RESUMEN

Fluctuating environments that consist of regular cycles of co-occurring stress are a common challenge faced by cellular populations. For a population to thrive in constantly changing conditions, an ability to coordinate a rapid cellular response is essential. Here, we identify a mutation conferring an arginine-to-histidine (Arg to His) substitution in the transcription terminator Rho. The rho R109H mutation frequently arose in Escherichia coli populations experimentally evolved under repeated long-term starvation conditions, during which the accumulation of metabolic waste followed by transfer into fresh media results in drastic environmental pH fluctuations associated with feast and famine. Metagenomic sequencing revealed that populations containing the rho mutation also possess putative loss-of-function mutations in ydcI, which encodes a recently characterized transcription factor associated with pH homeostasis. Genetic reconstructions of these mutations show that the rho allele confers plasticity via an alkaline-induced reduction of Rho function that, when found in tandem with a ΔydcI allele, leads to intracellular alkalization and genetic assimilation of Rho mutant function. We further identify Arg to His substitutions at analogous sites in rho alleles from species that regularly experience neutral to alkaline pH fluctuations in their environments. Our results suggest that Arg to His substitutions in Rho may serve to rapidly coordinate complex physiological responses through pH sensing and shed light on how cellular populations use environmental cues to coordinate rapid responses to complex, fluctuating environments.

Asunto(s)

Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Adaptación Fisiológica/genética , Mutación , Terminación de la Transcripción Genética , Regulación Bacteriana de la Expresión Génica , Factor Rho/metabolismo , Factor Rho/genética , Evolución Molecular

RESUMEN

Listeria pathogenicity island 1 (LIPI-1) is a genetic region containing a cluster of genes essential for virulence of the bacterial pathogen Listeria monocytogenes. Main virulence factors in LIPI-1 include long 5' untranslated regions (5'UTRs), among which is Rli51, a small RNA (sRNA) in the 5'UTR of the Zn-metalloprotease-coding mpl. So far, Rli51 function and molecular mechanisms have remained obscure. Here, we show that Rli51 exhibits a dual mechanism of regulation, functioning as a cis- and as a trans-acting sRNA. Under nutrient-rich conditions, rli51-mpl transcription is prematurely terminated, releasing a short 121-nucleotide-long sRNA. Rli51 is predicted to function as a transcription attenuator that can fold into either a terminator or a thermodynamically more stable antiterminator. We show that the sRNA Rli21/RliI binds to a single-stranded RNA loop in Rli51, which is essential to mediate premature transcription termination, suggesting that sRNA binding could stabilize the terminator fold. During intracellular infection, rli51 transcription is increased, which generates a higher abundance of the short Rli51 sRNA and allows for transcriptional read-through into mpl. Comparative intracellular bacterial transcriptomics in rli51-null mutants and the wild-type reference strain EGD-e suggests that Rli51 upregulates iron-scavenging proteins and downregulates virulence factors from LIPI-1. MS2 affinity purification confirmed that Rli51 binds transcripts of the heme-binding protein Lmo2186 and Lmo0937 in vivo. These results prove that Rli51 functions as a trans-acting sRNA in intracellular bacteria. Our research shows a growth condition-dependent mechanism of regulation for Rli51, preventing unintended mpl transcription in extracellular bacteria and regulating genes important for virulence in intracellular bacteria.

Asunto(s)

Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Listeria monocytogenes , ARN Bacteriano , ARN Pequeño no Traducido , Listeria monocytogenes/patogenicidad , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Islas Genómicas/genética , Transcripción Genética , Regiones no Traducidas 5' , Virulencia/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Humanos , Listeriosis/microbiología

RESUMEN

Eukaryotic genomes are widely transcribed by RNA polymerase II (pol II) both within genes and in intergenic regions. POL II elongation complexes comprising the polymerase, the DNA template and nascent RNA transcript must be extremely processive in order to transcribe the longest genes which are over 1 megabase long and take many hours to traverse. Dedicated termination mechanisms are required to disrupt these highly stable complexes. Transcription termination occurs not only at the 3' ends of genes once a full length transcript has been made, but also within genes and in promiscuously transcribed intergenic regions. Termination at these latter positions is termed "premature" because it is not triggered in response to a specific signal that marks the 3' end of a gene, like a polyA site. One purpose of premature termination is to remove polymerases from intergenic regions where they are "not wanted" because they may interfere with transcription of overlapping genes or the progress of replication forks. Premature termination has recently been appreciated to occur at surprisingly high rates within genes where it is speculated to serve regulatory or quality control functions. In this review I summarize current understanding of the different mechanisms of premature termination and its potential functions.

