A model for the Bacillus subtilis formylglycinamide ribonucleotide amidotransferase multiprotein complex

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), ATP, and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, P(i), and glutamate in the fourth step of the purine biosynthetic pathway. PurL exists in two forms: large PurL (lgPurL) is a single chain, multidomain enzyme of about 1300 amino acids, whereas small PurL (smPurL) contains about 800 amino acids but requires two additional gene products, PurS and PurQ, for activity. smPurL contains the ATP and FGAR binding sites, PurQ is a glutaminase, and the function of PurS is just now becoming understood. We determined the structure of Bacillus subtilis PurS in two different crystal forms P2(1) and C2 at 2.5 and 2.0 A resolution, respectively. PurS forms a tight dimer with a central six-stranded beta-sheet flanked by four helices. In both the P2(1) and the C2 crystal forms, the quaternary structure of PurS is a tetramer. The concave faces of the PurS dimers interact via the C-terminal region to form a twelve-stranded beta-barrel with a hydrophilic core. We used the structure of PurS together with the structure of lgPurL from Salmonella typhimurium to construct a model of the PurS/smPurL/PurQ complex. The HisH (glutaminase) domain of imidazole glycerol phosphate synthetase was used as an additional model of PurQ. The model shows stoichiometry of 2PurS/smPurL/PurQ using a PurS dimer or 4PurS/2smPurL/2PurQ using a PurS tetramer. Both models place key conserved residues at the ATP/FGAR binding site and at a structural ADP binding site. The homology model is consistent with biochemical studies on the reconstituted complex.     

Dissection of the network of interactions that links RNA processing with glycolysis in the Bacillus subtilis degradosome

The RNA degradosome is a multiprotein macromolecular complex that is involved in the degradation of messenger RNA in bacteria. The composition of this complex has been found to display a high degree of evolutionary divergence, which may reflect the adaptation of species to different environments. Recently, a degradosome-like complex identified in Bacillus subtilis was found to be distinct from those found in proteobacteria, the degradosomes of which are assembled around the unstructured C-terminus of ribonuclease E, a protein not present in B. subtilis. In this report, we have investigated in vitro the binary interactions between degradosome components and have characterized interactions between glycolytic enzymes, RNA-degrading enzymes, and those that appear to link these two cellular processes. The crystal structures of the glycolytic enzymes phosphofructokinase and enolase are presented and discussed in relation to their roles in the mediation of complex protein assemblies. Taken together, these data provide valuable insights into the structure and dynamics of the RNA degradosome, a fascinating and complex macromolecular assembly that links RNA degradation with central carbon metabolism.     

RNaseE and RNA helicase B play central roles in the cytoskeletal organization of the RNA degradosome

The RNA degradosome of Escherichia coli is a multiprotein complex that plays an essential role in normal RNA processing and decay. It was recently shown that the major degradosome constituents are organized in a coiled cytoskeletal-like structure that extends along the length of the cell. Here we show that the endoribonuclease E (RNaseE) and RNA helicase B (RhlB) components of the degradosome can each independently form coiled structures in the absence of the other degradosome proteins. In contrast, the cytoskeletal organization of the other degradosome proteins required the presence of the RNaseE or RhlB coiled elements. Although the RNaseE and RhlB structures were equally competent to support the helical organization of polynucleotide phosphorylase, the cytoskeletal-like organization of enolase occurred only in the presence of the RNaseE coiled structure. The results indicate that the RNA degradosome proteins are components of the bacterial cytoskeleton rather than existing as randomly distributed multiprotein complexes within the cell and suggest a model for the cellular organization of the components within the helical degradosomal structure.     

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 ribonuclease''s 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.     

Analysis of the RNA degradosome complex in Vibrio angustum S14

The RNA degradosome is built on the C-terminal half of ribonuclease E (RNase E) which shows high sequence variation, even amongst closely related species. This is intriguing given its central role in RNA processing and mRNA decay. Previously, we have identified RhlB (ATP-dependent DEAD-box RNA helicase)-binding, PNPase (polynucleotide phosphorylase)-binding and enolase-binding microdomains in the C-terminal half of Vibrio angustum S14 RNase E, and have shown through two-hybrid analysis that the PNPase and enolase-binding microdomains have protein-binding function. We suggest that the RhlB-binding, enolase-binding and PNPase-binding microdomains may be interchangeable between Escherichia coli and V. angustum S14 RNase E. In this study, we used two-hybrid techniques to show that the putative RhlB-binding microdomain can bind RhlB. We then used Blue Native-PAGE, a technique commonly employed in the separation of membrane protein complexes, in a study of the first of its kind to purify and analyse the RNA degradosome. We showed that the V. angustum S14 RNA degradosome comprises at least RNase E, RhlB, enolase and PNPase. Based on the results obtained from sequence analyses, two-hybrid assays, immunoprecipitation experiments and Blue Native-PAGE separation, we present a model for the V. angustum S14 RNA degradosome. We discuss the benefits of using Blue Native-PAGE as a tool to analyse the RNA degradosome, and the implications of microdomain-mediated RNase E interaction specificity.     

