Agamemnon J. Carpousis

The RNA degradosome and poly(A) polymerase of Escherichia coli are required in vivo for the degradation of small mRNA decay intermediates containing REP-stabilizers

In Escherichia coli, REP‐stabilizers are structural elements in polycistronic messages that protect 5′‐proximal cistrons from 3′→5′ exonucleolytic degradation. The stabilization of a protected cistron can be an important determinant in the level of gene expression. Our results suggest that RNase E, an endoribonuclease, initiates the degradation of REP‐stabilized mRNA. However, subsequent degradation of mRNA fragments containing a REP‐stabilizer poses a special challenge to the mRNA degradation machinery. Two enzymes, the DEAD‐box RNA helicase, RhlB and poly(A) polymerase (PAP) are required to facilitate the degradation of REP‐stabilizers by polynucleotide phosphorylase (PNPase). This is the first in vivo evidence that these enzymes are required for the degradation of REP‐stabilizers. Furthermore, our results show that REP degradation by RhlB and PNPase requires their association with RNase E as components of the RNA degradosome, thus providing the first in vivo evidence that this ribonucleolytic multienzyme complex is involved in the degradation of structured mRNA fragments.

The RNase E of Escherichia coli is a membrane-binding protein.

RNase E is an essential endoribonuclease involved in RNA processing and mRNA degradation. The N‐terminal half of the protein encompasses the catalytic domain; the C‐terminal half is the scaffold for the assembly of the multienzyme RNA degradosome. Here we identify and characterize ‘segment‐A’, an element in the beginning of the non‐catalytic region of RNase E that is required for membrane binding. We demonstrate in vitro that an oligopeptide corresponding to segment‐A has the propensity to form an amphipathic α‐helix and that it avidly binds to protein‐free phospholipid vesicles. We demonstrate in vitro and in vivo that disruption of segment‐A in full‐length RNase E abolishes membrane binding. Taken together, our results show that segment‐A is necessary and sufficient for RNase E binding to membranes. Strains in which segment‐A has been disrupted grow slowly. Since in vitro experiments show that phospholipid binding does not affect the ribonuclease activity of RNase E, the slow‐growth phenotype might arise from a defect involving processes such as accessibility to substrates or interactions with other membrane‐bound machinery. This is the first report demonstrating that RNase E is a membrane‐binding protein and that its localization to the inner cytoplasmic membrane is important for normal cell growth.

Polyphosphate kinase is a component of the Escherichia coli RNA degradosome

The Escherichia coli degradosome is a multienzyme complex with four major protein components: the endoribonuclease RNase E, the exoribonuclease PNPase, the RNA helicase RhlB and enolase. The first three of these proteins are known to have important functions in mRNA processing and degradation. In this work, we identify an additional component of the degradosome, polyphosphate kinase (PPK), which catalyses the reversible polymerization of the γ‐phosphate of ATP into polyphosphate (poly(P)). An E. coli strain deleted for the ppk gene showed increased stability of the ompA mRNA. Purified His‐tagged PPK was shown to bind RNA, and RNA binding was prevented by hydrolysable ATP. Chemical modification of RNA by PPK, for example the addition or removal of 3′ or 5′ terminal phosphates, could not be detected. However, polyphosphate was found to inhibit RNA degradation by the degradosome in vitro. This inhibition was overcome by the addition of ADP, required for the degradation of polyphosphate and for the regeneration of ATP by PPK in the degradosome. Thus, PPK in the degradosome appears to maintain an appropriate microenvironment, removing inhibitory polyphosphate and NDPs and regenerating ATP.

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

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.

Function in Escherichia coli of the non-catalytic part of RNase E: role in the degradation of ribosome-free mRNA

Summary RNase E contains a large non‐catalytic region that binds RNA and the protein components of the Escherichia coli RNA degradosome. The rne gene was replaced with alleles encoding deletions in the non‐catalytic part of RNase E. All the proteins are stable in vivo. RNase E activity was tested using a PT7–lacZ reporter gene, the message of which is particularly sensitive to degradation because translation is uncoupled from transcription. The non‐catalytic region has positive and negative effectors of mRNA degradation. Disrupting RhlB and enolase binding resulted in hypoactivity, whereas disrupting PNPase binding resulted in hyperactivity. Expression of the mutant proteins in vivo anticorrelates with activity showing that autoregulation compensates for defective function. There is no simple correlation between RNA binding and activity in vivo. An allele (rne131), expressing the catalytic domain alone, was put under Plac control. In contrast to rne+, low expression of rne131 severely affects growth. Even with autoregulation, all the mutants are less fit when grown in competition with wild type. Although the catalytic domain of RNase E is sufficient for viability, our work demonstrates that elements in the non‐catalytic part are necessary for normal activity in vivo.

