RNase III and RNase E Influence Posttranscriptional Regulatory Networks Involved in Virulence Factor Production, Metabolism, and Regulatory RNA Processing in Bordetella pertussis.

Bordetella pertussis has been shown to encode regulatory RNAs, yet the posttranscriptional regulatory circuits on which they act remain to be fully elucidated. We generated mutants lacking the endonucleases RNase III and RNase E and assessed their individual impact on the B. pertussis transcriptome. Transcriptome sequencing (RNA-Seq) analysis showed differential expression of ∼25% of the B. pertussis transcriptome in each mutant, with only 28% overlap between data sets. Both endonucleases exhibited substantial impact on genes involved in amino acid uptake (e.g., ABC transporters) and in virulence (e.g., the type III secretion system and the autotransporters vag8, tcfA, and brkA). Interestingly, mutations in RNase III and RNase E drove the stability of many transcripts, including those involved in virulence, in opposite directions, a result that was validated by qPCR and immunoblotting for tcfA and brkA. Of note, whereas similar mutations to RNase E in Escherichia coli have subtle effects on transcript stability, a striking >20-fold reduction in four gene transcripts, including tcfA and vag8, was observed in B. pertussis. We further compared our data set to the regulon controlled by the RNA chaperone Hfq to identify B. pertussis loci influenced by regulatory RNAs. This analysis identified ∼120 genes and 19 operons potentially regulated at the posttranscriptional level. Thus, our findings revealed how changes in RNase III- and RNase E-mediated RNA turnover influence pathways associated with virulence and cellular homeostasis. Moreover, we highlighted loci potentially influenced by regulatory RNAs, providing insights into the posttranscriptional regulatory networks involved in fine-tuning B. pertussis gene expression. IMPORTANCE Noncoding, regulatory RNAs in bacterial pathogens are critical components required for rapid changes in gene expression profiles. However, little is known about the role of regulatory RNAs in the growth and pathogenesis of Bordetella pertussis. To address this, mutants separately lacking ribonucleases central to regulatory RNA processing, RNase III and RNase E, were analyzed by RNA-Seq. Here, we detail the first transcriptomic analysis of the impact of altered RNA degradation in B. pertussis. Each mutant showed approximately 1,000 differentially expressed genes, with significant changes in the expression of pathways associated with metabolism, bacterial secretion, and virulence factor production. Our analysis suggests an important role for these ribonucleases during host colonization and provides insights into the breadth of posttranscriptional regulation in B. pertussis, further informing our understanding of B. pertussis pathogenesis.

Altering the divalent metal ion preference of RNase E.

RNase E is a major intracellular endoribonuclease in many bacteria and participates in most aspects of RNA processing and degradation. RNase E requires a divalent metal ion for its activity. We show that only Mg(2+) and Mn(2+) will support significant rates of activity in vitro against natural RNAs, with Mn(2+) being preferred. Both Mg(2+) and Mn(2+) also support cleavage of an oligonucleotide substrate with similar kinetic parameters for both ions. Salts of Ni(2+) and Zn(2+) permitted low levels of activity, while Ca(2+), Co(3+), Cu(2+), and Fe(2+) did not. A mutation to one of the residues known to chelate Mg(2+), D346C, led to almost complete loss of activity dependent on Mg(2+); however, the activity of the mutant enzyme was fully restored by the presence of Mn(2+) with kinetic parameters fully equivalent to those of wild-type enzyme. A similar mutation to the other chelating residue, D303C, resulted in nearly full loss of activity regardless of metal ion. The properties of RNase E D346C enabled a test of the ionic requirements of RNase E in vivo. Plasmid shuffling experiments showed that both rneD303C (i.e., the rne gene encoding a D-to-C change at position 303) and rneD346C were inviable whether or not the selection medium was supplied with MnSO4, implying that RNase E relies on Mg(2+) exclusively in vivo.

Streptomyces coelicolor polynucleotide phosphorylase can polymerize nucleoside diphosphates under phosphorolysis conditions, with implications for the degradation of structured RNAs.

