Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 10.737
Filtrar
1.
Nat Commun ; 14(1): 484, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36717560

RESUMO

Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that the stress-response σB factor from the human pathogen, Mycobacterium tuberculosis, induces the RNAP holoenzyme oligomerization into a supramolecular complex composed of eight RNAP units. Cryo-electron microscopy revealed a pseudo-symmetric structure of the RNAP octamer in which RNAP protomers are captured in an auto-inhibited state and display an open-clamp conformation. The structure shows that σB is sequestered by the RNAP flap and clamp domains. The transcriptional activator RbpA prevented octamer formation by promoting the initiation-competent RNAP conformation. Our results reveal that a non-conserved region of σ is an allosteric controller of transcription initiation and demonstrate how basal transcription factors can regulate gene expression by modulating the RNAP holoenzyme assembly and hibernation.


Assuntos
RNA Polimerases Dirigidas por DNA , Mycobacterium tuberculosis , Fator sigma , Humanos , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/metabolismo , Holoenzimas/metabolismo , Mycobacterium tuberculosis/genética , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
2.
J Bacteriol ; 205(1): e0031022, 2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36598485

RESUMO

Promoter recognition by the RNA polymerase (RNAP) holoenzyme is a key step in gene regulation. In Chlamydia trachomatis, a medically important obligate intracellular bacterium, σ66 allows the RNAP to initiate promoter-specific transcription throughout the chlamydial developmental cycle. Here, we investigated the intrinsic properties of σ66-specific promoters with emphasis on their role in the developmental gene expression of C. trachomatis. First, we examined whether promoters that contain a 5'-T(-15)G(-14)-3' (TG) motif upstream from the -10 element appear more often than others in genes that are preferentially expressed during the early, middle, or late stages of the C. trachomatis developmental cycle. We then determined the critical genetic elements that are required for transcription initiation in vitro. We also assessed the activity of promoters in the presence of Scc4, which can directly interact with σ66RNAP. Finally, we evaluated the promoter-specific dynamics during C. trachomatis infection using a reporter assay. These results reveal that the TG motif is an important determinant in certain early or late promoters. The TG promoters that have the -35 element are recognized by σ66RNAP and Scc4 differently from those lacking the -35 element. Based on these properties, the σ66-specific promoters can fall into three classes. Architectural diversity, behavioral plasticity, and the specific interplays between promoters and the σ66RNAP likely contribute to developmental gene transcription in C. trachomatis. IMPORTANCE Meticulous promoter elucidation is required to understand the foundations of transcription initiation. However, knowledge of promoter-specific transcription remains limited in C. trachomatis. This work underscores the structural and functional plasticity of σ66-specific promoters that are regulated by σ66RNAP, as well as their importance in the developmental gene regulation of C. trachomatis.


Assuntos
Chlamydia trachomatis , Escherichia coli , Chlamydia trachomatis/metabolismo , Escherichia coli/genética , Regiões Promotoras Genéticas , RNA Polimerases Dirigidas por DNA/metabolismo , Genes Controladores do Desenvolvimento , Fator sigma/metabolismo , Transcrição Genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
3.
Nat Commun ; 14(1): 195, 2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36635281

RESUMO

Bacteriophage T7 RNA polymerase (T7 RNAP) is widely used for synthesizing RNA molecules with synthetic modifications and unnatural base pairs (UBPs) for a variety of biotechnical and therapeutic applications. However, the molecular basis of transcription recognition of UBPs by T7 RNAP remains poorly understood. Here we focused on a representative UBP, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole 2-carbaldehyde (Pa), and investigated how the hydrophobic Ds-Pa pair is recognized by T7 RNAP. Our kinetic assays revealed that T7 RNAP selectively recognizes the Ds or Pa base in the templates and preferentially incorporates their cognate unnatural base nucleotide substrate (PaTP or DsTP) over natural NTPs. Our structural studies reveal that T7 RNAP recognizes the unnatural substrates at the pre-insertion state in a distinct manner compared to natural substrates. These results provide mechanistic insights into transcription recognition of UBP by T7 RNAP and provide valuable information for designing the next generation of UBPs.


