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2.
Nat Commun ; 12(1): 5523, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535646

RESUMO

RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.


Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Vírus/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Microscopia Crioeletrônica , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Fatores de Tempo , Proteínas Virais/metabolismo , Viroides/metabolismo
3.
Nat Commun ; 12(1): 5524, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535658

RESUMO

Recruitment of RNA polymerase and initiation factors to the promoter is the only known target for transcription activation and repression in archaea. Whether any of the subsequent steps towards productive transcription elongation are involved in regulation is not known. We characterised how the basal transcription machinery is distributed along genes in the archaeon Saccharolobus solfataricus. We discovered a distinct early elongation phase where RNA polymerases sequentially recruit the elongation factors Spt4/5 and Elf1 to form the transcription elongation complex (TEC) before the TEC escapes into productive transcription. TEC escape is rate-limiting for transcription output during exponential growth. Oxidative stress causes changes in TEC escape that correlate with changes in the transcriptome. Our results thus establish that TEC escape contributes to the basal promoter strength and facilitates transcription regulation. Impaired TEC escape coincides with the accumulation of initiation factors at the promoter and recruitment of termination factor aCPSF1 to the early TEC. This suggests two possible mechanisms for how TEC escape limits transcription, physically blocking upstream RNA polymerases during transcription initiation and premature termination of early TECs.


Assuntos
Regiões Promotoras Genéticas , Sulfolobus solfataricus/genética , Elongação da Transcrição Genética , Sistemas CRISPR-Cas/genética , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Estresse Oxidativo/genética , Análise de Regressão , Sulfolobus solfataricus/crescimento & desenvolvimento
4.
Nat Commun ; 12(1): 4951, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400637

RESUMO

The polyadenosine tail (poly[A]-tail) is a universal modification of eukaryotic messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). In budding yeast, Pap1-synthesized mRNA poly(A) tails enhance export and translation, whereas Trf4/5-mediated polyadenylation of ncRNAs facilitates degradation by the exosome. Using direct RNA sequencing, we decipher the extent of poly(A) tail dynamics in yeast defective in all relevant exonucleases, deadenylases, and poly(A) polymerases. Predominantly ncRNA poly(A) tails are 20-60 adenosines long. Poly(A) tails of newly transcribed mRNAs are 50 adenosine long on average, with an upper limit of 200. Exonucleolysis by Trf5-assisted nuclear exosome and cytoplasmic deadenylases trim the tails to 40 adenosines on average. Surprisingly, PAN2/3 and CCR4-NOT deadenylase complexes have a large pool of non-overlapping substrates mainly defined by expression level. Finally, we demonstrate that mRNA poly(A) tail length strongly responds to growth conditions, such as heat and nutrient deprivation.


Assuntos
Poli A/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Exossomos/metabolismo , Poliadenilação , Polinucleotídeo Adenililtransferase/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Nucleic Acids Res ; 49(15): 8777-8784, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365509

RESUMO

Transcribing RNA polymerase (RNAP) can fall into backtracking, phenomenon when the 3' end of the transcript disengages from the template DNA. Backtracking is caused by sequences of the nucleic acids or by misincorporation of erroneous nucleotides. To resume productive elongation backtracked complexes have to be resolved through hydrolysis of RNA. There is currently no consensus on the mechanism of catalysis of this reaction by Escherichia coli RNAP. Here we used Salinamide A, that we found inhibits RNAP catalytic domain Trigger Loop (TL), to show that the TL is required for RNA cleavage during proofreading of misincorporation events but plays little role during cleavage in sequence-dependent backtracked complexes. Results reveal that backtracking caused by misincorporation is distinct from sequence-dependent backtracking, resulting in different conformations of the 3' end of RNA within the active center. We show that the TL is required to transfer the 3' end of misincorporated transcript from cleavage-inefficient 'misincorporation site' into the cleavage-efficient 'backtracked site', where hydrolysis takes place via transcript-assisted catalysis and is largely independent of the TL. These findings resolve the controversy surrounding mechanism of RNA hydrolysis by E. coli RNA polymerase and indicate that the TL role in RNA cleavage has diverged among bacteria.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , RNA Mensageiro/metabolismo , Elongação da Transcrição Genética , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrólise , Clivagem do RNA
6.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445278