We have examined the ability of wild-type polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor and two mutant forms of the enzyme, N459D and C468A, to function in the polymerization of ADP and in the phosphorolysis of RNA substrates derived from the S. coelicolor rpsO-pnp operon. The wild-type enzyme was twice as active in polymerization as N459D and four times as active as C468A. The kcat/Km value for phosphorolysis of a structured RNA substrate by N459D was essentially the same as that observed for the wild-type enzyme, while C468A was 50% as active with this substrate. A mixture of all four common nucleoside diphosphates increased the kcat/Km for phosphorolysis of the structured substrate by the wild-type enzyme by a factor of 1.7 but did not affect phosphorolysis catalyzed by N459D or C468A. We conducted phosphorolysis of the structured substrate in the presence of nucleoside diphosphates and labeled the 3' ends of the products of those reactions using [(32)P]pCp. Digestion of the end-labeled RNAs and display of the products on a sequencing gel revealed that wild-type S. coelicolor PNPase was able to synthesize RNA 3' tails under phosphorolysis conditions while the N459D and C468A mutants could not. The wild-type enzyme did not add 3' tails to a substrate that already possessed an unstructured 3' tail. We propose a model in which the transient synthesis of 3' tails facilitates the phosphorolysis of structured substrates by Streptomyces PNPase.

Determinants in the rpsT mRNAs recognized by the 5'-sensor domain of RNase E.

RNase E plays a central role in processing virtually all classes of cellular RNA in many bacterial species. A characteristic feature of RNase E and its paralogue RNase G, as well as several other unrelated ribonucleases, is their preference for 5'-monophosphorylated substrates. The basis for this property has been explored in vitro. At limiting substrate, cleavage of the rpsT mRNA by RNase E (residues 1-529) is inefficient, requiring excess enzyme. The rpsT mRNA is cleaved sequentially in a 5' to 3' direction, with the initial cleavage(s) at positions 116/117 or 190/191 being largely driven by direct entry, independent of the 5'-terminus or the 5'-sensor domain of RNase E. Generation of the 147 nt 3'-limit product requires sequential cleavages that generate 5'-monophosphorylated termini on intermediates, and the 5'-sensor domain of RNase E. These requirements can be bypassed with limiting enzyme by deleting a stem-loop structure adjacent to the site of the major, most distal cleavage. Alternatively, this specific cleavage can be activated substantially by a 5'-phosphorylated oligonucleotide annealed 5' to the cleavage site. This finding suggests that monophosphorylated small RNAs may destabilize their mRNA targets by recruiting the 5-sensor domain of RNase E 'in trans'.

S1 and KH domains of polynucleotide phosphorylase determine the efficiency of RNA binding and autoregulation.

To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient pnp::lacZ fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using in vitro band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between ß-galactosidase levels reflecting autoregulation and apparent KD values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase.

RNase E: at the interface of bacterial RNA processing and decay.

RNase E is an essential endonuclease that is abundant in many bacteria and plays an important part in all aspects of RNA metabolism. It functions as part of a large macromolecular complex known as the RNA degradosome. Recent evidence suggests that this complex associates with the inner membrane of bacteria, an observation that challenges traditional models in which soluble RNases are proposed to randomly interact with RNAs in the cytosol. In this Review, I summarize the major roles of RNase E in RNA processing and decay and discuss the various mechanisms that regulate its activity. I also propose a new model to rationalize the mechanism of RNase E action in the context of its localization in the bacterial cell.

Roles of the 5'-phosphate sensor domain in RNase E.

Viable mutations affecting the 5'-phosphate sensor of RNase E, including R169Q or T170A, become lethal when combined with deletions removing part of the non-catalytic C-terminal domain of RNase E. The phosphate sensor is required for efficient autoregulation of RNase E synthesis as RNase E R169Q is strongly overexpressed with accumulation of proteolytic fragments. In addition, mutation of the phosphate sensor stabilizes the rpsT P1 mRNA as much as sixfold and slows the maturation of 16S rRNA. In contrast, the decay of other model mRNAs and the processing of several tRNA precursors are unaffected by mutations in the phosphate sensor. Our data point to the existence of overlapping mechanisms of substrate recognition by RNase E, which lead to a hierarchy of efficiencies with which its RNA targets are attacked.