RESUMEN

Binding of the bacterial Rho helicase to nascent transcripts triggers Rho-dependent transcription termination (RDTT) in response to cellular signals that modulate mRNA structure and accessibility of Rho utilization (Rut) sites. Despite the impact of temperature on RNA structure, RDTT was never linked to the bacterial response to temperature shifts. We show that Rho is a central player in the cold-shock response (CSR), challenging the current view that CSR is primarily a posttranscriptional program. We identify Rut sites in 5'-untranslated regions of key CSR genes/operons (cspA, cspB, cspG, and nsrR-rnr-yjfHI) that trigger premature RDTT at 37°C but not at 15°C. High concentrations of RNA chaperone CspA or nucleotide changes in the cspA mRNA leader reduce RDTT efficiency, revealing how RNA restructuring directs Rho to activate CSR genes during the cold shock and to silence them during cold acclimation. These findings establish a paradigm for how RNA thermosensors can modulate gene expression.

Asunto(s)

Regiones no Traducidas 5' , Respuesta al Choque por Frío , Proteínas de Escherichia coli , Escherichia coli , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano , Factor Rho , Factor Rho/metabolismo , Factor Rho/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Respuesta al Choque por Frío/genética , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Terminación de la Transcripción Genética , Frío , Transcripción Genética , Operón , Proteínas y Péptidos de Choque por Frío

RESUMEN

The transcription termination process is an important part of the gene expression process in the cell. It has been studied extensively, but many aspects of the mechanism are not well understood. The widespread availability of experimental RNA-seq data from high-throughput experiments provides a unique opportunity to infer the end of the transcription units genome wide. This data is available for both Rho-dependent and Rho-independent termination pathways that drive transcription termination in bacteria. Our book chapter gives an overview of the current knowledge of Rho-independent transcription termination mechanisms and the prediction approaches currently deployed to infer the termination sites. Thereafter, we describe our method that uses cluster hairpins to detect Rho-independent transcription termination sites. These clusters are a group of hairpins that lies at <15 bp from each other and are together capable of enforcing the termination process. The idea of a group of hairpins being extensively used for transcription termination is new, and results show that at least 52% of the total cases are of this type, while in the remaining cases, a single strong hairpin is capable of driving transcription termination. The reads derived from the RNA-seq data for corresponding bacteria have been used to validate the predicted sites. The predictions that match these RNA-seq derived sites have higher confidence, and we find almost 98% of the predicted sites, including alternate termination sites, to match the RNA-seq data. We discuss the features of predicted hairpins in detail for a better understanding of the Rho-independent transcription termination mechanism in bacteria. We also explain how users can use the tools developed by us to do transcription terminator predictions and design their experiments through genome-level visualization of the transcription termination sites from the precomputed INTERPIN database.

Asunto(s)

RNA-Seq , Terminación de la Transcripción Genética , RNA-Seq/métodos , Programas Informáticos , Biología Computacional/métodos , ARN Bacteriano/genética , Bacterias/genética , Análisis de Secuencia de ARN/métodos , Regiones Terminadoras Genéticas/genética , Regulación Bacteriana de la Expresión Génica

RESUMEN

The regulation of transcription by RNA polymerase II (RNAPII) underpins all cellular processes and is perturbed in thousands of diseases. In humans, RNAPII transcribes ∼20000 protein-coding genes and engages in apparently futile non-coding transcription at thousands of other sites. Despite being so ubiquitous, this transcription is usually attenuated soon after initiation and the resulting products are immediately degraded by the nuclear exosome. We and others have recently described a new complex, "Restrictor", which appears to control such unproductive transcription. Underpinned by the RNA binding protein, ZC3H4, Restrictor curtails unproductive/pervasive transcription genome-wide. Here, we discuss these recent discoveries and speculate on some of the many unknowns regarding Restrictor function and mechanism.