Assuntos
RNA Polimerases Dirigidas por DNA , Transcrição Genética , Pareamento de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas Virais , Bacteriófago T7/genética , Bacteriófago T7/metabolismo , RNA/química
4.
Nature ; 613(7945): 783-789, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36631609

RESUMO

Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.


Assuntos
DNA Bacteriano , RNA Polimerases Dirigidas por DNA , Escherichia coli , RNA Bacteriano , Terminação da Transcrição Genética , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/ultraestrutura , Regiões Terminadoras Genéticas/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/ultraestrutura
5.
J Cell Sci ; 136(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36594557

RESUMO

Transcription termination is the final step of a transcription cycle, which induces the release of the transcript at the termination site and allows the recycling of the polymerase for the next round of transcription. Timely transcription termination is critical for avoiding interferences between neighbouring transcription units as well as conflicts between transcribing RNA polymerases (RNAPs) and other DNA-associated processes, such as replication or DNA repair. Understanding the mechanisms by which the very stable transcription elongation complex is dismantled is essential for appreciating how physiological gene expression is maintained and also how concurrent processes that occur synchronously on the DNA are coordinated. Although the strategies employed by the different classes of eukaryotic RNAPs are traditionally considered to be different, novel findings point to interesting commonalities. In this Cell Science at a Glance and the accompanying poster, we review the current understanding about the mechanisms of transcription termination by the three eukaryotic RNAPs.


Assuntos
Eucariotos , Transcrição Genética , Eucariotos/genética , Eucariotos/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , DNA
6.
Sci Adv ; 8(51): eadd3479, 2022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36542713

RESUMO

Gene transcription is carried out by RNA polymerase (RNAP) and requires the conversion of the initial closed promoter complex, where DNA is double stranded, to a transcription-competent open promoter complex, where DNA is opened up. In bacteria, RNAP relies on σ factors for its promoter specificities. Using a special form of sigma factor (σ54), which forms a stable closed complex and requires its activator that belongs to the AAA+ ATPases (ATPases associated with diverse cellular activities), we obtained cryo-electron microscopy structures of transcription initiation complexes that reveal a previously unidentified process of DNA melting opening. The σ54 amino terminus threads through the locally opened up DNA and then becomes enclosed by the AAA+ hexameric ring in the activator-bound intermediate complex. Our structures suggest how ATP hydrolysis by the AAA+ activator could remove the σ54 inhibition while helping to open up DNA, using σ54 amino-terminal peptide as a pry bar.


Assuntos
RNA Polimerases Dirigidas por DNA , DNA , RNA Polimerase Sigma 54/genética , RNA Polimerase Sigma 54/química , RNA Polimerase Sigma 54/metabolismo , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , Transcrição Genética
7.
Phys Rev E ; 106(4-1): 044406, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36397483

RESUMO

Transcriptional pausing is highly regulated by the template DNA and nascent transcript sequences. Here, we propose a thermodynamic model of transcriptional pausing, based on the thermal energy of transcription bubbles and nascent RNA structures, to describe the kinetics of the reaction pathways between active translocation, elemental, backtracked, and hairpin-stabilized pauses. The model readily predicts experimentally detected pauses in high-resolution optical-tweezer measurements of transcription. Unlike other models, it also predicts the effect of tension and the GreA transcription factor on pausing.


Assuntos
RNA Polimerases Dirigidas por DNA , Transcrição Genética , RNA Polimerases Dirigidas por DNA/metabolismo , Conformação de Ácido Nucleico , Termodinâmica , Fatores de Transcrição
8.
Appl Environ Microbiol ; 88(22): e0093922, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36342148