Substrate binding and active site residues in RNases E and G: role of the 5'-sensor.

The paralogous endoribonucleases, RNase E and RNase G, play major roles in intracellular RNA metabolism in Escherichia coli and related organisms. To assay the relative importance of the principal RNA binding sites identified by crystallographic analysis, we introduced mutations into the 5'-sensor, the S1 domain, and the Mg(+2)/Mn(+2) binding sites. The effect of such mutations has been measured by assays of activity on several substrates as well as by an assay of RNA binding. RNase E R169Q and the equivalent mutation in RNase G (R171Q) exhibit the strongest reductions in both activity (the k(cat) decrease approximately 40- to 100-fold) and RNA binding consistent with a key role for the 5'-sensor. Our analysis also supports a model in which the binding of substrate results in an increase in catalytic efficiency. Although the phosphate sensor plays a key role in vitro, it is unexpectedly dispensable in vivo. A strain expressing only RNase E R169Q as the sole source of RNase E activity is viable, exhibits a modest reduction in doubling time and colony size, and accumulates immature 5 S rRNA. Our results point to the importance of alternative RNA binding sites in RNase E and to alternative pathways of RNA recognition.

Preparation of the Escherichia coli RNase E protein and reconstitution of the RNA degradosome.

The RNA degradosome is a multienzyme complex that plays a key role in the processing of stable RNAs, the degradation of mRNAs, and the action of small regulatory RNAs. Initially discovered in Escherichia coli, similar or related complexes are found in other bacteria. The core of the RNA degradosome is the essential endoribonuclease, RNase E. The C-terminus of this enzyme serves as a scaffold to which other components of the RNA degradosome bind. These ligands include the phosphorolytic 3'-exonuclease, polynucleotide phosphorylase, the DEAD-box RNA helicase, RhlB, and the glycolytic enzyme, enolase. In addition, the DEAD-box RNA helicases CsdA and RhlE and the RNA binding protein, Hfq, may bind to RNase E in place of one or more of the prototypical components. This chapter describes purification of RNase E (the Rne protein), reconstitution of a minimal degradosome that recapitulates the activity of authentic degradosomes, and methods for the assay of the reconstituted complex.

Kinetics of polynucleotide phosphorylase: comparison of enzymes from Streptomyces and Escherichia coli and effects of nucleoside diphosphates.

We examined the activity of polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli in phosphorolysis using substrates derived from the rpsO-pnp operon of S. coelicolor. The Streptomyces and E. coli enzymes were both able to digest a substrate with a 3' single-stranded tail although E. coli PNPase was more effective in digesting this substrate than were the Streptomyces enzymes. The kcat for the E. coli enzyme was ca. twofold higher than that observed with the S. coelicolor enzyme. S. coelicolor PNPase was more effective than its E. coli counterpart in digesting a substrate possessing a 3' stem-loop structure, and the Km for the E. coli enzyme was ca. twice that of the S. coelicolor enzyme. Electrophoretic mobility shift assays revealed an increased affinity of S. coelicolor PNPase for the substrate possessing a 3' stem-loop structure compared with the E. coli enzyme. We observed an effect of nucleoside diphosphates on the activity of the S. coelicolor PNPase but not the E. coli enzyme. In the presence of a mixture of 20 microM ADP, CDP, GDP, and UDP, the Km for the phosphorolysis of the substrate with the 3' stem-loop was some fivefold lower than the value observed in the absence of nucleoside diphosphates. No effect of nucleoside diphosphates on the phosphorolytic activity of E. coli PNPase was observed. To our knowledge, this is the first demonstration of an effect of nucleoside diphosphates, the normal substrates for polymerization by PNPase, on the phosphorolytic activity of that enzyme.

Role of RNA structure and susceptibility to RNase E in regulation of a cold shock mRNA, cspA mRNA.

