The RNase E of Escherichia coli has at least two binding sites for DEAD-box RNA helicases: Functional replacement of RhlB by RhlE

Laboratoire de Microbiologie et Génétique Moléculaires, UMR 5100, Centre National de la Recherche Scientifique (CNRS) et Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France.
Molecular Microbiology (Impact Factor: 4.42). 01/2005; 54(5):1422-30. DOI: 10.1111/j.1365-2958.2004.04361.x
Source: PubMed


The non-catalytic region of Escherichia coli RNase E contains a protein scaffold that binds to the other components of the RNA degradosome. Alanine scanning yielded a mutation, R730A, that disrupts the interaction between RNase E and the DEAD-box RNA helicase, RhlB. We show that three other DEAD-box helicases, SrmB, RhlE and CsdA also bind to RNase E in vitro. Their binding differs from that of RhlB because it is not affected by the R730A mutation. Furthermore, the deletion of residues 791-843, which does not affect RhlB binding, disrupts the binding of SrmB, RhlE and CsdA. Therefore, RNase E has at least two RNA helicase binding sites. Reconstitution of a complex containing the protein scaffold of RNase E, PNPase and RhlE shows that RhlE can furnish an ATP-dependent activity that facilitates the degradation of structured RNA by PNPase. Thus, RhlE can replace the function of RhlB in vitro. The results in the accompanying article show that CsdA can also replace RhlB in vitro. Thus, RhlB, RhlE and CsdA are interchangeable in in vitro RNA degradation assays.

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Available from: Isabelle Toesca, Feb 03, 2015
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    • "Under standard conditions of laboratory growth, RNase E interacts with RhlB, enolase and PNPase (red) to form the canonical RNA degradosome. Non-canonical protein interactions include Hfq (light blue) (Ikeda et al., 2011); CsdA, SrmB and RhlE (light green) (Khemici et al., 2004b; Prud'homme-Genereux et al., 2004); RraA (pink) (Gao et al., 2006); RraB (purple) (Gorna et al., 2010); MinD (orange) (Taghbalout and Rothfield, 2007); RapZ (blue) (Gopel et al., 2013); PAPI (poly(A)polymerase) (green) (Raynal and Carpousis, 1999; Carabetta et al., 2010). Other non-canonical interactions, whose binding sites have not been mapped, are indicated in gray: RNase II (Lu and Taghbalout, 2014); RNase R (Carabetta et al., 2010); GroEL (Miczak et al., 1996); DnaK, and polyphosphate kinase (PPK) (Blum et al., 1997); ribosomal proteins such as S1, L4, L17 (Feng et al., 2001; Singh et al., 2009; Tsai et al., 2012). "
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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.
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    • "The Caulobacter crescentus degradosome, for example, contains aconitase instead of enolase (Hardwick et al. 2011). In E. coli, RhlB, CsdA and RhlE helicases are interchangeable in vitro (Khemici et al. 2004), and RhlB can be replaced by CsdA in the cold (Prud'homme-Genereux et al. 2004). Furthermore, a number of proteins have been shown to bind non-stoichiometrically to the degradosome in E. coli, to modulate or regulate its activity . "
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    • "In vitro DeaD directly associates with RNase E – the main endonuclease and the scaffold of the degradosome complex (Prud'homme- Généreux et al., 2004). RhlB – the other degradosome associated helicase – is still detected in the cold shock adapted degradosome, suggesting that either both helicases associate with RNase E (Khemici et al., 2004) or cold shocked cells have a heterogeneous population of degradosomes (Prud'homme-Généreux et al., 2004). "
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