Poly(A) polymerase I of Escherichia coli: characterization of the catalytic domain, an RNA binding site and regions for the interaction with proteins involved in mRNA degradation

Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) superfamily that includes the eukaryotic PAPs and all the known tRNA CCA‐adding enzymes. Five highly conserved aspartic acids in the putative catalytic site of PAP I were changed to either alanine or proline, demonstrating their importance for polymerase activity. A glycine that is absolutely conserved in all Ntrs was also changed yielding a novel mutant protein in which ATP was wastefully hydrolysed in a primer‐independent reaction. This is the first work to characterize the catalytic site of a eubacterial PAP and, despite the conservation of certain sequences, we predict that the overall architecture of the eukaryotic and eubacterial active sites is likely to be different. Binding sites for RNase E, a component of the RNA degradosome, and RNA were mapped by North‐western and Far‐western blotting using truncated forms of PAP I. Additional protein–protein interactions were detected between PAP I and CsdA, RhlE and SrmB, suggesting an unexpected connection between PAP I and these E. coli DEAD box RNA helicases. These results show that the functional organization of PAP I is similar to the eukaryotic PAPs with an N‐terminal catalytic domain, a C‐terminal RNA binding domain and sites for the interaction with other protein factors.

The K-loop, a general feature of the Pyrococcus C/D guide RNAs, is an RNA structural motif related to the K-turn

The C/D guide RNAs predicted from the genomic sequences of three species of Pyrococcus delineate a family of small non-coding archaeal RNAs involved in the methylation of rRNA and tRNA. The C/D guides assemble into ribonucleoprotein (RNP) that contains the methyltransferase. The protein L7Ae, a key structural component of the RNP, binds to a Kink-turn (K-turn) formed by the C/D motif. The K-turn is a structure that consists of two RNA stems separated by a short asymmetric loop with a characteristic sharp bend (kink) between the two stems. The majority of the pyrococcal C/D guides contain a short 3 nt-spacer between the C′/D′ motifs. We show here that conserved terminal stem–loops formed by the C′/D′ motif of the Pyrococcus C/D RNAs are also L7Ae-binding sites. These stem–loops are related to the K-turn by sequence and structure, but they consist of a single stem closed by a terminal loop. We have named this structure the K-loop. We show that conserved non-canonical base pairs in the stem of the K-loop are necessary for L7Ae binding. For the C/D guides with a 3 nt-spacer we show that the sequence and length is also important. The K-loop could improve the stability of the C/D guide RNAs in Pyrococcal species, which are extreme hyperthermophiles.

From conformational chaos to robust regulation: the structure and function of the multi-enzyme RNA degradosome

The RNA degradosome is a massive multi-enzyme assembly that occupies a nexus in RNA metabolism and post-transcriptional control of gene expression in Escherichia coli and many other bacteria. Powering RNA turnover and quality control, the degradosome serves also as a machine for processing structured RNA precursors during their maturation. The capacity to switch between destructive and processing modes involves cooperation between degradosome components and is analogous to the process of RNA surveillance in other domains of life. Recruitment of components and cellular compartmentalisation of the degradosome are mediated through small recognition domains that punctuate a natively unstructured segment within a scaffolding core. Dynamic in conformation, variable in composition and non-essential under certain laboratory conditions, the degradosome has nonetheless been maintained throughout the evolution of many bacterial species, due most likely to its diverse contributions in global cellular regulation. We describe the role of the degradosome and its components in RNA decay pathways in E. coli, and we broadly compare these pathways in other bacteria as well as archaea and eukaryotes. We discuss the modular architecture and molecular evolution of the degradosome, its roles in RNA degradation, processing and quality control surveillance, and how its activity is regulated by non-coding RNA. Parallels are drawn with analogous machinery in organisms from all life domains. Finally, we conjecture on roles of the degradosome as a regulatory hub for complex cellular processes.

The social fabric of the RNA degradosome.

Bacterial transcripts each have a characteristic half-life, suggesting that the processes of RNA degradation work in an active and selective manner. Moreover, the processes are well controlled, thereby ensuring that degradation is orderly and coordinated. Throughout much of the bacterial kingdom, RNA degradation processes originate through the actions of assemblies of key RNA enzymes, known as RNA degradosomes. Neither conserved in composition, nor unified by common evolutionary ancestry, RNA degradosomes nonetheless can be found in divergent bacterial lineages, implicating a common requirement for the co-localisation of RNA metabolic activities. We describe how the cooperation of components in the representative degradosome of Escherichia coli may enable controlled access to transcripts, so that they have defined and programmable lifetimes. We also discuss how this cooperation contributes to precursor processing and to the riboregulation of intricate post-transcriptional networks in the control of gene expression. The E. coli degradosome interacts with the cytoplasmic membrane, and we discuss how this interaction may spatially organise the assembly and contribute to subunit cooperation and substrate capture. This article is part of a Special Issue entitled: RNA Decay mechanisms.

RNase II levels change according to the growth conditions: characterization of gmr, a new Escherichia coli gene involved in the modulation of RNase II.

In Escherichia coli, ribonucleases are effectors that rapidly modulate the levels of mRNAs for adaptation to a changing environment. Factors involved in the regulation of these ribonucleases can be relevant for mRNA stability. RNase II is one of the main ribonucleases responsible for exonucleolytic activity in E. coli extracts. We have identified and characterized a new E. coli gene, which was named gmr (gene modulating RNase II). The results demonstrate that a deletion of gmr can be associated with changes in RNase II levels and activity. Western analysis and exoribonuclease activity assays showed a threefold increase in RNase II in the gmr deletion strain. Gmr does not affect RNase II mRNA, but modulates RNase II at the level of protein stability. RNase II protein turnover is slower in the gmr deletion strain. We also show that RNase II levels change in different media, and that this regulation is abolished in a strain lacking gmr. The data presented here show that the regulation of ribonucleolytic activity can depend on growth conditions, and this regulation can be mediated by factors that are not RNases.