Anastasia J. Callaghan

Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover

The coordinated regulation of gene expression is required for homeostasis, growth and development in all organisms. Such coordination may be partly achieved at the level of messenger RNA stability, in which the targeted destruction of subsets of transcripts generates the potential for cross-regulating metabolic pathways. In Escherichia coli, the balance and composition of the transcript population is affected by RNase E, an essential endoribonuclease that not only turns over RNA but also processes certain key RNA precursors. RNase E cleaves RNA internally, but its catalytic power is determined by the 5′ terminus of the substrate, even if this lies at a distance from the cutting site. Here we report crystal structures of the catalytic domain of RNase E as trapped allosteric intermediates with RNA substrates. Four subunits of RNase E catalytic domain associate into an interwoven quaternary structure, explaining why the subunit organization is required for catalytic activity. The subdomain encompassing the active site is structurally congruent to a deoxyribonuclease, making an unexpected link in the evolutionary history of RNA and DNA nucleases. The structure explains how the recognition of the 5′ terminus of the substrate may trigger catalysis and also sheds light on the question of how RNase E might selectively process, rather than destroy, specific RNA precursors.

Crystal Structure of Escherichia coli Polynucleotide Phosphorylase Core Bound to RNase E, RNA and Manganese: Implications for Catalytic Mechanism and RNA Degradosome Assembly

Polynucleotide phosphorylase (PNPase) is a processive exoribonuclease that contributes to messenger RNA turnover and quality control of ribosomal RNA precursors in many bacterial species. In Escherichia coli, a proportion of the PNPase is recruited into a multi-enzyme assembly, known as the RNA degradosome, through an interaction with the scaffolding domain of the endoribonuclease RNase E. Here, we report crystal structures of E. coli PNPase complexed with the recognition site from RNase E and with manganese in the presence or in the absence of modified RNA. The homotrimeric PNPase engages RNase E on the periphery of its ring-like architecture through a pseudo-continuous anti-parallel β-sheet. A similar interaction pattern occurs in the structurally homologous human exosome between the Rrp45 and Rrp46 subunits. At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites. Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state. We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.

The Crystal Structure of the Escherichia coli RNase E Apoprotein and a Mechanism for RNA Degradation

Summary RNase E is an essential bacterial endoribonuclease involved in the turnover of messenger RNA and the maturation of structured RNA precursors in Escherichia coli. Here, we present the crystal structure of the E. coli RNase E catalytic domain in the apo-state at 3.3 Å. This structure indicates that, upon catalytic activation, RNase E undergoes a marked conformational change characterized by the coupled movement of two RNA-binding domains to organize the active site. The structural data suggest a mechanism of RNA recognition and cleavage that explains the enzymes preference for substrates possessing a 5′-monophosphate and accounts for the protective effect of a triphosphate cap for most transcripts. Internal flexibility within the quaternary structure is also observed, a finding that has implications for recognition of structured RNA substrates and for the mechanism of internal entry for a subset of substrates that are cleaved without 5′-end requirements.

The regulatory protein RraA modulates RNA-binding and helicase activities of the E. coli RNA degradosome.

The Escherichia coli endoribonuclease RNase E is an essential enzyme having key roles in mRNA turnover and the processing of several structured RNA precursors, and it provides the scaffold to assemble the multienzyme RNA degradosome. The activity of RNase E is inhibited by the protein RraA, which can interact with the ribonucleases degradosome-scaffolding domain. Here, we report that RraA can bind to the RNA helicase component of the degradosome (RhlB) and the two RNA-binding sites in the degradosome-scaffolding domain of RNase E. In the presence of ATP, the helicase can facilitate the exchange of RraA for RNA stably bound to the degradosome. Our data suggest that RraA can affect multiple components of the RNA degradosome in a dynamic, energy-dependent equilibrium. The multidentate interactions of RraA impede the RNA-binding and ribonuclease activities of the degradosome and may result in complex modulation and rerouting of degradosome activity.

Recognition of the 70S ribosome and polysome by the RNA degradosome in Escherichia coli

The RNA degradosome is a multi-enzyme assembly that contributes to key processes of RNA metabolism, and it engages numerous partners in serving its varied functional roles. Small domains within the assembly recognize collectively a diverse range of macromolecules, including the core protein components, the cytoplasmic lipid membrane, mRNAs, non-coding regulatory RNAs and precursors of structured RNAs. We present evidence that the degradosome can form a stable complex with the 70S ribosome and polysomes, and we demonstrate the proximity in vivo of ribosomal proteins and the scaffold of the degradosome, RNase E. The principal interactions are mapped to two, independent, RNA-binding domains from RNase E. RhlB, the RNA helicase component of the degradosome, also contributes to ribosome binding, and this is favoured through an activating interaction with RNase E. The catalytic activity of RNase E for processing 9S RNA (the ribosomal 5S RNA precursor) is repressed in the presence of the ribosome, whereas there is little affect on the cleavage of single-stranded substrates mediated by non-coding RNA, suggestings that the enzyme retains capacity to cleave unstructured substrates when associated with the ribosome. We propose that polysomes may act as antennae that enhance the rates of capture of the limited number of degradosomes, so that they become recruited to sites of active translation to act on mRNAs as they become exposed or tagged for degradation.

