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1.
bioRxiv ; 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38915573

RESUMO

Apart from its well-established role in initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remains largely unexplored. To determine the prevalence of TFIIB's role in termination, we performed GRO-seq analyses in sua7-1 mutant (TFIIB sua7-1 ) and the isogenic wild type (TFIIB WT ) strains of yeast. Almost a three-fold increase in readthrough of the poly(A)-termination signal was observed in TFIIB sua7-1 mutant compared to the TFIIB WT cells. Of all genes analyzed in this study, nearly 74% genes exhibited a statistically significant increase in terminator readthrough in the mutant. To gain an understanding of the mechanistic basis of TFIIB involvement in termination, we performed mass spectrometry of TFIIB, affinity purified from chromatin and soluble cellular fractions, from TFIIB sua7-1 and TFIIB WT cells. TFIIB purified from the chromatin fraction of TFIIB WT cells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with both CF1A and Rat1 termination complexes in TFIIB sua7-1 mutant. ChIP assay revealed that the recruitment of Pta1 subunit of CPF complex, Rna15 subunit of CF1 complex and Rat1 subunit of Rat1 complex registered nearly 90% decline in the mutant over wild type cells. The overall conclusion of these results is that TFIIB affects termination of transcription on a genome-wide scale, and TFIIB-termination factor interaction may play a crucial role in the process.

2.
Mol Biol Cell ; : mbcE23120493, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38865176

RESUMO

RNase E is the most common RNA decay nuclease in bacteria, setting the global mRNA decay rate and scaffolding formation of the RNA degradosome complex and BR-bodies. To properly set the global mRNA decay rate, RNase E from Escherichia coli and neighboring γ-proteobacteria were found to autoregulate RNase E levels via the decay of its mRNA's 5' UTR. While the 5' UTR is absent from other groups of bacteria in the Rfam database, we identified that the α-proteobacterium Caulobacter crescentus RNase E contains a similar 5' UTR structure that promotes RNase E autoregulation. In both bacteria, the C-terminal IDR of RNase E is required for proper autoregulation to occur, and this IDR is also necessary and sufficient for RNase E to phase-separate, generating BR-bodies. Using in vitro purified RNase E, we find that the IDR's ability to promote phase-separation correlates with enhanced 5' UTR cleavage, suggesting that phase-separation of RNase E with the 5' UTR enhances autoregulation. Finally, using growth competition experiments we find that a strain capable of autoregulation rapidly outcompetes a strain with a 5' UTR mutation that cannot autoregulate, suggesting autoregulation promotes optimal cellular fitness.

3.
bioRxiv ; 2024 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-38617242

RESUMO

Biomolecular condensates, such as the nucleoli or P-bodies, are non-membrane-bound assemblies of proteins and nucleic acids that facilitate specific cellular processes. Like eukaryotic P-bodies, the recently discovered bacterial ribonucleoprotein bodies (BR-bodies) organize the mRNA decay machinery, yet the similarities in molecular and cellular functions across species have been poorly explored. Here, we examine the functions of BR-bodies in the nitrogen-fixing endosymbiont Sinorhizobium meliloti, which colonizes the roots of compatible legume plants. Assembly of BR-bodies into visible foci in S. meliloti cells requires the C-terminal intrinsically disordered region (IDR) of RNase E, and foci fusion is readily observed in vivo, suggesting they are liquid-like condensates that form via mRNA sequestration. Using Rif-seq to measure mRNA lifetimes, we found a global slowdown in mRNA decay in a mutant deficient in BR-bodies, indicating that compartmentalization of the degradation machinery promotes efficient mRNA turnover. While BR-bodies are constitutively present during exponential growth, the abundance of BR-bodies increases upon cell stress, whereby they promote stress resistance. Finally, using Medicago truncatula as host, we show that BR-bodies enhance competitiveness during colonization and appear to be required for effective symbiosis, as mutants without BR-bodies failed to stimulate plant growth. These results suggest that BR-bodies provide a fitness advantage for bacteria during infection, perhaps by enabling better resistance against the host immune response.

4.
Cell Rep ; 42(10): 113229, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37815915

RESUMO

Bacterial ribonucleoprotein bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here, we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We find 111 BR-body-enriched proteins showing that BR-bodies are more complex than previously assumed. We identify five BR-body-enriched proteins that undergo RNA-dependent phase separation in vitro with a complex network of condensate mixing. We observe that some RNP condensates co-assemble with preferred directionality, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body-associated proteins have the capacity to dissolve the condensate. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing.