RESUMEN

RNA polymerase II (RNAPII) transcription initiates bidirectionally at many human protein-coding genes. Sense transcription usually dominates and leads to messenger RNA production, whereas antisense transcription rapidly terminates. The basis for this directionality is not fully understood. Here, we show that sense transcriptional initiation is more efficient than in the antisense direction, which establishes initial promoter directionality. After transcription begins, the opposing functions of the endonucleolytic subunit of Integrator, INTS11, and cyclin-dependent kinase 9 (CDK9) maintain directionality. Specifically, INTS11 terminates antisense transcription, whereas sense transcription is protected from INTS11-dependent attenuation by CDK9 activity. Strikingly, INTS11 attenuates transcription in both directions upon CDK9 inhibition, and the engineered recruitment of CDK9 desensitises transcription to INTS11. Therefore, the preferential initiation of sense transcription and the opposing activities of CDK9 and INTS11 explain mammalian promoter directionality.

Asunto(s)

Quinasa 9 Dependiente de la Ciclina , Regiones Promotoras Genéticas , Iniciación de la Transcripción Genética , Humanos , Quinasa 9 Dependiente de la Ciclina/metabolismo , Quinasa 9 Dependiente de la Ciclina/genética , Regulación de la Expresión Génica , Proteínas Nucleares , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Transcripción Genética , Factores de Elongación Transcripcional

RESUMEN

INTS11 and CPSF73 are metal-dependent endonucleases for Integrator and pre-mRNA 3'-end processing, respectively. Here, we show that the INTS11 binding partner BRAT1/CG7044, a factor important for neuronal fitness, stabilizes INTS11 in the cytoplasm and is required for Integrator function in the nucleus. Loss of BRAT1 in neural organoids leads to transcriptomic disruption and precocious expression of neurogenesis-driving transcription factors. The structures of the human INTS9-INTS11-BRAT1 and Drosophila dIntS11-CG7044 complexes reveal that the conserved C terminus of BRAT1/CG7044 is captured in the active site of INTS11, with a cysteine residue directly coordinating the metal ions. Inspired by these observations, we find that UBE3D is a binding partner for CPSF73, and UBE3D likely also uses a conserved cysteine residue to directly coordinate the active site metal ions. Our studies have revealed binding partners for INTS11 and CPSF73 that behave like cytoplasmic chaperones with a conserved impact on the nuclear functions of these enzymes.

Asunto(s)

Núcleo Celular , Citoplasma , Proteínas de Drosophila , Unión Proteica , Humanos , Animales , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Citoplasma/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Endonucleasas/metabolismo , Endonucleasas/genética , Células HEK293 , Neurogénesis/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Dominio Catalítico

RESUMEN

The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3' processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for ANTHESIS PROMOTING FACTOR 1 (APRF1), a homolog of yeast Swd2 and human WDR82, known to regulate RNA polymerase II (RNA Pol II) during transcription termination. APRF1 interacts with TYPE ONE SERINE/THREONINE PROTEIN PHOSPHATASE 4 (TOPP4) (yeast Glc7/human PP1) and LUMINIDEPENDENS (LD), the latter showing structural features found in Ref2/PNUTS, all components of the yeast and human phosphatase module of the CPF 3' end-processing machinery. LD has been shown to co-associate in vivo with the histone H3 K4 demethylase FLOWERING LOCUS D (FLD). This work shows how the APRF1/LD-mediated polyadenylation/termination process influences subsequent rounds of transcription by changing the local chromatin environment at FLC.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Cromatina , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Proteínas de Dominio MADS , ARN Polimerasa II , Terminación de la Transcripción Genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimología , Cromatina/metabolismo , Cromatina/genética , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Histonas/metabolismo , Histonas/genética , Histona Desacetilasas

RESUMEN

BACKGROUND & AIMS: Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown. METHODS: To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro-infected hepatocytes and in chronically infected patients. RESULTS: The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. CONCLUSIONS: Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication. IMPACT AND IMPLICATIONS: HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.