RESUMO

Epigenetics play a vital role in early embryo development. Offspring conceived via assisted reproductive technologies (ARTs) have a three times higher risk of epigenetic diseases than naturally conceived children. However, investigations into ART-associated placental histone modifications or sex-stratified analyses of ART-associated histone modifications remain limited. In the current study, we carried out immunohistochemistry, chip-sequence analysis, and a series of in vitro experiments. Our results demonstrated that placentas from intra-cytoplasmic sperm injection (ICSI), but not in vitro fertilization (IVF), showed global tri-methylated-histone-H3-lysine-4 (H3K4me3) alteration compared to those from natural conception. However, for acetylated-histone-H3-lysine-9 (H3K9ac) and acetylated-histone-H3-lysine-27 (H3K27ac), no significant differences between groups could be found. Further, sex -stratified analysis found that, compared with the same-gender newborn cord blood mononuclear cell (CBMC) from natural conceptions, CBMC from ICSI-boys presented more genes with differentially enriched H3K4me3 (n = 198) than those from ICSI-girls (n = 79), IVF-girls (n = 5), and IVF-boys (n = 2). We also found that varying oxygen conditions, RNA polymerase II subunit A (Polr2A), and lysine demethylase 5A (KDM5A) regulated H3K4me3. These findings revealed a difference between IVF and ICSI and a difference between boys and girls in H3K4me3 modification, providing greater insight into ART-associated epigenetic alteration.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Epigênese Genética , Histonas/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Caracteres Sexuais , Injeções de Esperma Intracitoplásmicas , Adulto , Feminino , Humanos , Recém-Nascido , Masculino , Metilação , Gravidez
7.
J Virol ; 95(20): e0059221, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34379509

RESUMO

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Vírus da Estomatite Vesicular Indiana/genética , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Encéfalo/virologia , COVID-19/imunologia , Linhagem Celular , Síndrome da Liberação de Citocina/prevenção & controle , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Imunogenicidade da Vacina , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Mesocricetus , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Th1/imunologia , Vacinas Sintéticas/imunologia , Vírus da Estomatite Vesicular Indiana/enzimologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
8.
Biomolecules ; 11(7)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206477

RESUMO

The coordination of bacterial genomic transcription involves an intricate network of interdependent genes encoding nucleoid-associated proteins (NAPs), DNA topoisomerases, RNA polymerase subunits and modulators of transcription machinery. The central element of this homeostatic regulatory system, integrating the information on cellular physiological state and producing a corresponding transcriptional response, is the multi-subunit RNA polymerase (RNAP) holoenzyme. In this review article, we argue that recent observations revealing DNA topoisomerases and metabolic enzymes associated with RNAP supramolecular complex support the notion of structural coupling between transcription machinery, DNA topology and cellular metabolism as a fundamental device coordinating the spatiotemporal genomic transcription. We analyse the impacts of various combinations of RNAP holoenzymes and global transcriptional regulators such as abundant NAPs, on genomic transcription from this viewpoint, monitoring the spatiotemporal patterns of couplons-overlapping subsets of the regulons of NAPs and RNAP sigma factors. We show that the temporal expression of regulons is by and large, correlated with that of cognate regulatory genes, whereas both the spatial organization and temporal expression of couplons is distinctly impacted by the regulons of NAPs and sigma factors. We propose that the coordination of the growth phase-dependent concentration gradients of global regulators with chromosome configurational dynamics determines the spatiotemporal patterns of genomic expression.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/metabolismo , Transcrição Genética , Bactérias/genética , Proteínas de Bactérias/genética , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Fatores de Transcrição/genética
9.
Nat Commun ; 12(1): 4503, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301927

RESUMO

Promoter-proximal pausing of RNA polymerase II is a key process regulating gene expression. In latent HIV-1 cells, it prevents viral transcription and is essential for latency maintenance, while in acutely infected cells the viral factor Tat releases paused polymerase to induce viral expression. Pausing is fundamental for HIV-1, but how it contributes to bursting and stochastic viral reactivation is unclear. Here, we performed single molecule imaging of HIV-1 transcription. We developed a quantitative analysis method that manages multiple time scales from seconds to days and that rapidly fits many models of promoter dynamics. We found that RNA polymerases enter a long-lived pause at latent HIV-1 promoters (>20 minutes), thereby effectively limiting viral transcription. Surprisingly and in contrast to current models, pausing appears stochastic and not obligatory, with only a small fraction of the polymerases undergoing long-lived pausing in absence of Tat. One consequence of stochastic pausing is that HIV-1 transcription occurs in bursts in latent cells, thereby facilitating latency exit and providing a rationale for the stochasticity of viral rebounds.