Assuntos
Proteoma , RNA , Proteoma/metabolismo , Proteômica , Ribonucleoproteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
5.
Microbiol Spectr ; 11(6): e0193423, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-37850787

RESUMO

IMPORTANCE: One of the most important control points in gene regulation is RNA stability, which determines the half-life of a transcript from its transcription until its degradation. Bacteria have evolved a sophisticated multi-enzymatic complex, the RNA degradosome, which is dedicated mostly to RNA turnover. The combined activity of RNase E and the other RNA degradosome enzymes provides an efficient pipeline for the complete degradation of RNAs. The DEAD-box RNA helicases are very often found in RNA degradosomes from phylogenetically distant bacteria, confirming their importance in unwinding structured RNA for subsequent degradation. This work showed that the absence of the RNA helicase RhlB in the free-living Alphaproteobacterium Caulobacter crescentus causes important changes in gene expression and cell physiology. These are probably due, at least in part, to inefficient RNA processing by the RNA degradosome, particularly at low-temperature conditions.


Assuntos
Caulobacter , Caulobacter/genética , Caulobacter/metabolismo , Temperatura , RNA/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Processamento Pós-Transcricional do RNA
6.
Sci Rep ; 13(1): 12937, 2023 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-37558691

RESUMO

Bacterial Ribonucleoprotein bodies (BR-bodies) play an essential role in organizing RNA degradation via phase separation in the cytoplasm of bacteria. BR-bodies mediate multi-step mRNA decay through the concerted activity of the endoribonuclease RNase E coupled with the 3'-5' exoribonuclease Polynucleotide Phosphorylase (PNPase). In vivo, studies indicated that the loss of PNPase recruitment into BR-bodies led to a significant build-up of RNA decay intermediates in Caulobacter crescentus. However, it remained unclear whether this is due to a lack of colocalized PNPase and RNase E within BR-bodies or whether PNPase's activity is stimulated within the BR-body. We reconstituted RNase E's C-terminal domain with PNPase towards a minimal BR-body in vitro to distinguish these possibilities. We found that PNPase's catalytic activity is accelerated when colocalized within the RNase E biomolecular condensates, partly due to scaffolding and mass action effects. In contrast, disruption of the RNase E-PNPase protein-protein interaction led to a loss of PNPase recruitment into the RNase E condensates and a loss of ribonuclease rate enhancement. We also found that RNase E's unique biomolecular condensate environment tuned PNPase's substrate specificity for poly(A) over poly(U). Intriguingly, a critical PNPase reactant, phosphate, reduces RNase E phase separation both in vitro and in vivo. This regulatory feedback ensures that under limited phosphate resources, PNPase activity is enhanced by recruitment into RNase E's biomolecular condensates.


Assuntos
Condensados Biomoleculares , Escherichia coli , Escherichia coli/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo
7.
J Bacteriol ; 205(5): e0042022, 2023 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-37092987

RESUMO

The initiation of translation in bacteria is thought to occur upon base pairing between the Shine-Dalgarno (SD) site in the mRNA and the anti-SD site in the rRNA. However, in many bacterial species, such as Caulobacter crescentus, a minority of mRNAs have SD sites. To examine the functional importance of SD sites in C. crescentus, we analyzed the transcriptome and found that more SD sites exist in the coding sequence than in the preceding start codons. To examine the function of SD sites in initiation, we designed a series of mutants with altered ribosome accessibility and SD content in translation initiation regions (TIRs) and in elongator AUG regions (EARs). A lack of mRNA structure content is required for initiation in TIRs, and, when introduced into EARs, can stimulate initiation, thereby suggesting that low mRNA structure content is a major feature that is required for initiation. SD sites appear to stimulate initiation in TIRs, which generally lack structure content, but SD sites only stimulate initiation in EARs if RNA secondary structures are destabilized. Taken together, these results suggest that the difference in secondary structure between TIRs and EARs directs ribosomes to start codons where SD base pairing can tune the efficiency of initiation, but SDs in EARs do not stimulate initiation, as they are blocked by stable secondary structures. This highlights the importance of studying translation initiation mechanisms in diverse bacterial species. IMPORTANCE Start codon selection is an essential process that is thought to occur via the base pairing of the rRNA to the SD site in the mRNA. This model is based on studies in E. coli, yet whole-genome sequencing revealed that SD sites are absent at start codons in many species. By examining the transcriptome of C. crescentus, we found more SD-AUG pairs in the CDS of mRNAs than preceding start codons, yet these internal sites do not initiate. Instead, start codon regions have lower mRNA secondary structure content than do internal SD-AUG regions. Therefore, we find that start codon selection is not controlled by the presence of SD sites, which tune initiation efficiency, but by lower mRNA structure content surrounding the start codon.