RESUMO

Controlled gene expression is crucial for engineering bacteria for basic and applied research. Inducible systems enable tight regulation of expression, wherein a small-molecule inducer causes the transcription factor to activate or repress transcriptional initiation. The T7 expression system is one of the most widely used inducible systems, particularly for high overexpression of proteins. However, it is well known that the highly active T7 RNA polymerase (RNAP) has several drawbacks, including toxicity to the host and substantial leaky expression in the absence of an inducer. Much work has been done to address these issues; current solutions require special strains or additional plasmids, making the system more complicated and less accessible. Here, we challenge the assumption that the T7 expression system is the best choice for obtaining high protein titers. We hypothesized that expression from strong inducible promoters expressed from high-copy plasmids could compete with expression levels obtained from T7 RNAP but that such promoters would possess improved control of transcription. Employing inducible systems from a toolbox we developed previously, we demonstrate that our plasmids consistently give higher outputs and greater fold changes over basal expression than the T7 system across rich and minimal media. In addition, we show that they outperformed the T7 system when we used an engineered metabolic pathway to produce lycopene. IMPORTANCE Genetic systems for protein overexpression are required tools in microbiological and biochemical research. Ideally, these systems include standardized genetic parts with predictable behavior, enabling the construction of stable expression systems in the host organism. Modularity of a genetic system is advantageous, so that the expression system can be easily moved into a host that best suits the needs of a given experiment. The T7 expression system lacks both predictability and stability and requires special host strains to function. Despite these limitations, it remains one of the most popular systems for protein overproduction. This study directly compared the T7 system to four inducible systems from our broad-host-range plasmid toolbox and demonstrated these alternative expression systems have distinct advantages over the T7. The systems are entirely plasmid-based and not constrained to a specific bacterial host, expanding the options for high-level protein expression across strains.


Assuntos
RNA Polimerases Dirigidas por DNA , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas , Regulação da Expressão Gênica
9.
Int J Mol Sci ; 23(21)2022 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-36361509

RESUMO

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen also infects several other vertebrate hosts. E. chaffeensis has a biphasic developmental cycle during its growth in vertebrate monocytes/macrophages and invertebrate tick cells. Host- and vector-specific differences in the gene expression from many genes of E. chaffeensis are well documented. It is unclear how the organism regulates gene expression during its developmental cycle and for its adaptation to vertebrate and tick host cell environments. We previously mapped promoters of several E. chaffeensis genes which are recognized by its only two sigma factors: σ32 and σ70. In the current study, we investigated in assessing five predicted E. chaffeensis transcription regulators; EcxR, CtrA, MerR, HU and Tr1 for their possible roles in regulating the pathogen gene expression. Promoter segments of three genes each transcribed with the RNA polymerase containing σ70 (HU, P28-Omp14 and P28-Omp19) and σ32 (ClpB, DnaK and GroES/L) were evaluated by employing multiple independent molecular methods. We report that EcxR binds to all six promoters tested. Promoter-specific binding of EcxR to several gene promoters results in varying levels of gene expression enhancement. This is the first detailed molecular characterization of transcription regulators where we identified EcxR as a gene regulator having multiple promoter-specific interactions.


Assuntos
Ehrlichia chaffeensis , Carrapatos , Animais , Humanos , Ehrlichia chaffeensis/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Monócitos/metabolismo , Fatores de Transcrição/metabolismo , Carrapatos/metabolismo
10.
Int J Mol Sci ; 23(21)2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36362266

RESUMO

Bacillus genetics need more versatile promoters for gene circuit engineering. UP elements are widely distributed in noncoding regions and interact with the α-subunit of RNA polymerase (RNAP). They can be applied as a standard element for synthetic biology. Characterization of the binding motif between UP elements and RNAP may assist with rational and effective engineering. In this study, 11 Bacillus constitutive promoters were screened for strength in Bacillus licheniformis. The motif in UP elements from a strong native promoter, PLan, was characterized. The influence of specific sequences on RNAP binding and expression strength was investigated both in vitro and in vivo. It was found that sequences up to 50 base pairs upstream of the consensus motif significantly contributed to α-CTD (the alpha subunit carboxy-terminal domain) association. Meanwhile, two repeats of a proximal subsite were able to more strongly activate the expression (by 8.2-fold) through strengthening interactions between UP elements and RNAP. Based the above molecular basis, a synthetic UP element, UP5-2P, was constructed and applied to nine wild-type promoters. Fluorescence polarization results demonstrated that it had an apparent effect on promoter-α-CTD interactions, and elevated expression strength was observed for all the engineered promoters. The highest improved core promoter, Pacpp, was more strongly activated by 7.4-fold. This work thus develops a novel strategy for Bacillus promoter engineering.


