Reconstitution of a minimal RNA degradosome demonstrates functional coordination between a 3' exonuclease and a DEAD-box RNA helicase

Coburn, Glen A.; Miao, Xin; Briant, Douglas J.; Mackie, George A.

doi:10.1101/gad.13.19.2594

Cited by 166 publications

(176 citation statements)

References 39 publications

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“…For example, PNPase and RNase E cooperate in the degradation of RNA I, an antisense regulator of replication of ColE1-type plasmids (25), and RhlB helicase can stimulate degradation of the malE-malF transcript by PNPase in vitro (1). Studies of reconstituted degradosomes in vitro (26,27) have yielded analogous results. However, degradosome proteins can remain unattached to RNase E (2, 11); even in bacteria that have the ability to form the assembled degradosome complex, only 5-10% of cellular enolase and 10-20% of PNPase are estimated to be present in the complex (1,11).…”

supporting

confidence: 52%

Global analysis of Escherichia coli RNA degradosome function using DNA microarrays

Bernstein

Lin

Cohen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

207

212

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RNase E, an essential endoribonuclease of Escherichia coli, interacts through its C-terminal region with multiple other proteins to form a complex termed the RNA degradosome. To investigate the degradosome's proposed role as an RNA decay machine, we used DNA microarrays to globally assess alterations in the steady-state abundance and decay of 4,289 E. coli mRNAs at single-gene resolution in bacteria carrying mutations in the degradosome constituents RNase E, polynucleotide phosphorylase, RhlB helicase, and enolase. Our results show that the functions of all four of these proteins are necessary for normal mRNA turnover. We identified specific transcripts and functionally distinguishable transcript classes whose half-life and abundance were affected congruently by multiple degradosome proteins, affected differentially by mutations in degradosome constituents, or not detectably altered by degradosome mutations. Our results, which argue that decay of some E. coli mRNAs in vivo depends on the action of assembled degradosomes, whereas others are acted on by degradosome proteins functioning independently of the complex, imply the existence of structural features or biochemical factors that target specific classes of mRNAs for decay by degradosomes.RNase E ͉ rhlB helicase ͉ enolase ͉ polynucleotide phosphorylase T he turnover of mRNA is a necessary component of normal genetic regulation within cells. In eubacteria, mRNA degradation results from the combined action of endo-and exoribonucleases. In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the RhlB RNA helicase, and the glycolytic enzyme enolase assemble on the C-terminal region of the endoribonuclease RNase E as constituents of a protein complex termed the RNA degradosome (refs. 1-4; for recent reviews, see refs. 5 and 6). Two heat shock proteins, GroEL and DnaK (4), and polyphosphate kinase (Ppk) (7) also are associated with degradosomes in substoichiometric amounts. The N-terminal half of RNase E, which contains the catalytic domain of the enzyme (8), is not sufficient for degradosome formation (3, 9) but can associate the degradosome protein complex with the cytoplasmic membrane (11).The E. coli ams͞rne locus, which encodes RNase E, is required for bulk RNA turnover (12, 13) as well as for the processing of 9S rRNA (14,15). Investigations of the decay of individual transcripts (16)(17)(18)(19) and global investigations of mRNA abundance in rne mutants (20) have indicated a broadly important role for this enzyme. However, the RNase E region that provides a scaffold for degradosome formation is not essential for cell survival and growth (17,21,22), and truncated RNase E proteins lacking this domain are active in vivo as well as in vitro (8,23).Although the degradosome commonly has been viewed as an RNA decay ''machine'' (e.g., ref. 24), until recently (11) it was not known whether assembled degradosomes actually exist as such in living cells. Moreover, whether degradosome formation is a significant factor in mRNA decay in vivo has been...

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supporting

confidence: 52%

Global analysis of Escherichia coli RNA degradosome function using DNA microarrays

Bernstein

Lin

Cohen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

207

212

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“…Other RNAdegrading complexes, including the exosome, 31,32 have been identi®ed in eukaryotic organisms and are not known to contain enolase. Also the observation that a functional minimal degradosome consisting of RNase E, PNPase and RhlB could be assembled in vitro 11,33 does not support the idea of a crucial role of enolase in RNA degradation. It seems more likely that enolase may play a structural role in the E. coli degradosome, and perhaps an allosteric role to affect the activity of its linked partners.…”

Section: Discussionmentioning

confidence: 84%

“…A minimal degradosome, assembled from all components except enolase, is capable of degrading RNA, indicating that enolase is not crucial for the complex's RNA degradation function in vitro. 11 However, it is possible that enolase might play a subtle structural role in the complete assembly or it could serve to couple glycolytic and RNA degradative processes in vivo through allosteric effects.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of the Escherichia coli RNA degradosome component enolase