Assuntos
Escherichia coli , Biossíntese de Proteínas , Códon de Iniciação , RNA Mensageiro/metabolismo , Escherichia coli/genética , Códon , Bactérias/genética , Iniciação Traducional da Cadeia Peptídica
8.
bioRxiv ; 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-38168245

RESUMO

RNase E is the most common RNA decay nuclease in bacteria, setting the global mRNA decay rate and scaffolding formation of the RNA degradosome complex and BR-bodies. To properly set the global mRNA decay rate, RNase E from Escherichia coli and neighboring γ-proteobacteria were found to autoregulate RNase E levels via the decay of its mRNA's 5' UTR. While the 5' UTR is absent from other groups of bacteria in the Rfam database, we identified that the α-proteobacterium Caulobacter crescentus RNase E contains a similar 5' UTR structure that promotes RNase E autoregulation. In both bacteria, the C-terminal IDR of RNase E is required for proper autoregulation to occur, and this IDR is also necessary and sufficient for RNase E to phase-separate, generating BR-bodies. Using in vitro purified RNase E, we find that the IDR's ability to promote phase-separation correlates with enhanced 5' UTR cleavage, suggesting that phase-separation of RNase E with the 5' UTR enhances autoregulation. Finally, using growth competition experiments we find that a strain capable of autoregulation rapidly outcompetes a strain with a 5' UTR mutation that cannot autoregulate, suggesting autoregulation promotes optimal cellular fitness.

9.
NAR Genom Bioinform ; 3(3): lqab081, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34568822

RESUMO

Bacterial translation is thought to initiate by base pairing of the 16S rRNA and the Shine-Dalgarno sequence in the mRNA's 5' untranslated region (UTR). However, transcriptomics has revealed that leaderless mRNAs, which completely lack any 5' UTR, are broadly distributed across bacteria and can initiate translation in the absence of the Shine-Dalgarno sequence. To investigate the mechanism of leaderless mRNA translation initiation, synthetic in vivo translation reporters were designed that systematically tested the effects of start codon accessibility, leader length, and start codon identity on leaderless mRNA translation initiation. Using these data, a simple computational model was built based on the combinatorial relationship of these mRNA features that can accurately classify leaderless mRNAs and predict the translation initiation efficiency of leaderless mRNAs. Thus, start codon accessibility, leader length, and start codon identity combine to define leaderless mRNA translation initiation in bacteria.

10.
Curr Opin Microbiol ; 61: 91-98, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33878678

RESUMO

While bacteria typically lack membrane bound organelles, the mechanisms of subcellular organization have been unclear. Bacteria have recently been found to harbor membraneless organelles containing enzymes of many biochemical pathways. These organelles, called biomolecular condensates, have been found to commonly form through the process of liquid-liquid phase separation and are typically enriched in nucleic acid binding proteins. Interestingly, eukaryote and bacterial transcription and RNA decay machinery have been found to form biomolecular condensates. Additionally, DEAD Box ATPases from eukaryotes and bacteria have also been found to modulate biomolecular condensates. The shared ability of RNA metabolic enzymes to assemble into biomolecular condensates across domains suggests that this mode of subcellular organization aids in the control of RNA metabolism.


Assuntos
Organelas , RNA Bacteriano , Bactérias/genética , Células Eucarióticas , Estabilidade de RNA , RNA Bacteriano/genética
11.
STAR Protoc ; 1(3): 100205, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33377099

RESUMO

Bacterial RNP bodies (BR bodies) contain the mRNA decay machinery, but the collection of associated RNAs and proteins are poorly defined. Here, we present a protocol for the rapid differential centrifugation-based enrichment of BR bodies from Caulobacter crescentus cells. As native BR bodies are highly labile and dissociate by degrading internal mRNAs, an active site mutant of RNase E, which blocks dissolution of BR bodies, allows BR-body stabilization during enrichment. For complete details on the use and execution of this protocol, please refer to Al-Husini et al. (2020).