Degradation of the cspA mRNA in vivo is very rapid at temperatures greater than 30 degrees C and is moderately dependent on RNase E. Investigations in vitro show that degradosomes prepared from normal or cold-shocked cultures cleave the cspA mRNA preferentially at a single site in vitro between two stem-loops approximately 24 residues 3' to the termination codon and approximately 31 residues from the 3' end. The site of cleavage is independent of the temperature and largely independent of the phosphorylation status of the 5' end of cspA mRNA. A 5' stem-loop, potential occlusion of the initiation and termination codons, temperature-dependent translational efficiency, and the position of the RNase E cleavage site can explain the differential stability of the cspA mRNA.

Function of the conserved S1 and KH domains in polynucleotide phosphorylase.

We have examined the roles of the conserved S1 and KH RNA binding motifs in the widely dispersed prokaryotic exoribonuclease polynucleotide phosphorylase (PNPase). These domains can be released from the enzyme by mild proteolysis or by truncation of the gene. Using purified recombinant enzymes, we have assessed the effects of specific deletions on RNA binding, on activity against a synthetic substrate under multiple-turnover conditions, and on the ability of truncated forms of PNPase to form a minimal RNA degradosome with RNase E and RhlB. Deletion of the S1 domain reduces the apparent activity of the enzyme by almost 70-fold under low-ionic-strength conditions and limits the enzyme to digest a single substrate molecule. Activity and product release are substantially regained at higher ionic strengths. This deletion also reduces the affinity of the enzyme for RNA, without affecting the enzyme's ability to bind to RNase E. Deletion of the KH domain produces similar, but less severe, effects, while deletion of both the S1 and KH domains accentuates the loss of activity, product release, and RNA binding but has no effect on binding to RNase E. We propose that the S1 domain, possibly arrayed with the KH domain, forms an RNA binding surface that facilitates substrate recognition and thus indirectly potentiates product release. The present data as well as prior observations can be rationalized by a two-step model for substrate binding.

Physical and functional interactions among RNase E, polynucleotide phosphorylase and the cold-shock protein, CsdA: evidence for a 'cold shock degradosome'.

Escherichia coli contains at least five ATP-dependent DEAD-box RNA helicases which may play important roles in macromolecular metabolism, especially in translation and mRNA decay. Here we demonstrate that one member of this family, CsdA, whose expression is induced by cold shock, interacts physically and functionally with RNase E. Three independent approaches show that after a shift of cultures to 15 degrees C, CsdA co-purifies with RNase E and other components of the RNA degradosome. Moreover, functional assays using reconstituted minimal degradosomes prepared from purified components in vitro show that CsdA can fully replace the resident RNA helicase of the RNA degradosome, RhlB. In addition, under these conditions, CsdA displays RNA-dependent ATPase activity. Taken together, our data are consistent with a model in which CsdA accumulates during the early stages of cold acclimatization and subsequently assembles into degradosomes with RNase E synthesized in cold-adapted cultures. These findings show that the RNA degradosome is a flexible macromolecular machine capable of adapting to altered environmental conditions.

Structural characterization of the RNase E S1 domain and identification of its oligonucleotide-binding and dimerization interfaces.

S1 domains occur in four of the major enzymes of mRNA decay in Escherichia coli: RNase E, PNPase, RNase II, and RNase G. Here, we report the structure of the S1 domain of RNase E, determined by both X-ray crystallography and NMR spectroscopy. The RNase E S1 domain adopts an OB-fold, very similar to that found with PNPase and the major cold shock proteins, in which flexible loops are appended to a well-ordered five-stranded beta-barrel core. Within the crystal lattice, the protein forms a dimer stabilized primarily by intermolecular hydrophobic packing. Consistent with this observation, light-scattering, chemical crosslinking, and NMR spectroscopic measurements confirm that the isolated RNase E S1 domain undergoes a specific monomer-dimer equilibrium in solution with a K(D) value in the millimolar range. The substitution of glycine 66 with serine dramatically destabilizes the folded structure of this domain, thereby providing an explanation for the temperature-sensitive phenotype associated with this mutation in full-length RNase E. Based on amide chemical shift perturbation mapping, the binding surface for a single-stranded DNA dodecamer (K(D)=160(+/-40)microM) was identified as a groove of positive electrostatic potential containing several exposed aromatic side-chains. This surface, which corresponds to the conserved ligand-binding cleft found in numerous OB-fold proteins, lies distal to the dimerization interface, such that two independent oligonucleotide-binding sites can exist in the dimeric form of the RNase E S1 domain. Based on these data, we propose that the S1 domain serves a dual role of dimerization to aid in the formation of the tetrameric quaternary structure of RNase E as described by Callaghan et al. in 2003 and of substrate binding to facilitate RNA hydrolysis by the adjacent catalytic domains within this multimeric enzyme.

