to the production of type I interferons in response to exposure of cells to external stimuli. One of these pathways comprises RNA polymerase (P...The innate immune system has numerous signal transduction pathways that lead to the production of type I interferons in response to exposure of cells to external stimuli. One of these pathways comprises RNA polymerase (Pol) III that senses common DNA viruses, such as cytomegalovirus, vaccinia, herpes simplex virus-1 and varicella zoster virus. This polymerase detects and transcribes viral genomic regions to generate AU-rich transcripts that bring to the induction of type I interferons. Remarkably, Pol III is also stimulated by foreign non-viral DNAs and expression of one of its subunits is induced by an RNA virus, the Sindbis virus. Moreover, a protein subunit of RNase P, which is known to associate with Pol III in initiation complexes, is induced by viral infection. Accordingly, alliance of the two tRNA enzymes in innate immunity merits a consideration.
Multisubunit RNA polymerase (Pol) complexes are the core machinery for gene expression in eukaryotes. The enzymes Pol I, Pol II and Pol III transcribe distinct subsets of nuclear genes. This family of nuclear RNA polymer...Multisubunit RNA polymerase (Pol) complexes are the core machinery for gene expression in eukaryotes. The enzymes Pol I, Pol II and Pol III transcribe distinct subsets of nuclear genes. This family of nuclear RNA polymerases expanded in terrestrial plants by the duplication of Pol II subunit genes. Two Pol II-related enzymes, Pol IV and Pol V, are highly specialized in the production of regulatory, non-coding RNAs. Pol IV and Pol V are the central players of RNA-directed DNA methylation (RdDM), an RNA interference pathway that represses transposable elements (TEs) and selected genes. Genetic and biochemical analyses of Pol IV/V subunits are now revealing how these enzymes evolved from ancestral Pol II to sustain non-coding RNA biogenesis in silent chromatin. Intriguingly, Pol IV-RdDM regulates genes that influence flowering time, reproductive development, stress responses and plant-pathogen interactions. Pol IV target genes vary among closely related taxa, indicating that these regulatory circuits are often species-specific. Data from crops like maize, rice, tomato and suggest that dynamic repositioning of TEs, accompanied by Pol IV targeting to TE-proximal genes, leads to the reprogramming of plant gene expression over short evolutionary timescales.
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· 2020 Oct · PMID 33151112
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A large number of distal -regulatory elements (REs) have been annotated in the human genome, which plays a central role in orchestrating spatiotemporal gene expression. Since many REs regulate non-adjacent genes, long-ra...A large number of distal -regulatory elements (REs) have been annotated in the human genome, which plays a central role in orchestrating spatiotemporal gene expression. Since many REs regulate non-adjacent genes, long-range RE-promoter interactions are an important factor in the functional characterization of the engaged REs. In this regard, recent studies have demonstrated that identification of long-range target genes can decipher the effect of genetic mutations residing within REs on abnormal gene expression. In addition, investigation of altered long-range REs-promoter interactions induced by chromosomal rearrangements has revealed their critical roles in pathogenic gene expression. In this review, we briefly discuss how the analysis of 3D chromatin structure can help us understand the functional impact of REs harboring disease-associated genetic variants and how chromosomal rearrangements disrupting topologically associating domains can lead to pathogenic gene expression.
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· 2020 Oct · PMID 33112729
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Increasingly sophisticated biochemical and genetic techniques are unraveling the regulatory factors and mechanisms that control gene expression in the Archaea. While some similarities in regulatory strategies are univers...Increasingly sophisticated biochemical and genetic techniques are unraveling the regulatory factors and mechanisms that control gene expression in the Archaea. While some similarities in regulatory strategies are universal, archaeal-specific regulatory strategies are emerging to complement a complex patchwork of shared archaeal-bacterial and archaeal-eukaryotic regulatory mechanisms employed in the archaeal domain. The prokaryotic archaea encode core transcription components with homology to the eukaryotic transcription apparatus and also share a simplified eukaryotic-like initiation mechanism, but also deploy tactics common to bacterial systems to regulate promoter usage and influence elongation-termination decisions. We review the recently established complete archaeal transcription cycle, highlight recent findings of the archaeal transcription community and detail the expanding post-initiation regulation imposed on archaeal transcription.
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· 2020 Oct · PMID 33054514
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Chromatin accessibility is generally perceived as a common property of active regulatory elements where transcription factors are recruited via DNA-specific interactions and other physico-chemical properties to regulate...Chromatin accessibility is generally perceived as a common property of active regulatory elements where transcription factors are recruited via DNA-specific interactions and other physico-chemical properties to regulate gene transcription. Recent work in the context of mitosis provides less trivial and potentially more interesting relationships than previously anticipated.
Plants have adapted to tolerate and survive constantly changing environmental conditions by reprogramming gene expression in response to stress or to drive developmental transitions. Among the many signals that plants pe...Plants have adapted to tolerate and survive constantly changing environmental conditions by reprogramming gene expression in response to stress or to drive developmental transitions. Among the many signals that plants perceive, light and temperature are of particular interest due to their intensely fluctuating nature which is combined with a long-term seasonal trend. Whereas specific receptors are key in the light-sensing mechanism, the identity of plant thermosensors for high and low temperatures remains far from fully addressed. This review aims at discussing common as well as divergent characteristics of gene expression regulation in plants, controlled by light and temperature. Light and temperature signaling control the abundance of specific transcription factors, as well as the dynamics of co-transcriptional processes such as RNA polymerase elongation rate and alternative splicing patterns. Additionally, sensing both types of cues modulates gene expression by altering the chromatin landscape and through the induction of long non-coding RNAs (lncRNAs). However, while light sensing is channeled through dedicated receptors, temperature can broadly affect chemical reactions inside plant cells. Thus, direct thermal modifications of the transcriptional machinery add another level of complexity to plant transcriptional regulation. Besides the rapid transcriptome changes that follow perception of environmental signals, plant developmental transitions and acquisition of stress tolerance depend on long-term maintenance of transcriptional states (active or silenced genes). Thus, the rapid transcriptional response to the signal (Phase I) can be distinguished from the long-term memory of the acquired transcriptional state (Phase II - remembering the signal). In this review we discuss recent advances in light and temperature signal perception, integration and memory in , focusing on transcriptional regulation and highlighting the contrasting and unique features of each type of cue in the process.
Most living organisms possess an internal timekeeping mechanism known as the circadian clock, which enhances fitness by synchronizing the internal timing of biological processes with diurnal and seasonal environmental ch...Most living organisms possess an internal timekeeping mechanism known as the circadian clock, which enhances fitness by synchronizing the internal timing of biological processes with diurnal and seasonal environmental changes. In plants, the pace of these biological rhythms relies on oscillations in the expression level of hundreds of genes tightly controlled by a group of core clock regulators and co-regulators that engage in transcriptional and translational feedback loops. In the last decade, the role of several core clock genes in the control of defense responses has been addressed, and a growing amount of evidence demonstrates that circadian regulation is relevant for plant immunity. A reciprocal connection between these pathways was also established following the observation that in , as well as in crop species like tomato, plant-pathogen interactions trigger a reconfiguration of the circadian transcriptional network. In this review, we summarize the current knowledge regarding the interaction between the circadian clock and biotic stress responses at the transcriptional level, and discuss the relevance of this crosstalk in the plant-pathogen evolutionary arms race. A better understanding of these processes could aid in the development of genetic tools that improve traditional breeding practices, enhancing tolerance to plant diseases that threaten crop yield and food security all around the world.