Assuntos
Regulação Viral da Expressão Gênica , Infecções por HIV/genética , HIV-1/genética , Regiões Promotoras Genéticas/genética , Latência Viral/genética , Algoritmos , RNA Polimerases Dirigidas por DNA/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Células HeLa , Humanos , Modelos Genéticos , Processos Estocásticos , Fatores de Tempo , Ativação Viral/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
10.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202438

RESUMO

The chloroplast is a semi-autonomous organelle with its own genome. The expression of chloroplast genes depends on both chloroplasts and the nucleus. Although many nucleus-encoded proteins have been shown to localize in chloroplasts and are essential for chloroplast gene expression, it is not clear whether transcription factors can regulate gene expression in chloroplasts. Here we report that the transcription factor NAC102 localizes in both chloroplasts and nucleus in Arabidopsis. Specifically, NAC102 localizes in chloroplast nucleoids. Yeast two-hybrid assay and co-immunoprecipitation assay suggested that NAC102 interacts with chloroplast RNA polymerases. Furthermore, overexpression of NAC102 in chloroplasts leads to reduced chloroplast gene expression and chlorophyll content, indicating that NAC102 functions as a repressor in chloroplasts. Our study not only revealed that transcription factors are new regulators of chloroplast gene expression, but also discovered that transcription factors can function in chloroplasts in addition to the canonical organelle nucleus.


Assuntos
Cloroplastos/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Cloroplastos , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Ligação Proteica , Transporte Proteico
11.
Genes Cells ; 26(8): 611-626, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34081835

RESUMO

Serum/glucocorticoid-regulated kinase 1 (SGK1) is predominantly expressed in endothelial cells of mouse embryos, and Sgk1 null mice show embryonic lethality due to impaired vascular formation. However, how the SGK1 expression is controlled in developing vasculature remains unknown. In this study, we first identified a proximal endothelial enhancer through lacZ reporter mouse analyses. The mouse Sgk1 proximal enhancer was narrowed down to the 5' region of the major transcription initiation site, while a human corresponding region possessed relatively weak activity. We then searched for distal enhancer candidates using in silico analyses of publicly available databases for DNase accessibility, RNA polymerase association and chromatin modification. A region approximately 500 kb distant from the human SGK1 gene was conserved in the mouse, and the mouse and human genomic fragments drove transcription restricted to embryonic endothelial cells. Minimal fragments of both proximal and distal enhancers had consensus binding elements for the ETS transcription factors, which were essential for the responsiveness to ERG, FLI1 and ETS1 proteins in luciferase assays and the endothelial lacZ reporter expression in mouse embryos. These results suggest that endothelial SGK1 expression in embryonic vasculature is maintained through at least two ETS-regulated enhancers located in the proximal and distal regions.


Assuntos
Endotélio Vascular/metabolismo , Elementos Facilitadores Genéticos , Proteínas Imediatamente Precoces/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Cromatina/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/embriologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Camundongos , Proteínas Oncogênicas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Sítio de Iniciação de Transcrição , Regulador Transcricional ERG/metabolismo
12.
Nucleic Acids Res ; 49(13): 7732-7739, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34181731

RESUMO

Bacteriophage ΦKZ (PhiKZ) is the archetype of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. ΦKZ is an incredibly interesting virus, expressing many systems that the host already possesses. On infection, it forms a 'nucleus', erecting a barrier around its genome to exclude host endonucleases and CRISPR-Cas systems. ΦKZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. ΦKZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial ß/ß' subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of ΦKZ nvRNAP to better than 3.0 Å, shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, the position, structural homology and biochemical role of which imply that it has evolved from an ancestral homologue to σ-factor.


