scholarly journals Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus

2017 ◽  
Author(s):  
Camille A. Bayas ◽  
Jiarui Wang ◽  
Marissa K. Lee ◽  
Jared M. Schrader ◽  
Lucy Shapiro ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Rna ◽  
Spatial Organization ◽  
Caulobacter Crescentus ◽  
Subcellular Location ◽  
Single Particle Tracking ◽  
Rrna Synthesis ◽  
Rnase E ◽  
Ribosomal Protein L1 ◽  
Rna Degradosome

We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single particle tracking and super-resolution 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 E. coli, where RNase E co-localizes 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. Moreover, RNase E cluster positions correlate with the subcellular location of chromosomal loci of two highly transcribed ribosomal RNA genes, suggesting that RNase E’s function in ribosomal RNA 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.

scholarly journals Spatial organization and dynamics of RNase E and ribosomes inCaulobacter crescentus

2018 ◽  
Vol 115 (16) ◽  
pp. E3712-E3721 ◽  
Author(s):  
Camille A. Bayas ◽  
Jiarui Wang ◽  
Marissa K. Lee ◽  
Jared M. Schrader ◽  
Lucy Shapiro ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Spatial Organization ◽  
Caulobacter Crescentus ◽  
Subcellular Location ◽  
Single Particle Tracking ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rnase E ◽  
Ribosomal Protein L1 ◽  
Rna Degradosome

We report the dynamic spatial organization ofCaulobacter crescentusRNase 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 inEscherichia coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes inCaulobacter, 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 inCaulobacter, with chromosomal readout serving as the template for this organization.

scholarly journals Association of the Cold Shock DEAD-Box RNA Helicase RhlE to the RNA Degradosome in Caulobacter crescentus

2017 ◽  
Vol 199 (13) ◽  
Author(s):  
Angel A. Aguirre ◽  
Alexandre M. Vicente ◽  
Steven W. Hardwick ◽  
Daniela M. Alvelos ◽  
Ricardo R. Mazzon ◽  
...  
Keyword(s):  
Low Temperature ◽  
Caulobacter Crescentus ◽  
Immunoblot Analysis ◽  
Rna Helicase ◽  
Rnase E ◽  
Rna Helicases ◽  
Content Type ◽  
Dead Box ◽  
Rna Degradosome ◽  
The Dead

ABSTRACT In diverse bacterial lineages, multienzyme assemblies have evolved that are central elements of RNA metabolism and RNA-mediated regulation. The aquatic Gram-negative bacterium Caulobacter crescentus, which has been a model system for studying the bacterial cell cycle, has an RNA degradosome assembly that is formed by the endoribonuclease RNase E and includes the DEAD-box RNA helicase RhlB. Immunoprecipitations of extracts from cells expressing an epitope-tagged RNase E reveal that RhlE, another member of the DEAD-box helicase family, associates with the degradosome at temperatures below those optimum for growth. Phenotype analyses of rhlE, rhlB, and rhlE rhlB mutant strains show that RhlE is important for cell fitness at low temperature and its role may not be substituted by RhlB. Transcriptional and translational fusions of rhlE to the lacZ reporter gene and immunoblot analysis of an epitope-tagged RhlE indicate that its expression is induced upon temperature decrease, mainly through posttranscriptional regulation. RNase E pulldown assays show that other proteins, including the transcription termination factor Rho, a second DEAD-box RNA helicase, and ribosomal protein S1, also associate with the degradosome at low temperature. The results suggest that the RNA degradosome assembly can be remodeled with environmental change to alter its repertoire of helicases and other accessory proteins. IMPORTANCE DEAD-box RNA helicases are often present in the RNA degradosome complex, helping unwind secondary structures to facilitate degradation. Caulobacter crescentus is an interesting organism to investigate degradosome remodeling with change in temperature, because it thrives in freshwater bodies and withstands low temperature. In this study, we show that at low temperature, the cold-induced DEAD-box RNA helicase RhlE is recruited to the RNA degradosome, along with other helicases and the Rho protein. RhlE is essential for bacterial fitness at low temperature, and its function may not be complemented by RhlB, although RhlE is able to complement for rhlB loss. These results suggest that RhlE has a specific role in the degradosome at low temperature, potentially improving adaptation to this condition.

scholarly journals Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly

Open Biology ◽  
2012 ◽  
Vol 2 (4) ◽  
pp. 120028 ◽  
Author(s):  
Steven W. Hardwick ◽  
Tobias Gubbey ◽  
Isabelle Hug ◽  
Urs Jenal ◽  
Ben F. Luisi
Keyword(s):  
Active Sites ◽  
Rna Binding ◽  
Caulobacter Crescentus ◽  
Free Form ◽  
Central Channel ◽  
Rnase E ◽  
Polynucleotide Phosphorylase ◽  
Catalytic Core ◽  
Rna Degradosome ◽  
Substrate Channelling

Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3′–5′ directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3′ end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a ‘splayed’ conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3′ end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.

scholarly journals RNA polymerase organizes into distinct spatial clusters independent of ribosomal RNA transcription in E. coli

2018 ◽  
Author(s):  
Xiaoli Weng ◽  
Christopher H. Bohrer ◽  
Kelsey Bettridge ◽  
Arvin Cesar Lagda ◽  
Cedric Cagliero ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Growth Condition ◽  
Ribosomal Rna ◽  
Spatial Organization ◽  
Large Fraction ◽  
Rrna Synthesis ◽  
Rich Medium ◽  
E Coli ◽  
The Rich ◽  
Molecular Components

AbstractRecent studies have shown that RNA polymerase (RNAP) is spatially organized into distinct clusters in E. coli and B. subtilis cells. Spatially organized molecular components in prokaryotic systems imply compartmentalization without the use of membranes, which may offer new insights into pertinent functions and regulations. However, the function of RNAP clusters and whether its formation is driven by active ribosomal RNA (rRNA) transcription remain elusive. In this work, we investigated the spatial organization of RNAP in E. coli cells using quantitative superresolution imaging. We observed that RNAP formed large, distinct clusters under a rich medium growth condition and preferentially located in the center of the nucleoid. Two-color superresolution colocalization imaging showed that under the rich medium growth condition, nearly all RNAP clusters were active in synthesizing rRNA, suggesting that rRNA synthesis may be spatially separated from mRNA synthesis that most likely occurs at the nucleoid periphery. Surprisingly, a large fraction of RNAP clusters persisted under conditions in which rRNA synthesis was reduced or abolished, or when only one out of the seven rRNA operons (rrn) remained. Furthermore, when gyrase activity was inhibited, we observed a similar rRNA synthesis level, but multiple dispersed, smaller rRNA and RNAP clusters occupying not only the center but also the periphery of the nucleoid, comparable to an expanded nucleoid. These results suggested that RNAP was organized into active transcription centers for rRNA synthesis under the rich medium growth condition; their presence and spatial organization, however, were independent of rRNA synthesis activity under the conditions used but were instead influenced by the structure and characteristics of the underlying nucleoid. Our work opens the door for further investigations of the function and molecular nature of RNAP clusters and points to a potentially new mechanism of transcription regulation by the spatial organization of individual molecular components.

scholarly journals Specific Binding of Escherichia coliRibosomal Protein S1 to boxA Transcriptional Antiterminator RNA

1998 ◽  
Vol 180 (8) ◽  
pp. 2248-2252 ◽  
Author(s):  
Jeremy Mogridge ◽  
Jack Greenblatt
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Protein ◽  
Ribosomal Rna ◽  
Specific Binding ◽  
Rrna Synthesis ◽  
Bacteriophage Λ ◽  
N Protein ◽  
Ribosomal Protein S1

ABSTRACT We show that ribosomal protein S1 specifically binds theboxA transcriptional antiterminator RNAs of bacteriophage λ and the Escherichia coli ribosomal RNA operons. Although S1 competes with the NusB-S10 antitermination complex for binding to boxA, it does not affect antitermination by the λ N protein in vitro, and its role, if any, in rRNA synthesis is still unknown.

Spatial organization enhances versatility and specificity in cyclic di-GMP signaling

2020 ◽  
Vol 401 (12) ◽  
pp. 1323-1334
Author(s):  
Sandra Kunz ◽  
Peter L. Graumann
Keyword(s):  
Protein Interactions ◽  
Cell Cycle Progression ◽  
Spatial Organization ◽  
Caulobacter Crescentus ◽  
Second Messenger ◽  
Protein Protein Interactions ◽  
Spatial Cues ◽  
Signaling Specificity ◽  
Regulatory Circuits ◽  
Second Messenger Signaling

AbstractThe second messenger cyclic di-GMP regulates a variety of processes in bacteria, many of which are centered around the decision whether to adopt a sessile or a motile life style. Regulatory circuits include pathogenicity, biofilm formation, and motility in a wide variety of bacteria, and play a key role in cell cycle progression in Caulobacter crescentus. Interestingly, multiple, seemingly independent c-di-GMP pathways have been found in several species, where deletions of individual c-di-GMP synthetases (DGCs) or hydrolases (PDEs) have resulted in distinct phenotypes that would not be expected based on a freely diffusible second messenger. Several recent studies have shown that individual signaling nodes exist, and additionally, that protein/protein interactions between DGCs, PDEs and c-di-GMP receptors play an important role in signaling specificity. Additionally, subcellular clustering has been shown to be employed by bacteria to likely generate local signaling of second messenger, and/or to increase signaling specificity. This review highlights recent findings that reveal how bacteria employ spatial cues to increase the versatility of second messenger signaling.

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