Structural elucidation of a novel mechanism for the bacteriophage-based inhibition of the RNA degradosome.

In all domains of life, the catalysed degradation of RNA facilitates rapid adaptation to changing environmental conditions, while destruction of foreign RNA is an important mechanism to prevent host infection. We have identified a virus-encoded protein termed gp37/Dip, which directly binds and inhibits the RNA degradation machinery of its bacterial host. Encoded by giant phage фKZ, this protein associates with two RNA binding sites of the RNase E component of the Pseudomonas aeruginosa RNA degradosome, occluding them from substrates and resulting in effective inhibition of RNA degradation and processing. The 2.2 Å crystal structure reveals that this novel homo-dimeric protein has no identifiable structural homologues. Our biochemical data indicate that acidic patches on the convex outer surface bind RNase E. Through the activity of Dip, фKZ has evolved a unique mechanism to down regulate a key metabolic process of its host to allow accumulation of viral RNA in infected cells.Fonds Wetenschappelijk Onderzoek (Grant ID: G.0599.11); Agentschap voor Innovatie door Wetenschap en Technologie (Grant ID: SBO 100042); Fonds Wetenschappelijk Onderzoek (Scholarship); Wellcome Trust (Scholarship); Onderzoeksraad, KU Leuven (Grant ID: GOA Bacteriophage Biosystems); Onderzoeksraad, KU Leuven (Grant ID: CREA/09/017)This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.7554/eLife.1641

RNase E and the High-Fidelity Orchestration of RNA Metabolism.

The bacterial endoribonuclease RNase E occupies a pivotal position in the control of gene expression, as its actions either commit transcripts to an irreversible fate of rapid destruction or unveil their hidden functions through specific processing. Moreover, the enzyme contributes to quality control of rRNAs. The activity of RNase E can be directed and modulated by signals provided through regulatory RNAs that guide the enzyme to specific transcripts that are to be silenced. Early in its evolutionary history, RNase E acquired a natively unfolded appendage that recruits accessory proteins and RNA. These accessory factors facilitate the activity of RNase E and include helicases that remodel RNA and RNA-protein complexes, and polynucleotide phosphorylase, a relative of the archaeal and eukaryotic exosomes. RNase E also associates with enzymes from central metabolism, such as enolase and aconitase. RNase E-based complexes are diverse in composition, but generally bear mechanistic parallels with eukaryotic machinery involved in RNA-induced gene regulation and transcript quality control. That these similar processes arose independently underscores the universality of RNA-based regulation in life. Here we provide a synopsis and perspective of the contributions made by RNase E to sustain robust gene regulation with speed and accuracy.Wellcome Trus

Characterization of the RNA Degradosome of Pseudoalteromonas haloplanktis: Conservation of the RNase E-RhlB Interaction in the Gammaproteobacteria ▿

The degradosome is a multienzyme complex involved in mRNA degradation in Escherichia coli. The essential endoribonuclease RNase E contains a large noncatalytic region necessary for protein-protein interactions with other components of the RNA degradosome. Interacting proteins include the DEAD-box RNA helicase RhlB, the glycolytic enzyme enolase, and the exoribonuclease PNPase. Pseudoalteromonas haloplanktis, a psychrotolerant gammaproteobacterium distantly related to E. coli, encodes homologs of each component of the RNA degradosome. In P. haloplanktis, RNase E associates with RhlB and PNPase but not enolase. Plasmids expressing P. haloplanktis RNase E (Ph-RNase E) can complement E. coli strains lacking E. coli RNase E (Ec-RNase E). Ph-RNase E, however, does not confer a growth advantage to E. coli at low temperature. Ph-RNase E has a heterologous protein-protein interaction with Ec-RhlB but not with Ec-enolase or Ec-PNPase. The Ph-RNase E binding sites for RhlB and PNPase were mapped by deletion analysis. The PNPase binding site is located at the C-terminal end of Ph-RNase E at the same position as that in Ec-RNase E, but the sequence of the site is not conserved. The sequence of the RhlB binding site in Ph-RNase E is related to the sequence in Ec-RNase E. Together with the heterologous interaction between Ph-RNase E and Ec-RhlB, our results suggest that the underlying structural motif for the RNase E-RhlB interaction is conserved. Since the activity of Ec-RhlB requires its physical interaction with Ec-RNase E, conservation of the underlying structural motif over a large evolutionary distance could be due to constraints involved in the control of RhlB activity

Gene autoregulation by 3’ UTR-derived bacterial small RNAs

Negative feedback regulation, that is the ability of a gene to repress its own synthesis, is the most abundant regulatory motif known to biology. Frequently reported for transcriptional regulators, negative feedback control relies on binding of a transcription factor to its own promoter. Here, we report a novel mechanism for gene autoregulation in bacteria relying on small regulatory RNA (sRNA) and the major endoribonuclease, RNase E. TIER-seq analysis (transiently-inactivating-an-endoribonuclease-followed-by-RNA-seq) revealed similar to 25,000 RNase E-dependent cleavage sites in Vibrio cholerae, several of which resulted in the accumulation of stable sRNAs. Focusing on two examples, OppZ and CarZ, we discovered that these sRNAs are processed from the 3' untranslated region (3' UTR) of the oppABCDF and carAB operons, respectively, and basepair with their own transcripts to inhibit translation. For OppZ, this process also triggers Rho-dependent transcription termination. Our data show that sRNAs from 3' UTRs serve as autoregulatory elements allowing negative feedback control at the post-transcriptional level