Assuntos
RNA Polimerases Dirigidas por DNA/química , Fagos de Pseudomonas/enzimologia , Proteínas Virais/química , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Regiões Promotoras Genéticas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Virais/metabolismo
13.
J Mol Biol ; 433(15): 167100, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34119489

RESUMO

Bacterial NusG associates with RNA polymerase (RNAP) through its N-terminal domain, while the C-terminal domain (CTD) forms dynamic interactions with Rho, S10, NusB and NusA to affect transcription elongation. While virtually all bacteria encode for a core NusG, many also synthesize paralogs that transiently bind RNAP to alter expression of targeted genes. Yet, despite the importance of the genes they regulate, most of the subfamilies of NusG paralogs (e.g., UpxY, TaA, ActX and LoaP) have not been investigated in depth. Herein, we discover that LoaP requires a small RNA hairpin located within the 5' leader region of its targeted operons. LoaP binds the RNA element with nanomolar affinity and high specificity, in contrast to other NusG proteins, which have not been shown to exhibit RNA-binding activity. These data reveal a sequence feature that can be used to identify LoaP-regulated operons. This discovery also expands the repertoire of macromolecular interactions exhibited by the NusG CTD during transcription elongation to include an RNA ligand.


Assuntos
Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Regiões 5' não Traduzidas , Bacillus/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Conformação Molecular , Óperon , Domínios Proteicos , RNA Bacteriano/química
14.
Nucleic Acids Res ; 49(12): 7088-7102, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34157109

RESUMO

RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase-α, ß, ß'. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the ß or ß' subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed a severe decrease in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Endorribonucleases/fisiologia , RNA Mensageiro/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Endorribonucleases/genética , Evolução Molecular , Deleção de Genes , Duplicação Gênica , Genes Bacterianos , Homeostase , Mutação , Supressão Genética , Transcrição Genética , Transcriptoma
15.
Nat Commun ; 12(1): 3367, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099688

RESUMO

In plants, RNA-directed DNA methylation (RdDM) is a well-known de novo DNA methylation pathway that involves two plant-specific RNA polymerases, Pol IV and Pol V. In this study, we discovered and characterized an RdDM factor, RDM15. Through DNA methylome and genome-wide siRNA analyses, we show that RDM15 is required for RdDM-dependent DNA methylation and siRNA accumulation at a subset of RdDM target loci. We show that RDM15 contributes to Pol V-dependent downstream siRNA accumulation and interacts with NRPE3B, a subunit specific to Pol V. We also show that the C-terminal tudor domain of RDM15 specifically recognizes the histone 3 lysine 4 monomethylation (H3K4me1) mark. Structure analysis of RDM15 in complex with the H3K4me1 peptide showed that the RDM15 tudor domain specifically recognizes the monomethyllysine through an aromatic cage and a specific hydrogen bonding network; this chemical feature-based recognition mechanism differs from all previously reported monomethyllysine recognition mechanisms. RDM15 and H3K4me1 have similar genome-wide distribution patterns at RDM15-dependent RdDM target loci, establishing a link between H3K4me1 and RDM15-mediated RdDM in vivo. In summary, we have identified and characterized a histone H3K4me1-specific binding protein as an RdDM component, and structural analysis of RDM15 revealed a chemical feature-based lower methyllysine recognition mechanism.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Metilação de DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Histonas/metabolismo , RNA Interferente Pequeno/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Regulação da Expressão Gênica de Plantas , Lisina/metabolismo , Metilação , Plantas Geneticamente Modificadas , Ligação Proteica , Conformação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Sequenciamento Completo do Genoma/métodos
16.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069057

RESUMO

Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity.


Assuntos
Bacteriófago T7/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Mutação , Nucleotídeos/análise , Moldes Genéticos , Proteínas Virais/metabolismo , Sistema Livre de Células , RNA Polimerases Dirigidas por DNA/genética , Transcrição Genética , Proteínas Virais/genética
17.
RNA ; 27(9): 1017-1024, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34131025

RESUMO

Connections between distinct catalytic RNA motifs through networks of mutations that retain catalytic function (neutral networks) were likely central to the evolution of biocatalysis. Despite suggestions that functional RNAs collectively form an interconnected web of neutral networks, little evidence has emerged to demonstrate the existence of such intersecting networks in naturally occurring RNAs. Here we show that neutral networks of two naturally occurring, seemingly unrelated endonucleolytic ribozymes, the hammerhead (HH) and hairpin (HP), intersect. Sequences at the intersection of these networks exhibit catalytic functions corresponding to both ribozymes by potentially populating both catalytic folds and enable a smooth crossover between the two. Small and structurally simple endonucleolytic motifs like the HH ribozyme could, through mutational walks along their neutral networks, encounter novel catalytic phenotypes, and structurally flexible, bifunctional sequences at the intersection of these networks could have acted as nodes for evolutionary diversification in an RNA world. Considering the simplicity and small size of the HH ribozyme, we propose that this self-cleaving motif could have been a precursor to other more complex endonucleolytic ribozymes. More generally, our results suggest that RNAs that possess distinct sequences, structures, and catalytic functions, can potentially share evolutionary history through mutational connections in sequence space.