Assuntos
Bacillus , Bacillus/genética , Bacillus/metabolismo , Transcrição Genética , Sequência de Bases , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas
11.
Biophys J ; 121(22): 4299-4310, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36230000

RESUMO

RNA polymerase II (RNAP II) synthesizes RNA by reading the DNA code. During transcription initiation, RNAP II opens the double-stranded DNA to expose the DNA template to the active site. The molecular interactions driving and controlling DNA opening are not well understood. We used all-atom steered molecular dynamics simulations to derive a continuous pathway of DNA opening in human RNAP II, involving a 55 Å DNA strand displacement and a nearly 360° DNA helix rotation. To drive such large-scale transitions, we used a combination of RMSD-based collective variables, a newly designed rotational coordinate, and a path collective variable. The simulations reveal extensive interactions of the DNA with three conserved protein loops near the active site, namely with the rudder, fork loop 1, and fork loop 2. According to the simulations, DNA-protein interactions support DNA opening by a twofold mechanism; they catalyze DNA opening by attacking Watson-Crick hydrogen bonds, and they stabilize the open DNA bubble by the formation of a wide set of DNA-protein salt bridges.


Assuntos
RNA Polimerases Dirigidas por DNA , RNA Polimerase II , Humanos , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transcrição Genética
12.
Cell Rep ; 41(3): 111507, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36261005

RESUMO

Collisions between transcribing RNA polymerases and DNA replication forks are disruptive. The threat of collisions is particularly acute during the rapid early embryonic cell cycles of Drosophila when S phase occupies the entirety of interphase. We hypothesize that collision-avoidance mechanisms safeguard this early transcription. Real-time imaging of endogenously tagged RNA polymerase II (RNAPII) and a reporter for nascent transcripts in unperturbed embryos shows clustering of RNAPII at around 2 min after mitotic exit, followed by progressive dispersal as associated nascent transcripts accumulate later in interphase. Abrupt inhibition of various steps in DNA replication, including origin licensing, origin firing, and polymerization, suppresses post-mitotic RNAPII clustering and transcription in nuclear cycles. We propose that replication dependency defers the onset of transcription so that RNAPII transcribes behind advancing replication forks. The resulting orderly progression can explain how early embryos circumvent transcription-replication conflicts to express essential developmental genes.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fase S
13.
Cell Rep ; 41(3): 111492, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36261020

RESUMO

Transcription induces a wave of DNA supercoiling, altering the binding affinity of RNA polymerases and reshaping the biochemical landscape of gene regulation. As supercoiling rapidly diffuses, transcription dynamically reshapes the regulation of proximal genes, forming a complex feedback loop. However, a theoretical framework is needed to integrate biophysical regulation with biochemical transcriptional regulation. To investigate the role of supercoiling-mediated feedback within multi-gene systems, we model transcriptional regulation under the influence of supercoiling-mediated polymerase dynamics, allowing us to identify patterns of expression that result from physical inter-gene coupling. We find that gene syntax-the relative ordering and orientation of genes-defines the expression profiles, variance, burst dynamics, and inter-gene correlation of two-gene systems. Furthermore, supercoiling can enhance or weaken biochemical regulation. Our results suggest that supercoiling couples behavior between neighboring genes, providing a regulatory mechanism that tunes transcriptional variance in engineered gene networks and explains the behavior of co-localized native circuits.


Assuntos
DNA Super-Helicoidal , Transcrição Genética , DNA Super-Helicoidal/genética , Retroalimentação , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , DNA
14.
PLoS One ; 17(10): e0273746, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36282801