Assuntos
Caulobacter crescentus/metabolismo , Centrifugação com Gradiente de Concentração/métodos , Ribonucleoproteínas/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/patogenicidade , Separação Celular/métodos , Centrifugação/métodos , Endorribonucleases/metabolismo , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Ribonucleoproteínas/metabolismo
12.
Microbiol Resour Announc ; 9(44)2020 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-33122415

RESUMO

Caulobacter crescentus is a model alphaproteobacterium with a well-studied genetic network controlling its cell cycle. Essential for such studies is an accurate map of the expressed features of its genome. Here, we provide an updated map of the expressed RNAs by integrative analysis of 5' global rapid amplification of cDNA ends, transcriptome sequencing, rifampicin treatment RNA sequencing, and RNA end-enriched sequencing data sets.

13.
Wiley Interdiscip Rev RNA ; 11(6): e1599, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32445438

RESUMO

In bacteria, mRNA decay is controlled by megadalton scale macromolecular assemblies called, "RNA degradosomes," composed of nucleases and other RNA decay associated proteins. Recent advances in bacterial cell biology have shown that RNA degradosomes can assemble into phase-separated structures, termed bacterial ribonucleoprotein bodies (BR-bodies), with many analogous properties to eukaryotic processing bodies and stress granules. This review will highlight the functional role that BR-bodies play in the mRNA decay process through its organization into a membraneless organelle in the bacterial cytoplasm. This review will also highlight the phylogenetic distribution of BR-bodies across bacterial species, which suggests that these phase-separated structures are broadly distributed across bacteria, and in evolutionarily related mitochondria and chloroplasts. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Export and Localization > RNA Localization RNA Turnover and Surveillance > Regulation of RNA Stability.


Assuntos
Bactérias/química , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Bactérias/metabolismo , Cloroplastos/metabolismo , Mitocôndrias/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/isolamento & purificação
14.
Mol Cell ; 78(4): 670-682.e8, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32343944

RESUMO

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.


Assuntos
Caulobacter crescentus/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Complexos Multienzimáticos/metabolismo , Organelas/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/crescimento & desenvolvimento , Endorribonucleases/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Humanos , Complexos Multienzimáticos/genética , Organelas/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Helicases/genética , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
15.
Elife ; 82019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-31609203

RESUMO

The bacterium Myxococcus xanthus exhibits a complex multicellular life cycle. In the presence of nutrients, cells prey cooperatively. Upon starvation, they enter a developmental cycle wherein cells aggregate to produce macroscopic fruiting bodies filled with resistant myxospores. We used RNA-Seq technology to examine the transcriptome of the 96 hr developmental program. These data revealed that 1415 genes were sequentially expressed in 10 discrete modules, with expression peaking during aggregation, in the transition from aggregation to sporulation, or during sporulation. Analysis of genes expressed at each specific time point provided insights as to how starving cells obtain energy and precursors necessary for assembly of fruiting bodies and into developmental production of secondary metabolites. This study offers the first global view of developmental transcriptional profiles and provides important tools and resources for future studies.


Assuntos
Proteínas de Bactérias/genética , Myxococcus xanthus/genética , Transcriptoma/genética , Regulação Bacteriana da Expressão Gênica/genética , Transdução de Sinais/genética , Esporos Bacterianos/genética
16.
mSystems ; 4(4)2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138672

RESUMO

Caulobacter crescentus is a model for the bacterial cell cycle which culminates in asymmetric cell division, yet little is known about the absolute levels of protein synthesis of the cellular parts needed to complete the cell cycle. Here we utilize ribosome profiling to provide absolute measurements of mRNA translation in C. crescentus, providing an important resource with quantitative genome-wide measurements of protein output across individual genes. Analysis of protein synthesis rates revealed ∼4.5% of cellular protein synthesis is for genes related to vitamin B12 import (btuB) and B12-independent methionine biosynthesis (metE) when grown in common growth media lacking B12 While its facultative B12 lifestyle provides a fitness advantage in the absence of B12, we find that it provides a fitness disadvantage of the cells in the presence of B12, potentially explaining why many Caulobacter species have lost the metE gene and become obligates for B12 IMPORTANCE Caulobacter crescentus is a model system of the bacterial cell cycle culminating in asymmetric cell division, with each daughter cell inheriting a distinct set of proteins. While a genetic network of master transcription factors coordinates the cell cycle timing of transcription for nearly 20% of Caulobacter genes, we lack knowledge of how many of each protein "part" encoded in the genome are synthesized. Therefore, to determine the absolute production rates across the genome, we performed ribosome profiling, providing, for the first time, a quantitative resource with measurements of each protein "part" needed to generate daughter cells. This resource furthers the goal of a systems-level understanding of the genetic network controlling asymmetric cell division. To highlight the utility of this data set, we probe the protein synthesis cost of a B12 utilization pathway and provide new insights into Caulobacter's adaptation to its natural environments.

