Reconstitution and Analysis of the Multienzyme Escherichia coli RNA Degradosome

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Journal of Molecular Biology (Impact Factor: 4.33). 08/2008; 382(4):870-83. DOI: 10.1016/j.jmb.2008.07.059
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ABSTRACT The Escherichia coli RNA degradosome is a multienzyme assembly that functions in transcript turnover and maturation of structured RNA precursors. We have developed a procedure to reconstitute the RNA degradosome from recombinant components using modular coexpression vectors. The reconstituted assembly can be purified on a scale that has enabled biochemical and biophysical analyses, and we compare the properties of recombinant and cell-extracted RNA degradosomes. We present evidence that auxiliary protein components can be recruited to the 'superprotomer' core of the assembly through a dynamic equilibrium involving RNA intermediaries. We discuss the implications for the regulation of RNA degradosome function in vivo.

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Available from: Maria Wiktoria Górna, Mar 04, 2014
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    • "Based on experimental evidence, it has been proposed that the stoichiometry of protomers is 1:1:2:1 for RNase E : PNPase : enolase : RhlB respectively. However, stoichiometry is clearly sensitive to conditions of reconstitution and growth (Liou et al., 2001; Mauri and Deho, 2008; Worrall et al., 2008; Dominguez-Malfavon et al., 2013). In rich media, there appears to be an excess of PNPase and enolase, suggesting that a proportion of these enzymes are not complexed with RNase E. In contrast, RhlB, which was recently shown to be encoded by a leaderless mRNA (Clarke et al., 2015), is not detectable in minimal media suggesting down regulation depending on growth conditions (Dominguez-Malfavon et al., 2013). "
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    ABSTRACT: Ribonuclease E (RNase E) of Escherichia coli, which is the founding member of a widespread family of proteins in Bacteria and Chloroplasts, is a fascinating enzyme that still has not revealed all its secrets. RNase E is an essential single-strand specific endoribonuclease that is involved in the processing and degradation of nearly every transcript in E. coli. A striking enzymatic property is a preference for substrates with a 5' monophosphate end although recent work explains how RNase E can overcome the protection afforded by the 5' triphosphate end of a primary transcript. Other features of E. coli RNase E include its interaction with enzymes involved in RNA degradation to form the multienzyme RNA degradosome and its localization to the inner cytoplasmic membrane. The N-terminal catalytic core of the RNase E protomer associates to form a tetrameric holoenzyme. Each RNase E protomer has a large C-terminal Intrinsically Disordered (ID) noncatalytic region that contains sites for interactions with protein components of the RNA degradosome as well as RNA and phospholipid bilayers. In this review, RNase E homologs have been classified into five types based on their primary structure. A recent analysis has shown that Type I RNase E in the γ-Proteobacteria forms an orthologous group of proteins that has been inherited vertically. The RNase E catalytic core and a large ID noncatalytic region containing an RNA binding motif and a Membrane Targeting Sequence (MTS) are universally conserved features of these orthologs. Although the ID noncatalytic region has low composition and sequence complexity, it is possible to map microdomains, which are Short Linear Motifs (SLiMs) that are sites of interaction with protein and other ligands. Throughout Bacteria, the composition of the multienzyme RNA degradosome varies with species, but interactions with exoribonucleases (PNPase, RNase R), glycolytic enzymes (enolase, aconitase) and RNA helicases (DEAD-box proteins, Rho) are common. Plasticity in RNA degradosome composition is due to rapid evolution of RNase E microdomains. Characterization of the RNase E-PNPase interaction in α-Proteobacteria, γ-Proteobacteria and Cyanobacteria suggests that it arose independently several times during evolution, thus conferring an advantage in control and coordination of RNA processing and degradation. This article is protected by copyright. All rights reserved.
    Molecular Microbiology 06/2015; 97(6). DOI:10.1111/mmi.13095 · 4.42 Impact Factor
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    • "RNase E and polynucleotide phosphorylase (PNPase) are two major components of the RNA degradation process [7] [8]. "
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    ABSTRACT: The RNA chaperone Hfq in bacteria stabilizes sRNAs by protecting them from the attack of ribonucleases. Upon release from Hfq, sRNAs are preferably degraded by PNPase. PNPase usually forms multienzyme ribonucleolytic complexes with endoribonuclease E and/or RNA helicase RhlB to facilitate the degradation of the structured RNA. However, whether PNPase activity on Hfq-free sRNAs is associated with the assembly of RNase E or RhlB has yet to be determined. Here we examined the roles of the main endoribonucleases, exoribonucleases, and ancillary RNA-modifying enzymes in the degradation of Y. pestis RyhB in the absence of Hfq. Expectedly, the transcript levels of both RyhB1 and RyhB2 increase only after inactivating PNPase, which confirms the importance of PNPase in sRNA degradation. By contrast, the signal of RyhB becomes barely perceptible after inactivating of RNase III, which may be explained by the increase in PNPase levels resulting from the exemption of pnp mRNA from RNase III processing. No significant changes are observed in RyhB stability after deletion of either the PNPase-binding domain of RNase E or rhlB. Therefore, PNPase acts as a major enzyme of RyhB degradation independent of PNPase-containing RNase E and RhlB assembly in the absence of Hfq.
    04/2014; 2014:798918. DOI:10.1155/2014/798918
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    • "Analysis of degradosome assembly in strains expressing FLAG-tagged RNase E variants of 598, and 701 amino-acids lacking the carboxy-end region, shows there is no interaction with the canonical components [5]. Although resident components of RNAD, like the PNPase appear not to be exchanged under normal growth conditions once they are engaged in the complex [6] [7], the RNAD is believed to be a dynamic complex, capable of exchanging resident components under certain conditions. For instance, it has been reported that other enzymes such as the cold-shock RNA helicase CsdA is isolated from RNAD-like complexes at low temperature, and it can substitute RhlB in vitro in assays testing functional RNAD-like complexes . "
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    ABSTRACT: We report an analysis in vivo of the RNA Degradosome assembly of Escherichia coli. Employing fluorescence microscopy imaging and fluorescence energy transfer (FRET) measurements, we present evidence for in vivo pairwise interactions between RNase E-PNPase (polynucleotide phosphorylase), and RNase E-Enolase. These interactions are absent in a mutant strain with genomically encoded RNase E that lacks the C-terminal half, supporting the role of the carboxy-end domain as the scaffold for the degradosome. We also present data of in vivo pairwise interactions between Enolase-PNPase and Enolase-RhlB. The data support a model for the RNA Degradosome (RNAD), in which the RNase E carboxy-end is proximal to PNPase, more distant to Enolase, and more than 10 nm from RhlB helicase. Our measurements were made in strains with mono-copy chromosomal fusions of the RNAD enzymes with fluorescent proteins, allowing measurement of the expression of the different proteins under different growth and stress conditions.
    Biochimie 08/2013; 95(11). DOI:10.1016/j.biochi.2013.07.022 · 2.96 Impact Factor
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