Asunto(s)

ARN Helicasas DEAD-box , Virus de la Hepatitis B , Hepatocitos , ARN Viral , Replicación Viral , Humanos , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Hepatocitos/virología , Hepatocitos/metabolismo , Replicación Viral/genética , ARN Viral/genética , Regulación Viral de la Expresión Génica , Terminación de la Transcripción Genética , Hepatitis B Crónica/virología , Hepatitis B Crónica/genética , Hepatitis B Crónica/metabolismo , Poliadenilación , Transactivadores/genética , Transactivadores/metabolismo

RESUMEN

N6-methyladenosine (m6A) is a crucial RNA modification that regulates diverse biological processes in human cells, but its co-transcriptional deposition and functions remain poorly understood. Here, we identified the RNA helicase DDX21 with a previously unrecognized role in directing m6A modification on nascent RNA for co-transcriptional regulation. DDX21 interacts with METTL3 for co-recruitment to chromatin through its recognition of R-loops, which can be formed co-transcriptionally as nascent transcripts hybridize onto the template DNA strand. Moreover, DDX21's helicase activity is needed for METTL3-mediated m6A deposition onto nascent RNA following recruitment. At transcription termination regions, this nexus of actions promotes XRN2-mediated termination of RNAPII transcription. Disruption of any of these steps, including the loss of DDX21, METTL3, or their enzymatic activities, leads to defective termination that can induce DNA damage. Therefore, we propose that the R-loop-DDX21-METTL3 nexus forges the missing link for co-transcriptional modification of m6A, coordinating transcription termination and genome stability.

Asunto(s)

Adenosina , Adenosina/análogos & derivados , ARN Helicasas DEAD-box , Exorribonucleasas , Inestabilidad Genómica , Metiltransferasas , Estructuras R-Loop , ARN Polimerasa II , Terminación de la Transcripción Genética , Humanos , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Adenosina/metabolismo , Adenosina/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Células HEK293 , Cromatina/metabolismo , Cromatina/genética , Daño del ADN , Células HeLa , ARN/metabolismo , ARN/genética , Transcripción Genética , Metilación de ARN

RESUMEN

We compare the WebGeSTer and INtrinsic transcription TERmination hairPIN (INTERPIN) databases used for intrinsic transcription termination (ITT) site prediction in bacteria. The former deploys inverted nucleotide repeat detection for identification of RNA hairpin, while the latter a pair-potential function - the hairpin energy score evaluation being identical for both. We find INTERPIN more sensitive than WebGeSTer with about 6% and 51% additional predictions for ITTs in chromosomal and plasmid operons, respectively. INTERPIN hairpins are relatively shorter in length with ungapped stem, and even located in AT-rich segments, compared to GC-rich longer hairpins with a gapped stem in WebGeSTer. The GC%, length, and energy score from INTERPIN transcription units (TUs) are best inter-correlated while the lowest energy single hairpins from WebGeSTer, considered suitable for ITT, being the worst. Around 72% TUs from the two databases overlap, and ∼60% of all alternate ITT sites downstream of TUs overlap, of which 65% are cluster hairpins. This helps highlight hairpin features that can be used to identify termination sites in bacteria across different prediction methods. Overall, the pair-potential-function-based hairpins screened appear to be more consistent with the kinetic and thermodynamics processes of ITT known to date.Communicated by Ramaswamy H. Sarma.

RESUMEN

In eukaryotic cells, transcription, translation, and mRNA degradation occur in distinct subcellular regions. How these mRNA processes are organized in bacteria, without employing membrane-bound compartments, remains unclear. Here, we present generalizable principles underlying coordination between these processes in bacteria. In Escherichia coli, we found that co-transcriptional degradation is rare for mRNAs except for those encoding inner membrane proteins, due to membrane localization of the main ribonuclease, RNase E. We further found, by varying ribosome binding sequences, that translation affects mRNA stability not because ribosomes protect mRNA from degradation, but because low translation leads to premature transcription termination in the absence of transcription-translation coupling. Extending our analyses to Bacillus subtilis and Caulobacter crescentus, we established subcellular localization of RNase E (or its homolog) and premature transcription termination in the absence of transcription-translation coupling as key determinants that explain differences in transcriptional and translational coupling to mRNA degradation across genes and species.

RESUMEN

BACKGROUND: Splicing factors are vital for the regulation of RNA splicing, but some have also been implicated in regulating transcription. The underlying molecular mechanisms of their involvement in transcriptional processes remain poorly understood. RESULTS: Here, we describe a direct role of splicing factor RBM22 in coordinating multiple steps of RNA Polymerase II (RNAPII) transcription in human cells. The RBM22 protein widely occupies the RNAPII-transcribed gene locus in the nucleus. Loss of RBM22 promotes RNAPII pause release, reduces elongation velocity, and provokes transcriptional readthrough genome-wide, coupled with production of transcripts containing sequences from downstream of the gene. RBM22 preferentially binds to the hyperphosphorylated, transcriptionally engaged RNAPII and coordinates its dynamics by regulating the homeostasis of the 7SK-P-TEFb complex and the association between RNAPII and SPT5 at the chromatin level. CONCLUSIONS: Our results uncover the multifaceted role of RBM22 in orchestrating the transcriptional program of RNAPII and provide evidence implicating a splicing factor in both RNAPII elongation kinetics and termination control.

