Running rings around RNA: a superfamily of phosphate-dependent RNases.

The exosome of Saccharomyces cerevisiae and the degradosome of Escherichia coli are multienzyme complexes involved in the degradation of mRNA. Both contain enzymes that are similar to the phosphate-dependent exoribonuclease RNase PH. These enzymes are phosphorylases that degrade RNA from the 3'-end. A recent X-ray crystallographic study of the polynucleotide phosphorylase (PNPase) from Streptomyces antibioticus reveals, for the first time, the atomic structure of a member of the RNase PH superfamily. Here, information from the structure of PNPase is used to address two related issues. First, the structure supports the idea that PNPase, which is a trimer of multidomain subunits, arose by duplication of a gene encoding an RNase PH-like enzyme. Second, the structure might explain how RNase PH-like enzymes associate into oligomeric rings that degrade RNA in a processive reaction.     

Crystal structure of Escherichia coli PNPase: central channel residues are involved in processive RNA degradation

Bacterial polynucleotide phosphorylase (PNPase) plays a major role in mRNA turnover by the degradation of RNA from the 3′- to 5′-ends. Here, we determined the crystal structures of the wild-type and a C-terminal KH/S1 domain-truncated mutant (ΔKH/S1) of Escherichia coli PNPase at resolutions of 2.6 Å and 2.8 Å, respectively. The six RNase PH domains of the trimeric PNPase assemble into a ring-like structure containing a central channel. The truncated mutant ΔKH/S1 bound and cleaved RNA less efficiently with an eightfold reduced binding affinity. Thermal melting and acid-induced trimer dissociation studies, analyzed by circular dichroism and dynamic light scattering, further showed that ΔKH/S1 formed a less stable trimer than the full-length PNPase. The crystal structure of ΔKH/S1 is more expanded, containing a slightly wider central channel than that of the wild-type PNPase, suggesting that the KH/S1 domain helps PNPase to assemble into a more compact trimer, and it regulates the channel size allosterically. Moreover, site-directed mutagenesis of several arginine residues in the channel neck regions produced defective PNPases that either bound and cleaved RNA less efficiently or generated longer cleaved oligonucleotide products, indicating that these arginines were involved in RNA binding and processive degradation. Taking these results together, we conclude that the constricted central channel and the basic-charged residues in the channel necks of PNPase play crucial roles in trapping RNA for processive exonucleolytic degradation.     

Conserved domains in polynucleotide phosphorylase among eubacteria

Polynucleotide phosphorylase (PNPase) is a polynucleotide nucleotidyl transferase (E. C. 2.7.7.8) that is involved in mRNA degradation in prokaryotes. PNPase structure analysis has been performed in Streptomyces antibioticus; this revealed the presence of five domains: two ribonuclease PH (RPH)-like (pnp1 and pnp2), one alpha helical, one KH, and one S1 domains. The trimeric nature of this enzyme was also confirmed. In this work, we have investigated conserved domains or subdomains in bacterial PNPases (55), for this structure-based sequence homology analysis between predicted amino acid sequences from bacterial PNPases and that of S. antibioticus was performed. Our findings indicate that while pnp2 (% similarity average S = 84/% identity average I = 22), KH (S = 74.3%/I = 5.3%), S1 (S = 71.3%/I = 1.2%); and pnp1 (S = 52.8%/I = 0.3%) domain; structure and sequence are well conserved among different bacteria, alpha helical domain (S = 39.5%/I = 0) although conservation of the structure is somewhat maintained, the sequence is not conserved at all. Implications of such findings in PNPase activity will be discussed.     

Crystal structure of the phosphorolytic exoribonuclease RNase PH from Bacillus subtilis and implications for its quaternary structure and tRNA binding

RNase PH is a member of the family of phosphorolytic 3' --> 5' exoribonucleases that also includes polynucleotide phosphorylase (PNPase). RNase PH is involved in the maturation of tRNA precursors and especially important for removal of nucleotide residues near the CCA acceptor end of the mature tRNAs. Wild-type and triple mutant R68Q-R73Q-R76Q RNase PH from Bacillus subtilis have been crystallized and the structures determined by X-ray diffraction to medium resolution. Wild-type and triple mutant RNase PH crystallize as a hexamer and dimer, respectively. The structures contain a rare left-handed beta alpha beta-motif in the N-terminal portion of the protein. This motif has also been identified in other enzymes involved in RNA metabolism. The RNase PH structure and active site can, despite low sequence similarity, be overlayed with the N-terminal core of the structure and active site of Streptomyces antibioticus PNPase. The surface of the RNase PH dimer fit the shape of a tRNA molecule.     

Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly

Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3'-5' directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3' end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a 'splayed' conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.     