The quaternary structure of RNase G from Escherichia coli.

RNase G is the endoribonuclease responsible for forming the mature 5' end of 16S rRNA. This enzyme shares 35% identity with and 50% similarity to the N-terminal 470 amino acids encompassing the catalytic domain of RNase E, the major endonuclease in Escherichia coli. In this study, we developed non-denaturing purifications for overexpressed RNase G. Using mass spectrometry and N-terminal sequencing, we unambiguously identified the N-terminal sequence of the protein and found that translation is initiated at the second of two potential start sites. Using velocity sedimentation and oxidative cross-linking, we determined that RNase G exists largely as a dimer in equilibrium with monomers and higher multimers. Moreover, dimerization is required for activity. Four of the six cysteine residues of RNase G were mutated to serine. No single cysteine to serine mutation resulted in a complete loss of cross-linking, dimerization or activity. However, multiple mutations in a highly conserved cluster of cysteines, including C405 and C408, resulted in a partial loss of activity and a shift in the distribution of RNase G multimers towards monomers. We propose that many of the cysteines in RNase G lie on its surface and define, in part, the subunit-subunit interface.

Overexpression and purification of untagged polynucleotide phosphorylases.

We report here the development of new, straightforward procedures for the purification of bacterial polynucleotide phosphorylases (PNPases). The pnp genes from Streptomyces antibioticus, Streptomyces coelicolor, and Escherichia coli were overexpressed using the vectors pET11 and pET11A in E. coli BL21(DE3)pLysS. The enzymes were purified to apparent homogeneity after phosphorolysis in crude extracts followed by anion exchange and hydrophobic interaction chromatography. Yields of 5-15mg per liter of culture were obtained and the enzymes contained only small amounts of contaminating RNA as estimated from the A(280/260) ratios of purified preparations. All three enzymes were active in both the polymerization and phosphorolysis reactions normally catalyzed by PNPases. Incubation under phosphorolysis conditions but in the absence of potassium phosphate indicated that the enzymes were free of phosphate-independent nuclease activity. We suggest that the approaches described here may be applied generally to the overexpression and purification of eubacterial polynucleotide phosphorylases.

Ectopic RNase E sites promote bypass of 5'-end-dependent mRNA decay in Escherichia coli.

In Escherichia coli, 5'-terminal stem-loops form major impediments to mRNA decay, yet conditions that determine their effectiveness or the use of alternative decay pathway(s) are unclear. A synthetic 5'-terminal hairpin stabilizes the rpsT mRNA sixfold. This stabilization is dependent on efficient translational initiation and ribosome transit through at least two-thirds of the coding sequence past a major RNase E cleavage site in the rpsT mRNA. Insertion of a 12-15 residue 'ectopic' RNase E cleavage site from either the rne leader or 9S pre-rRNA into the 5'-non-coding region of the rpsT mRNA significantly reduces the stabilizing effect of the terminal stem-loop, dependent on RNase E. A similar insertion into the rpsT coding sequence is partially destabilizing. These findings demonstrate that RNase E can bypass an interaction with the 5'-terminus, and exploit an alternative 'internal entry' pathway. We propose a model for degradation of the rpsT mRNA, which explains the hierarchy of protection afforded by different 5'-termini, the use of internal entry for bypass of barriers to decay, 'ectopic sites' and the role of translating ribosomes.