RESUMO

The elementary steps of transcription as catalyzed by E. coli RNA polymerase during one and two rounds of the nucleotide addition cycle (NAC) were resolved in rapid kinetic studies. Modelling of stopped-flow kinetic data of pyrophosphate release in a coupled enzyme assay during one round of the NAC indicates that the rate of pyrophosphate release is significantly less than that for nucleotide incorporation. Upon modelling of the stopped-flow kinetic data for pyrophosphate release during two rounds of the NAC, it was observed that the presence of the next nucleotide for incorporation increases the rate of release of the first pyrophosphate equivalent; incorrect nucleotides for incorporation had no effect on the rate of pyrophosphate release. Although the next nucleotide for incorporation increases the rate of pyrophosphate release, it is still significantly less than the rate of incorporation of the first nucleotide. The results from the stopped-flow kinetic studies were confirmed by using quench-flow followed by thin-layer chromatography (QF-TLC) with only the first nucleotide for incorporation labeled on the gamma phosphate with 32P to monitor pyrophosphate release. Collectively, the results are consistent with an NTP-driven model for the NAC in which the binding of the next cognate nucleotide for incorporation causes a synergistic conformational change in the enzyme that triggers the more rapid release of pyrophosphate, translocation of the enzyme along the DNA template strand and nucleotide incorporation.


Assuntos
Escherichia coli , Nucleotídeos , Nucleotídeos/metabolismo , Escherichia coli/metabolismo , Difosfatos/metabolismo , Cinética , Transcrição Genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , DNA/metabolismo
15.
Sci Rep ; 12(1): 17882, 2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36284144

RESUMO

The mining of genomes from non-cultivated microorganisms using metagenomics is a powerful tool to discover novel proteins and other valuable biomolecules. However, function-based metagenome searches are often limited by the time-consuming expression of the active proteins in various heterologous host systems. We here report the initial characterization of novel single-subunit bacteriophage RNA polymerase, EM1 RNAP, identified from a metagenome data set obtained from an elephant dung microbiome. EM1 RNAP and its promoter sequence are distantly related to T7 RNA polymerase. Using EM1 RNAP and a translation-competent Escherichia coli extract, we have developed an efficient medium-throughput pipeline and protocol allowing the expression of metagenome-derived genes and the production of proteins in cell-free system is sufficient for the initial testing of the predicted activities. Here, we have successfully identified and verified 12 enzymes acting on bis(2-hydroxyethyl) terephthalate (BHET) in a completely clone-free approach and proposed an in vitro high-throughput metagenomic screening method.


Assuntos
Metagenoma , Proteínas do Complexo da Replicase Viral , Sistema Livre de Células/metabolismo , RNA Viral/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Metagenômica/métodos , Escherichia coli/genética , Escherichia coli/metabolismo
16.
Mol Cell ; 82(20): 3885-3900.e10, 2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36220101

RESUMO

RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination.


Assuntos
RNA Polimerases Dirigidas por DNA , RNA , RNA/genética , RNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Genética , DNA , Bactérias/genética , Bactérias/metabolismo , Conformação de Ácido Nucleico
17.
Methods Enzymol ; 675: 159-192, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36220269

RESUMO

Synchronized transcription elongation complexes (TECs) are a fundamental tool for investigating the biochemical properties of RNA polymerases (RNAPs) and nascent RNA. We recently developed a standardized system for isolating high-purity synchronized E. coli RNAP TECs from an in vitro transcription reaction. Our system uses a custom 5' leader sequence, called C3-SC1 to immobilize synchronized TECs on magnetic beads so that free DNA and non-productive transcription complexes can be depleted. The synchronized elongation complexes isolated by our procedure, called C3-SC1TECs, are >98% active, >95% pure, and can be used in both solid-phase and solution-based transcription assays. The yield of the procedure relative to input DNA is ~11% when C3-SC1TECs are isolated for solid-phase assays and ~8% when C3-SC1TECs are isolated for solution-based assays. Here we describe protocols for purifying C3-SC1TECs, and for assessing the activity, homogeneity, and yield of C3-SC1TEC preparations.