Dimeric assembly of human Suv3 helicase promotes its RNA unwinding function in mitochondrial RNA degradosome for RNA decay

Human Suv3 is a unique homodimeric helicase that constitutes the major component of the mitochondrial degradosome to work cooperatively with exoribonuclease PNPase for efficient RNA decay. However, the molecular mechanism of how Suv3 is assembled into a homodimer to unwind RNA remains elusive. Here, we show that dimeric Suv3 preferentially binds to and unwinds DNA–DNA, DNA–RNA, and RNA–RNA duplexes with a long 3′ overhang (≥10 nucleotides). The C‐terminal tail (CTT)‐truncated Suv3 (Suv3ΔC) becomes a monomeric protein that binds to and unwinds duplex substrates with ~six to sevenfold lower activities relative to dimeric Suv3. Only dimeric Suv3, but not monomeric Suv3ΔC, binds RNA independently of ATP or ADP, and is capable of interacting with PNPase, indicating that dimeric Suv3 assembly ensures its continuous association with RNA and PNPase during ATP hydrolysis cycles for efficient RNA degradation. We further determined the crystal structure of the apo‐form of Suv3ΔC, and SAXS structures of dimeric Suv3 and PNPase–Suv3 complex, showing that dimeric Suv3 caps on the top of PNPase via interactions with S1 domains, and forms a dumbbell‐shaped degradosome complex with PNPase. Overall, this study reveals that Suv3 is assembled into a dimeric helicase by its CTT for efficient and persistent RNA binding and unwinding to facilitate interactions with PNPase, promote RNA degradation, and maintain mitochondrial genome integrity and homeostasis.     

The Escherichia coli major exoribonuclease RNase II is a component of the RNA degradosome

Multiprotein complexes that carry out RNA degradation and processing functions are found in cells from all domains of life. In Escherichia coli, the RNA degradosome, a four-protein complex, is required for normal RNA degradation and processing. In addition to the degradosome complex, the cell contains other ribonucleases that also play important roles in RNA processing and/or degradation. Whether the other ribonucleases are associated with the degradosome or function independently is not known. In the present work, IP (immunoprecipitation) studies from cell extracts showed that the major hydrolytic exoribonuclease RNase II is associated with the known degradosome components RNaseE (endoribonuclease E), RhlB (RNA helicase B), PNPase (polynucleotide phosphorylase) and Eno (enolase). Further evidence for the RNase II-degradosome association came from the binding of RNase II to purified RNaseE in far western affinity blot experiments. Formation of the RNase II–degradosome complex required the degradosomal proteins RhlB and PNPase as well as a C-terminal domain of RNaseE that contains binding sites for the other degradosomal proteins. This shows that the RNase II is a component of the RNA degradosome complex, a previously unrecognized association that is likely to play a role in coupling and coordinating the multiple elements of the RNA degradation pathways.     

A novel zinc-binding fold in the helicase interaction domain of the Bacillus subtilis DnaI helicase loader

The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication. While the C-terminal domain of DnaI belongs to the structurally well-characterized AAA+ family of ATPases, the structure of the N-terminal domain, DnaI-N, has no homology to a known structure. Three-dimensional structure determination by nuclear magnetic resonance (NMR) spectroscopy shows that DnaI presents a novel fold containing a structurally important zinc ion. Surface plasmon resonance experiments indicate that DnaI-N is largely responsible for binding of DnaI to the hexameric helicase from B. stearothermophilus, which is a close homologue of the corresponding much less stable B. subtilis helicase.     

The yeast mitochondrial degradosome. Its composition,interplay between RNA helicase and RNase activities and the role in mitochondrial RNA metabolism

The yeast mitochondrial degradosome (mtEXO) is an NTP-dependent exoribonuclease involved in mitochondrial RNA metabolism. Previous purifications suggested that it was composed of three subunits. Our results suggest that the degradosome is composed of only two large subunits: an RNase and a RNA helicase encoded by nuclear genes DSS1 and SUV3, respectively, and that it co-purifies with mitochondrial ribosomes. We have found that the purified degradosome has RNA helicase activity that precedes and is essential for exoribonuclease activity of this complex. The degradosome RNase activity is necessary for mitochondrial biogenesis but in vitro the degradosome without RNase activity is still able to unwind RNA. In yeast strains lacking degradosome components there is a strong accumulation of mitochondrial mRNA and rRNA precursors not processed at 3'- and 5'-ends. The observed accumulation of precursors is probably the result of lack of degradation rather than direct inhibition of processing. We suggest that the degradosome is a central part of a mitochondrial RNA surveillance system responsible for degradation of aberrant and unprocessed RNAs.     

The formylglycinamide ribonucleotide amidotransferase complex from Bacillus subtilis: metabolite-mediated complex formation

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP- and glutamine-dependent formation of formylglycinamidine ribonucleotide, ADP, P(i), and glutamate in the fourth step of de novo purine biosynthesis. Like all amidotransferases (ATs), FGAR-AT is proposed to channel ammonia between a glutaminase and AT domain. In Gram-negative bacteria and eukaryotes, FGAR-AT is a single approximately 140 kDa protein. In archae and Gram-positive bacteria, the FGAR-AT is formed from three proteins: PurS (10 kDa), PurQ (25 kDa, a glutaminase), and smPurL (80 kDa, an AT). This is the only known AT to require a third structural component (PurS) for activity. Here we report the first purification and biochemical characterization of a three-component AT from Bacillus subtilis. Efforts to isolate an intact FGAR-AT focused initially on coexpression of PurS, smPurL, and PurQ. However, all attempts to purify the complex resulted in separation of the constituent proteins. PurS, smPurL, and PurQ were therefore separately expressed and purified to homogeneity. PurQ had a glutaminase activity of 0.002 s(-1), and smPurL had an ammonia-dependent AT activity of 0.044 s(-1). Reconstitution of PurS, smPurL, and PurQ at a ratio of 2:1:1 gave an activity of 2.49 s(-1), similar to that previously reported for the Escherichia coli 140 kDa FGAR-AT (5.00 s(-1)). PurS was essential for the glutamine-dependent FGAR-AT activity. Surprisingly, activity was found to be absolutely dependent on the presence of Mg2+ and ADP, and a stable FGAR-AT complex of 2PurS/1smPurL/1PurQ was detected only in the presence of Mg2+, ADP, and glutamine. The implications of these observations are discussed with respect to ammonia channeling.