Asunto(s)

Factor B de Elongación Transcripcional Positiva , ARN Polimerasa II , Humanos , Cromatina , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN Polimerasa II/metabolismo , Empalme del ARN , Factores de Empalme de ARN/genética , Transcripción Genética , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismo

RESUMEN

Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin is stalled by transcription, the impact of transcription on condensin, which is enriched at highly expressed genes in many species, remains unclear. Using degrons of Rpb1 or the torpedo nuclease Dhp1XRN2 to either deplete or displace RNAPII on chromatin in fission yeast metaphase cells, we show that RNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cis and hinders its ability to fold mitotic chromatin and to support chromosome segregation, consistent with the stalling of a loop extruder. Transcription termination by Dhp1 limits such a hindrance. Our results shed light on the integrated functioning of condensin, and we argue that a tight control of transcription underlies mitotic chromosome assembly by loop-extruding condensin.

Asunto(s)

Adenosina Trifosfatasas , Segregación Cromosómica , Complejos Multiproteicos , Schizosaccharomyces , Proteínas de Unión al ADN/genética , Cromatina , Cromosomas , ADN , Schizosaccharomyces/genética , ARN Polimerasa II/genética , Mitosis , Proteínas de Ciclo Celular/genética

RESUMEN

Introduction: African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that encodes its own host-like RNA polymerase (RNAP) and factors required to produce mature mRNA. The formation of accurate mRNA 3' ends by ASFV RNAP depends on transcription termination, likely enabled by a combination of sequence motifs and transcription factors, although these are poorly understood. The termination of any RNAP is rarely 100% efficient, and the transcriptional "readthrough" at terminators can generate long mRNAs which may interfere with the expression of downstream genes. ASFV transcriptome analyses reveal a landscape of heterogeneous mRNA 3' termini, likely a combination of bona fide termination sites and the result of mRNA degradation and processing. While short-read sequencing (SRS) like 3' RNA-seq indicates an accumulation of mRNA 3' ends at specific sites, it cannot inform about which promoters and transcription start sites (TSSs) directed their synthesis, i.e., information about the complete and unprocessed mRNAs at nucleotide resolution. Methods: Here, we report a rigorous analysis of full-length ASFV transcripts using long-read sequencing (LRS). We systematically compared transcription termination sites predicted from SRS 3' RNA-seq with 3' ends mapped by LRS during early and late infection. Results: Using in-vitro transcription assays, we show that recombinant ASFV RNAP terminates transcription at polyT stretches in the non-template strand, similar to the archaeal RNAP or eukaryotic RNAPIII, unaided by secondary RNA structures or predicted viral termination factors. Our results cement this T-rich motif (U-rich in the RNA) as a universal transcription termination signal in ASFV. Many genes share the usage of the same terminators, while genes can also use a range of terminators to generate transcript isoforms varying enormously in length. A key factor in the latter phenomenon is the highly abundant terminator readthrough we observed, which is more prevalent during late compared with early infection. Discussion: This indicates that ASFV mRNAs under the control of late gene promoters utilize different termination mechanisms and factors to early promoters and/or that cellular factors influence the viral transcriptome landscape differently during the late stages of infection.

Asunto(s)

Virus de la Fiebre Porcina Africana , Porcinos , Animales , Virus de la Fiebre Porcina Africana/genética , Transcripción Genética , ARN Polimerasas Dirigidas por ADN , ARN Mensajero/genética , ARN

RESUMEN

RNA polymerases are the central enzymes of gene expression and function frequently in either a head-on or co-directional manner on the busy DNA track. Whether and how these collisions between RNA polymerases contribute to transcriptional regulation is mysterious. Increasing evidence from biochemical and single-molecule studies suggests that RNA polymerase collisions function as an important regulator to fine-tune transcription, rather than creating deleterious "traffic jams". This review summarizes the recent progress on elucidating the consequences of RNA polymerase collisions during transcription and highlights the significance of cooperation and coordination between RNA polymerases.