Assuntos
DNA/genética , Sequências Repetidas Invertidas , RNA Catalítico/metabolismo , Transcrição Genética , Pareamento de Bases , Biocatálise , Sistema Livre de Células , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Radioisótopos de Fósforo , Mutação Puntual , RNA Catalítico/química , RNA Catalítico/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
18.
J Bacteriol ; 203(14): e0001721, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-33972352

RESUMO

Spores of firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple firmicute species and relative mRNA levels determined by transcriptome sequencing (RNA-seq). Determination of RNA levels in single spores allowed calculation of RNA nucleotides/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at >1/spore. Bacillus subtilis, Bacillus atrophaeus, and Clostridioides difficile spores had 49, 42, and 51 mRNAs at >1/spore, and numbers of mRNAs at ≥1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores and ∼4-fold higher in Bacillus megaterium spores. In all species, some to many abundant spore mRNAs (i) were transcribed by RNA polymerase with forespore-specific σ factors, (ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores, and (iii) were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by reverse transcriptase quantitative PCR (RT-qPCR) analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate. IMPORTANCE Only ∼6% of mRNAs in spores of six firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other Bacillales spore mRNAs are transcribed under the control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that firmicute spores' abundant mRNAs are not translated when spores germinate but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.


Assuntos
Firmicutes/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Esporos Bacterianos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Firmicutes/enzimologia , Firmicutes/metabolismo , RNA Bacteriano/genética , RNA Mensageiro/genética , Esporos Bacterianos/metabolismo
19.
Methods Mol Biol ; 2317: 49-76, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028762

RESUMO

Chloroplasts, the sites of photosynthesis and sources of reducing power, are at the core of the success story that sets apart autotrophic plants from most other living organisms. Along with their fellow organelles (e.g., amylo-, chromo-, etio-, and leucoplasts), they form a group of intracellular biosynthetic machines collectively known as plastids. These plant cell constituents have their own genome (plastome), their own (70S) ribosomes, and complete enzymatic equipment covering the full range from DNA replication via transcription and RNA processive modification to translation. Plastid RNA synthesis (gene transcription) involves the collaborative activity of two distinct types of RNA polymerases that differ in their phylogenetic origin as well as their architecture and mode of function. The existence of multiple plastid RNA polymerases is reflected by distinctive sets of regulatory DNA elements and protein factors. This complexity of the plastid transcription apparatus thus provides ample room for regulatory effects at many levels within and beyond transcription. Research in this field offers insight into the various ways in which plastid genes, both singly and groupwise, can be regulated according to the needs of the entire cell. Furthermore, it opens up strategies that allow to alter these processes in order to optimize the expression of desired gene products.


Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Plastídeos/genética , Regiões Promotoras Genéticas , Transcrição Genética , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Plantas/genética , Plastídeos/metabolismo
20.
J Biol Chem ; 297(1): 100812, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34023383

RESUMO

In vitro studies of transcription frequently require the preparation of defined elongation complexes. Defined transcription elongation complexes (TECs) are typically prepared by constructing an artificial transcription bubble from synthetic oligonucleotides and RNA polymerase. This approach is optimal for diverse applications but is sensitive to nucleic acid length and sequence and is not compatible with systems where promoter-directed initiation or extensive transcription elongation is crucial. To complement scaffold-directed approaches for TEC assembly, I have developed a method for preparing promoter-initiated Escherichia coli TECs using a purification strategy called selective photoelution. This approach combines TEC-dependent sequestration of a biotin-triethylene glycol transcription stall site with photoreversible DNA immobilization to enrich TECs from an in vitro transcription reaction. I show that selective photoelution can be used to purify TECs that contain a 273-bp DNA template and 194-nt structured RNA. Selective photoelution is a straightforward and robust procedure that, in the systems assessed here, generates precisely positioned TECs with >95% purity and >30% yield. TECs prepared by selective photoelution can contain complex nucleic acid sequences and will therefore likely be useful for investigating RNA structure and function in the context of RNA polymerases.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Luz , Fenômenos Magnéticos , Microesferas , Elongação da Transcrição Genética , Pareamento de Bases , Biotina/química , Regiões Promotoras Genéticas , RNA/química , Estreptavidina/química
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