Assuntos
Escherichia coli , Transcrição Genética , DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA/química
18.
Methods Enzymol ; 675: 207-233, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36220271

RESUMO

Transcription is the first and most highly regulated step in gene expression. Experimental techniques for monitoring transcription are, thus, important for studying gene expression and gene regulation as well as for translational research and drug development. Fluorescence methods are often superior to other techniques for real-time monitoring of biochemical processes. Green fluorescent proteins have long served as valuable tools for studying the process of translation. Here we present two methods that utilize fluorescent light-up RNA aptamers (FLAPs), the RNA mimics of green fluorescent proteins, to monitoring transcription and co-transcriptional RNA folding. FLAPs adopt defined three-dimensional folds that bind low molecular weight compounds called fluorogens with concomitant increase in fluorescence by many folds. FLAPs provide a strong fluorescence signal with low background that allows monitoring of transcription in real time in vitro and in vivo. However, it takes several seconds for RNA polymerase to synthesize FLAPs and the subsequent folding of the fluorogen-binding platform takes additional seconds or minutes. Here we show that Broccoli-FLAP is well suited for monitoring the rate of transcription initiation in a multi-round setup that mitigates the slow rate of the FLAP maturation. Furthermore, we demonstrate that a relatively slow and inefficient folding of iSpinach-FLAP can be taken advantage of for monitoring the action of RNA folding chaperones.


Assuntos
Aptâmeros de Nucleotídeos , Aptâmeros de Nucleotídeos/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , RNA , Dobramento de RNA
19.
Nat Commun ; 13(1): 5887, 2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-36202846

RESUMO

The development of a fertilized egg to an embryo requires the proper temporal control of gene expression. During cell differentiation, timing is often controlled via cascades of transcription factors (TFs). However, in early development, transcription is often inactive, and many TF levels stay constant, suggesting that alternative mechanisms govern the observed rapid and ordered onset of gene expression. Here, we find that in early embryonic development access of maternally deposited nuclear proteins to the genome is temporally ordered via importin affinities, thereby timing the expression of downstream targets. We quantify changes in the nuclear proteome during early development and find that nuclear proteins, such as TFs and RNA polymerases, enter the nucleus sequentially. Moreover, we find that the timing of nuclear proteins' access to the genome corresponds to the timing of downstream gene activation. We show that the affinity of proteins to importin is a major determinant in the timing of protein entry into embryonic nuclei. Thus, we propose a mechanism by which embryos encode the timing of gene expression in early development via biochemical affinities. This process could be critical for embryos to organize themselves before deploying the regulatory cascades that control cell identities.


Assuntos
Núcleo Celular , Proteoma , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Feminino , Genoma , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , Gravidez , Proteoma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
RNA ; 28(12): 1643-1658, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36198425

RESUMO

The E. coli 6S RNA is an RNA polymerase (RNAP) inhibitor that competes with σ70-dependent DNA promoters for binding to RNAP holoenzyme (RNAP:σ70). The 6S RNA when bound is then used as a template to synthesize a short product RNA (pRNA; usually 13-nt-long). This pRNA changes the 6S RNA structure, triggering the 6S RNA:pRNA complex to release and allowing DNA-dependent housekeeping gene expression to resume. In high nutrient conditions, 6S RNA turnover is extremely rapid but becomes very slow in low nutrient environments. This leads to a large accumulation of inhibited RNAP:σ70 in stationary phase. As pRNA initiates synthesis with ATP, we and others have proposed that the 6S RNA release rate strongly depends on ATP levels as a proxy for sensing the cellular metabolic state. By purifying endogenous 6S RNA:pRNA complexes using RNA Mango and using reverse transcriptase to generate pRNA-cDNA chimeras, we demonstrate that 6S RNA:pRNA formation can be simultaneous with 6S RNA 5' maturation. More importantly, we find a dramatic accumulation of capped pRNAs during stationary phase. This indicates that ATP levels in stationary phase are low enough for noncanonical initiator nucleotides (NCINs) such as NAD+ and NADH to initiate pRNA synthesis. In vitro, mutation of the conserved 6S RNA template sequence immediately upstream of the pRNA transcriptional start site can increase or decrease the pRNA capping efficiency, suggesting that evolution has tuned the biological 6S RNA sequence for an optimal capping rate. NCIN-initiated pRNA synthesis may therefore be essential for cell viability in low nutrient conditions.


Assuntos
Escherichia coli , Nucleotídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Nucleotídeos/metabolismo , Transcrição Genética , Conformação de Ácido Nucleico , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator sigma/genética , Fator sigma/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...