Helen A. Vincent

Substrate Recognition and Catalysis by the Exoribonuclease RNase R

RNase R is a processive, 3′ to 5′ hydrolytic exoribonuclease that together with polynucleotide phosphorylase plays an important role in the degradation of structured RNAs. However, RNase R differs from other exoribonucleases in that it can by itself degrade RNAs with extensive secondary structure provided that a single-stranded 3′ overhang is present. Using a variety of specifically designed substrates, we show here that a 3′ overhang of at least 7 nucleotides is required for tight binding and activity, whereas optimum binding and activity are achieved when the overhang is 10 or more nucleotides in length. In contrast, duplex RNAs with no overhang or with a 4-nucleotide overhang bind extremely poorly to RNase R and are inactive as substrates. A duplex RNA with a 10-nucleotide 5′ overhang also is not a substrate. Interestingly, this molecule is bound only weakly, indicating that RNase R does not simply recognize single-stranded RNA, but the RNA must thread into the enzyme with 3′ to 5′ polarity. We also show that ribose moieties are required for recognition of the substrate as a whole since RNase R is unable to bind or degrade single-stranded DNA. However, RNA molecules with deoxyribose or dideoxyribose residues at their 3′ termini can be bound and degraded. Based on these data and a homology model of RNase R, derived from the structure of the closely related enzyme, RNase II, we present a model for how RNase R interacts with its substrates and degrades RNA.

Insights Into How RNase R Degrades Structured RNA: Analysis of the Nuclease Domain

RNase R readily degrades highly structured RNA, whereas its paralogue, RNase II, is unable to do so. Furthermore, the nuclease domain of RNase R, devoid of all canonical RNA-binding domains, is sufficient for this activity. RNase R also binds RNA more tightly within its catalytic channel than does RNase II, which is thought to be important for its unique catalytic properties. To investigate this idea further, certain residues within the nuclease domain channel of RNase R were changed to those found in RNase II. Among the many examined, we identified one amino acid residue, R572, that has a significant role in the properties of RNase R. Conversion of this residue to lysine, as found in RNase II, results in weaker substrate binding within the nuclease domain channel, longer limit products, increased activity against a variety of substrates and a faster substrate on-rate. Most importantly, the mutant encounters difficulty in degrading structured RNA, pausing within a double-stranded region. Additional studies show that degradation of structured substrates is dependent upon temperature, suggesting a role for thermal breathing in the mechanism of action of RNase R. On the basis of these data, we propose a model in which tight binding within the nuclease domain allows RNase R to capitalize on the natural thermal breathing of an RNA duplex to degrade structured RNAs.

The roles of individual domains of RNase R in substrate binding and exoribonuclease activity. The nuclease domain is sufficient for digestion of structured RNA

RNase R and RNase II are the two representatives from the RNR family of processive, 3′ to 5′ exoribonucleases in Escherichia coli. Although RNase II is specific for single-stranded RNA, RNase R readily degrades through structured RNA. Furthermore, RNase R appears to be the only known 3′ to 5′ exoribonuclease that is able to degrade through double-stranded RNA without the aid of a helicase activity. Consequently, its functional domains and mechanism of action are of great interest. Using a series of truncated RNase R proteins we show that the cold-shock and S1 domains contribute to substrate binding. The cold-shock domains appear to play a role in substrate recruitment, whereas the S1 domain is most likely required to position substrates for efficient catalysis. Most importantly, the nuclease domain alone, devoid of the cold-shock and S1 domains, is sufficient for RNase R to bind and degrade structured RNAs. Moreover, this is a unique property of the nuclease domain of RNase R because this domain in RNase II stalls as it approaches a duplex. We also show that the nuclease domain of RNase R binds RNA more tightly than the nuclease domain of RNase II. This tighter binding may help to explain the difference in catalytic properties between RNase R and RNase II.

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.

Inhibition of homologous phosphorolytic ribonucleases by citrate may represent an evolutionarily conserved communicative link between RNA degradation and central metabolism

Abstract Ribonucleases play essential roles in all aspects of RNA metabolism, including the coordination of post-transcriptional gene regulation that allows organisms to respond to internal changes and environmental stimuli. However, as inherently destructive enzymes, their activity must be carefully controlled. Recent research exemplifies the repertoire of regulatory strategies employed by ribonucleases. The activity of the phosphorolytic exoribonuclease, polynucleotide phosphorylase (PNPase), has previously been shown to be modulated by the Krebs cycle metabolite citrate in Escherichia coli. Here, we provide evidence for the existence of citrate-mediated inhibition of ribonucleases in all three domains of life. In silico molecular docking studies predict that citrate will bind not only to bacterial PNPases from E. coli and Streptomyces antibioticus, but also PNPase from human mitochondria and the structurally and functionally related archaeal exosome complex from Sulfolobus solfataricus. Critically, we show experimentally that citrate also inhibits the exoribonuclease activity of bacterial, eukaryotic and archaeal PNPase homologues in vitro. Furthermore, bioinformatics data, showing key citrate-binding motifs conserved across a broad range of PNPase homologues, suggests that this regulatory mechanism may be widespread. Overall, our data highlight a communicative link between ribonuclease activity and central metabolism that may have been conserved through the course of evolution.

An improved method for surface immobilisation of RNA: application to small non-coding RNA-mRNA pairing.

Characterisation of RNA and its intermolecular interactions is increasing in importance as the inventory of known RNA functions continues to expand. RNA-RNA interactions are central to post-transcriptional gene regulation mechanisms in bacteria, and the interactions of bacterial small non-coding RNAs (sRNAs) with their mRNA targets are the subject of much current research. The technology of surface plasmon resonance (SPR) is an attractive approach to studying these interactions since it is highly sensitive, and allows interaction measurements to be recorded in real-time. Whilst a number of approaches exist to label RNAs for surface-immobilisation, the method documented here is simple, quick, efficient, and utilises the high-affinity streptavidin-biotin interaction. Specifically, we ligate a biotinylated nucleotide to the 3′ end of RNA using T4 RNA ligase. Although this is a previously recognised approach, we have optimised the method by our discovery that the incorporation of four or more adenine nucleotides at the 3′ end of the RNA (a poly-A-tail) is required in order to achieve high ligation efficiencies. We use this method within the context of investigating small non-coding RNA (sRNA) - mRNA interactions through the application of surface technologies, including quantitative SPR assays. We first focus on validating the method using the recently characterised Escherichia coli sRNA-mRNA pair, MicA-ompA, specifically demonstrating that the addition of the poly-A-tail to either RNA does not affect its subsequent binding interactions with partner molecules. We then apply this method to investigate the novel interactions of a Vibrio cholerae Qrr sRNA with partner mRNAs, hapR and vca0939; RNA-RNA pairings that are important in mediating pathogenic virulence. The calculated binding parameters allow insights to be drawn regarding sRNA-mRNA interaction mechanisms.