Polynucleotide phosphorylase activity may be modulated by metabolites in Escherichia coli

RNA turnover is an essential element of cellular homeostasis and response to environmental change. Whether the ribonucleases that mediate RNA turnover can respond to cellular metabolic status is an unresolved question. Here we present evidence that the Krebs cycle metabolite citrate affects the activity of Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that cellular metabolism is affected widely by PNPase activity. An E. coli strain that requires PNPase for viability has suppressed growth in the presence of increased citrate concentration. Transcriptome analysis reveals a PNPase-mediated response to citrate, and PNPase deletion broadly impacts on the metabolome. In vitro, citrate directly binds and modulates PNPase activity, as predicted by crystallographic data. Binding of metal-chelated citrate in the active site at physiological concentrations appears to inhibit enzyme activity. However, metal-free citrate is bound at a vestigial active site, where it stimulates PNPase activity. Mutagenesis data confirmed a potential role of this vestigial site as an allosteric binding pocket that recognizes metal-free citrate. Collectively, these findings suggest that RNA degradative pathways communicate with central metabolism. This communication appears to be part of a feedback network that may contribute to global regulation of metabolism and cellular energy efficiency.

Hfq binding changes the structure of Escherichia coli small noncoding RNAs OxyS and RprA, which are involved in the riboregulation of rpoS.

OxyS and RprA are two small noncoding RNAs (sRNAs) that modulate the expression of rpoS, encoding an alternative sigma factor that activates transcription of multiple Escherichia coli stress-response genes. While RprA activates rpoS for translation, OxyS down-regulates the transcript. Crucially, the RNA binding protein Hfq is required for both sRNAs to function, although the specific role played by Hfq remains unclear. We have investigated RprA and OxyS interactions with Hfq using biochemical and biophysical approaches. In particular, we have obtained the molecular envelopes of the Hfq-sRNA complexes using small-angle scattering methods, which reveal key molecular details. These data indicate that Hfq does not substantially change shape upon complex formation, whereas the sRNAs do. We link the impact of Hfq binding, and the sRNA structural changes induced, to transcript stability with respect to RNase E degradation. In light of these findings, we discuss the role of Hfq in the opposing regulatory functions played by RprA and OxyS in rpoS regulation.

The low-resolution solution structure of Vibrio cholerae Hfq in complex with Qrr1 sRNA

In Vibrio cholerae, the RNA binding protein and chaperone Hfq (VcHfq) facilitates the pairing of the quorum regulatory RNA (Qrr) small regulatory RNAs (sRNAs) to the 5′ untranslated regions of the mRNAs for a number of global regulators that modulate the expression of virulence genes. This Qrr-mediated sRNA circuit is an attractive antimicrobial target, but characterization at the molecular level is required for this to be realized. Here, we investigate the interactions between VcHfq and the Qrr sRNAs using a variety of biochemical and biophysical techniques. We show that the ring-shaped VcHfq hexamer binds the Qrrs with 1:1 stoichiometry through its proximal face, and the molecular envelope of the VcHfq-Qrr complex is experimentally determined from small angle scattering data to present the first structural glimpse of a Hfq-sRNA complex. This structure reveals that the VcHfq protein does not change shape on complex formation but the RNA does, suggesting that a chaperone role for VcHfq is a critical part of the VcHfq-Qrr interaction. Overall, these studies enhance our understanding of VcHfq-Qrr interactions.

Characterization of Vibrio cholerae Hfq Provides Novel Insights into the Role of the Hfq C-Terminal Region

Hfq is a bacterial RNA binding protein that facilitates small RNA-mediated posttranscriptional gene regulation. In Vibrio cholerae, Hfq and four Hfq-dependent small RNAs are essential for the expression of virulence genes, but little is known about this mechanism at the molecular level. To better understand V. cholerae Hfq structure and mechanism, we characterized the protein, alongside Escherichia coli Hfq for comparison, using biochemical and biophysical techniques. The N-terminal domain (NTD) of the two proteins is highly conserved, but the C-terminal regions (CTRs) vary in both sequence and length. Small-angle X-ray scattering studies showed that both proteins adopt a star-shaped hexameric structure in which the conserved NTD adopts the expected Sm fold while the variable CTR is disordered and extends radially outwards from the folded core. Despite their structural similarity, SDS-PAGE stability assays and collision-induced dissociation mass spectrometry revealed that the V. cholerae hexamer is less stable than that of E. coli. We propose that this is due to minor differences between the intersubunit interface formed by the NTDs and the ability of the E. coli CTR to stabilize this interface. However, based on electrophoretic mobility shift assays, the divergent CTRs do appear to perform a common function with regard to RNA-binding specificity. Overall, the similarities and differences in the fundamental properties of V. cholerae and E. coli Hfq provide insight into their assembly and molecular mechanisms.

The First Small-Molecule Inhibitors of Members of the Ribonuclease E Family

The Escherichia coli endoribonuclease RNase E is central to the processing and degradation of all types of RNA and as such is a pleotropic regulator of gene expression. It is essential for growth and was one of the first examples of an endonuclease that can recognise the 5′-monophosphorylated ends of RNA thereby increasing the efficiency of many cleavages. Homologues of RNase E can be found in many bacterial families including important pathogens, but no homologues have been identified in humans or animals. RNase E represents a potential target for the development of new antibiotics to combat the growing number of bacteria that are resistant to antibiotics in use currently. Potent small molecule inhibitors that bind the active site of essential enzymes are proving to be a source of potential drug leads and tools to dissect function through chemical genetics. Here we report the use of virtual high-throughput screening to obtain small molecules predicted to bind at sites in the N-terminal catalytic half of RNase E. We show that these compounds are able to bind with specificity and inhibit catalysis of Escherichia coli and Mycobacterium tuberculosis RNase E and also inhibit the activity of RNase G, a paralogue of RNase E.