Asunto(s)

ARN Polimerasas Dirigidas por ADN , Transcripción Genética , ADN/metabolismo , ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Transcripción Genética/genética

RESUMEN

Clostridioides difficile is an intestinal pathogen that exhibits phase variation of flagella and toxins through inversion of the flagellar (flg) switch controlling flagellar and toxin gene expression. The transcription termination factor Rho preferentially inhibits swimming motility of bacteria with the 'flg-OFF' switch sequence. How C. difficile Rho mediates this selectivity was unknown. C. difficile Rho contains an N-terminal insertion domain (NID) which is found in a subset of Rho orthologues and confers diverse functions. Here we determined how Rho distinguishes between flg-ON and -OFF mRNAs and the roles of the NID and other domains of C. difficile Rho. Using in vitro ATPase assays, we determined that Rho specifically binds a region containing the left inverted repeat of the flg switch, but only of flg-OFF mRNA, indicating that differential termination is mediated by selective Rho binding. Using a suite of in vivo and in vitro assays in C. difficile, we determined that the NID is essential for Rho termination of flg-OFF mRNA, likely by influencing the ability to form stable hexamers, and the RNA binding domain is critical for flg-OFF specific termination. This work gives insight into the novel mechanism by which Rho interacts with flg mRNA to mediate phase variation of flagella and toxins in C. difficile and broadens our understanding of Rho-mediated termination in an organism with an AT-rich genome.

Asunto(s)

Proteínas Bacterianas , Toxinas Bacterianas , Clostridioides difficile , Regulación Bacteriana de la Expresión Génica , Variación de la Fase , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Flagelos/genética , Flagelos/metabolismo , ARN Mensajero/metabolismo

RESUMEN

Bacterial riboswitches are molecular structures that play a crucial role in controlling gene expression to maintain cellular balance. The Escherichia coli lysC riboswitch has been previously shown to regulate gene expression through translation initiation and mRNA decay. Recent research suggests that lysC gene expression is also influenced by Rho-dependent transcription termination. Through a series of in silico, in vitro, and in vivo experiments, we provide experimental evidence that the lysC riboswitch directly and indirectly modulates Rho transcription termination. Our study demonstrates that Rho-dependent transcription termination plays a significant role in the cotranscriptional regulation of lysC expression. Together with previous studies, our work suggests that lysC expression is governed by a lysine-sensing riboswitch that regulates translation initiation, transcription termination, and mRNA degradation. Notably, both Rho and RNase E target the same region of the RNA molecule, implying that RNase E may degrade Rho-terminated transcripts, providing a means to selectively eliminate these incomplete messenger RNAs. Overall, this study sheds light on the complex regulatory mechanisms used by bacterial riboswitches, emphasizing the role of transcription termination in the control of gene expression and mRNA stability.

Asunto(s)

Riboswitch , Riboswitch/genética , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Transcripción Genética , Bacterias/genética , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/metabolismo

RESUMEN

The RNA/DNA helicase senataxin (SETX) has been involved in multiple crucial processes related to genome expression and integrity such us transcription termination, the regulation of transcription-replication conflicts and the resolution of R-loops. SETX has been the focus of numerous studies since the discovery that mutations in its coding gene are the root cause of two different neurodegenerative diseases: Ataxia with Oculomotor Apraxia type 2 (AOA2) and a juvenile form of Amyotrophic Lateral Sclerosis (ALS4). A plethora of cellular phenotypes have been described as the result of SETX deficiency, yet the precise molecular function of SETX as well as the molecular pathways leading from SETX mutations to AOA2 and ALS4 pathologies have remained unclear. However, recent data have shed light onto the biochemical activities and biological roles of SETX, thus providing new clues to understand the molecular consequences of SETX mutation. In this review we summarize near two decades of scientific effort to elucidate SETX function, we discuss strengths and limitations of the approaches and models used thus far to investigate SETX-associated diseases and suggest new possible research avenues for the study of AOA2 and ALS4 pathogenesis.

Asunto(s)

Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Humanos , ARN Helicasas/genética , ARN Helicasas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Enfermedades Neurodegenerativas/genética , Transcripción Genética , Mutación , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismo , ARN