17.
Methods Enzymol ; 612: 443-465, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30502952

RESUMO

Bacterial cell division is the result of a productive round of the cell cycle to yield two daughter cells. The cell cycle is highly coordinated in Caulobacter crescentus where it is driven by a cell cycle gene-regulatory network that coordinates gene expression with the major cell cycle events such as chromosome replication and cell division. Recent ribosomes profiling data showed that 484 genes undergo changes in translation efficiency during the cell cycle, suggesting a broad role for translational control in cell cycle regulation. In this chapter, we focus on how to perform ribosome profiling to measure the translation efficiency across cellular mRNAs at key stages in the Caulobacter cell cycle. This methodology relies on the high-yield ludox gradient synchronization of Caulobacter cells followed by ribosome profiling to measure ribosome density and total RNA-seq to measure mRNA levels.


Assuntos
Caulobacter crescentus/citologia , Caulobacter crescentus/metabolismo , Ciclo Celular/fisiologia , Ribossomos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Ciclo Celular/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
18.
Mol Cell ; 71(6): 1027-1039.e14, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30197298

RESUMO

Ribonucleoprotein (RNP) granules play an important role in organizing eukaryotic mRNA metabolism via liquid-liquid phase separation (LLPS) of mRNA decay factors into membrane-less organelles in the cytoplasm. Here we show that the bacterium Caulobacter crescentus Ribonuclease (RNase) E assembles RNP LLPS condensates that we term bacterial RNP-bodies (BR-bodies), similar to eukaryotic P-bodies and stress granules. RNase E requires RNA to assemble a BR-body, and disassembly requires RNA cleavage, suggesting BR-bodies provide localized sites of RNA degradation. The unstructured C-terminal domain of RNase E is both necessary and sufficient to assemble the core of the BR-body, is functionally conserved in related α-proteobacteria, and influences mRNA degradation. BR-bodies are rapidly induced under cellular stresses and provide enhanced cell growth under stress. To our knowledge, Caulobacter RNase E is the first bacterial protein identified that forms LLPS condensates, providing an effective strategy for subcellular organization in cells lacking membrane-bound compartments.


Assuntos
Caulobacter crescentus/metabolismo , Grânulos Citoplasmáticos/fisiologia , Ribonucleoproteínas/metabolismo , Alphaproteobacteria/metabolismo , Caulobacter crescentus/genética , Grânulos Citoplasmáticos/metabolismo , Endorribonucleases/metabolismo , Extração Líquido-Líquido , Complexos Multienzimáticos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , Estabilidade de RNA
19.
Curr Opin Chem Biol ; 46: 138-145, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30059836

RESUMO

In order to function on the ribosome with uniform rate and adequate accuracy, each bacterial tRNA has evolved to have a characteristic sequence and set of modifications that compensate for the differing physical properties of its esterified amino acid and its codon-anticodon interaction. The sequence of the T-stem of each tRNA compensates for the differential effect of the esterified amino acid on the binding and release of EF-Tu during decoding. The sequence and modifications in the anticodon loop and core of tRNA impact the codon-anticodon strength and the ability of the tRNA to bend during codon recognition. These discoveries impact the design of tRNAs for the efficient and accurate incorporation of unnatural amino acids into proteins using bacterial translation systems.


Assuntos
Aminoácidos/genética , Bactérias/genética , RNA Bacteriano/genética , RNA de Transferência/genética , Ribossomos/genética , Aminoácidos/química , Bactérias/química , Códon/genética , Evolução Molecular , Modelos Moleculares , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Bacteriano/química , RNA de Transferência/química , Ribossomos/química
20.
Proc Natl Acad Sci U S A ; 115(16): E3712-E3721, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29610352

RESUMO

We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single-particle tracking and superresolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in Escherichia coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E's function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.


Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/enzimologia , Endorribonucleases/metabolismo , Proteínas de Bactérias/análise , Caulobacter crescentus/metabolismo , Caulobacter crescentus/ultraestrutura , Ciclo Celular , Polaridade Celular , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/ultraestrutura , Endorribonucleases/análise , Regulação Bacteriana da Expressão Gênica , Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Complexos Multienzimáticos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , RNA Bacteriano/biossíntese , RNA Bacteriano/genética , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , Ribossomos/metabolismo , Imagem Individual de Molécula/métodos , Frações Subcelulares/enzimologia , Moldes Genéticos , Transcrição Gênica
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