Kühnel

Luisi

2001

Journal of Molecular Biology

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The crystal structure of Escherichia coli enolase (EC 4.2.1.11, phosphopyruvate hydratase), which is a component of the RNA degradosome, has been determined at 2.5 A Ê . There are four molecules in the asymmetric unit of the C2 cell, and in one of the molecules,¯exible loops close onto the active site. This closure mimics the conformation of the substratebound intermediate. A comparison of the structure of the E. coli enolase with the eukaryotic enolase structures available (lobster and yeast) indicates a high degree of conservation of the hydrophobic core and the subunit interface of this homodimeric enzyme. The dimer interface is enriched in charged residues compared with other protein homodimers, which may explain our observations from analytical ultracentrifugation that dimerisation is affected by ionic strength. The putative role of enolase in the RNA degradosome is discussed; although it was not possible to ascribe a speci®c role to it, a structural role is possible.

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“…The stimulating effect of RNase E on RhlB does not require the RNA-binding sites of the ribonuclease that flank the helicase interaction site (18), suggesting that the boost of ATPase activity is not caused indirectly by increased recruitment of RNA. Indeed, we observe that the RNase E/RhlB interaction actually decreases the RNA affinity of the helicase.…”

mentioning

confidence: 99%

Allosteric Activation of the ATPase Activity of the Escherichia coli RhlB RNA Helicase

Worrall

Howe

McKay

et al. 2008

Journal of Biological Chemistry

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Helicase B (RhlB) is one of the five DEAD box RNA-dependent ATPases found in Escherichia coli. Unique among these enzymes, RhlB requires an interaction with the partner protein RNase E for appreciable ATPase and RNA unwinding activities. To explore the basis for this activating effect, we have generated a di-cistronic vector that overexpresses a complex comprising RhlB and its recognition site within RNase E, corresponding to residues 696 -762. Complex formation has been characterized by isothermal titration calorimetry, revealing an avid, enthalpyfavored interaction between the helicase and RNase E-(696 -762) with an equilibrium binding constant (K a ) of at least 1 ؋ 10 8 M ؊1 . We studied ATPase activity of mutants with substitutions within the ATP binding pocket of RhlB and on the putative interaction surface that mediates recognition of RNase E. For comparisons, corresponding mutations were prepared in two other E. coli DEAD box ATPases, RhlE and SrmB. Strikingly, substitutions at a phenylalanine near the Q-motif found in DEAD box proteins boosts the ATPase activity of RhlB in the absence of RNA, but completely inhibits it in its presence. The data support the proposal that the protein-protein and RNA-binding surfaces both communicate allosterically with the ATPase catalytic center. We conjecture that this communication may govern the mechanical power and efficiency of the helicases, and is tuned in individual helicases in accordance with cellular function.RNA helicases are a diverse set of proteins found in all three kingdoms of life that possess the ability to unwind short stretches of RNA duplexes in reactions that require the hydrolysis of nucleoside triphosphates (1). They are the largest group of enzymes in eukaryotic RNA metabolism and are involved in virtually all aspects of cellular RNA manipulation including transcription, splicing, RNA nuclear export, and ribosome biogenesis (2, 3). The DEAD box helicases are grouped in helicase superfamily 2, whose members contain nine conserved motifs, including the Walker B motif DEAD from which they take their common name (Fig. 1). Escherichia coli contains five genes encoding for DEAD box proteins; csdA (formerly called deaD), dbpA, rhlB, rhlE, and srmB (4). The gene products have a common core of 350 -400 amino acids, consisting of two domains that have the same fold found in the DNA-recombination protein RecA. Flanking this two-domain core are variable regions that are thought to be responsible for the differing properties and functions of the five helicases (4).DEAD box helicases can unwind short RNA duplexes that are no more than two helical turns in length (ϳ22 bp), and some helicases can displace avidly bound proteins from RNA (1, 5). A role of DEAD box RNA helicases as nonspecific RNA unfolding enzymes has been described (6). Yang and Jankowsky (7) have elegantly shown that DEAD box helicases use a single strand RNA region to facilitate loading onto duplex RNA where only a few base pairs are disrupted in an ATP-dependent manner, leading to destabiliza...

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Reconstitution of a minimal RNA degradosome demonstrates functional coordination between a 3' exonuclease and a DEAD-box RNA helicase

Cited by 166 publications

References 39 publications

Global analysis of Escherichia coli RNA degradosome function using DNA microarrays

Global analysis of Escherichia coli RNA degradosome function using DNA microarrays

Crystal structure of the Escherichia coli RNA degradosome component enolase

Allosteric Activation of the ATPase Activity of the Escherichia coli RhlB RNA Helicase

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