Mutational analysis of polynucleotide phosphorylase from Escherichia coli

Polynucleotide phosphorylase (PNPase), a homotrimeric exoribonuclease present in bacteria, is involved in mRNA degradation. In Escherichia coli, expression of this enzyme is autocontrolled at the translational level. We introduced about 30 mutations in the pnp gene by site-directed mutagenesis, most of them in phylogenetically conserved residues, and determined their effects on the three catalytic activities of PNPase, phosphorolysis, polymerisation and phosphate exchange, as well as on the efficiency of translational repression. The data are presented and discussed in the light of the crystallographic structure of PNPase from Streptomyces antibioticus. The results show that both PNPase activity and the presence of the KH and S1 RNA-binding domains are required for autocontrol. Deletions of these RNA-binding domains do not abolish any of the three catalytic activities, indicating that they are contained in a domain independent of the catalytic centre. Moreover, the catalytic centre was located around the tungsten-binding site identified by crystallography. Some mutations affect the three catalytic activities differently, an observation consistent with the presence of different subsites.     

Domain analysis of the chloroplast polynucleotide phosphorylase reveals discrete functions in RNA degradation,polyadenylation, and sequence homology with exosome proteins

The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events, including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation. In spinach chloroplasts, the latter two steps of polyadenylation and exonucleolytic degradation are performed by the same phosphorolytic and processive enzyme, polynucleotide phosphorylase (PNPase). An analysis of its amino acid sequence shows that the protein is composed of two core domains related to RNase PH, two RNA binding domains (KH and S1), and an alpha-helical domain. The amino acid sequence and domain structure is largely conserved between bacteria and organelles. To define the molecular mechanism that controls the two opposite activities of this protein in the chloroplast, the ribonuclease, polymerase, and RNA binding properties of each domain were analyzed. The first core domain, which was predicted to be inactive in the bacterial enzymes, was active in RNA degradation but not in polymerization. Surprisingly, the second core domain was found to be active in degrading polyadenylated RNA only, suggesting that nonpolyadenylated molecules can be degraded only if tails are added, apparently by the same protein. The poly(A) high-binding-affinity site was localized to the S1 domain. The complete spinach chloroplast PNPase, as well as versions containing the core domains, complemented the cold sensitivity of an Escherichia coli PNPase-less mutant. Phylogenetic analyses of the two core domains showed that the two domains separated very early, resulting in the evolution of the bacterial and organelle PNPases and the exosome proteins found in eukaryotes and some archaea.     

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 Hexamer RraA from Pseudomonas Aeruginosa Reveals Six Conserved Protein–Protein Interaction Sites

RNase E functions as the rate-limiting enzyme in the global mRNA metabolism as well as in the maturation of functional RNAs. The endoribonuclease, binding to the PNPase trimer, the RhlB monomer, and the enolase dimer, assembles into an RNA degradosome necessary for effective RNA metabolism. The RNase E processing is found to be negatively regulated by the protein modulator RraA which appears to work by interacting with the non-catalytic region of the endoribonuclease and significantly reduce the interaction between RNase E and PNPase, RhlB and enolase of the RNA degradosome. Here we report the crystal structure of RraA from P. aeruginosa to a resolution of 2.0 ?. The overall architecture of RraA is very similar to other known RraAs, which are highly structurally conserved. Gel filtration and dynamic light scattering experiments suggest that the protein regulator is arranged as a hexamer, consistent with the crystal packing of "a dimer of trimer" arrangement. Structure and sequence conservation analysis suggests that the hexamer RraA contains six putative charged protein-protein interaction sites which may serve as binding sites for RNase E.     

Genetic analysis of polynucleotide phosphorylase structure and functions

Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity.     

Domain organization of Salmonella typhimurium formylglycinamide ribonucleotide amidotransferase revealed by X-ray crystallography

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent conversion of formylglycinamide ribonucleotide (FGAR) and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, P(i), and glutamate in the fourth step of the purine biosynthetic pathway. In eukaryotes and Gram-negative bacteria, FGAR-AT is encoded by the purL gene as a multidomain protein with a molecular mass of about 140 kDa. In Gram-positive bacteria and archaebacteria FGAR-AT is a complex of three proteins: PurS, PurL, and PurQ. We have determined the structure of FGAR-AT (PurL) from Salmonella typhimurium at 1.9 A resolution using X-ray crystallography. PurL is the last remaining enzyme in the purine biosynthetic pathway to have its structure determined. The structure reveals four domains: an N-terminal domain structurally homologous to a PurS dimer, a linker region, an FGAM synthetase domain homologous to an aminoimidazole ribonucleotide synthetase (PurM) dimer, and a triad glutaminase domain. The domains are intricately linked by interdomain interactions and peptide connectors. The fold common to PurM and the central region of PurL represents a superfamily for which HypE, SelD, and ThiL are predicted to be members. A structural ADP molecule was found bound to a site related to the putative active site by pseudo-2-fold symmetry and two sulfate ions were found at the putative active site. These observations and the structural similarities between PurM and StPurL were used to model the substrates FGAR and ATP in the StPurL active site. A glutamylthioester intermediate was found in the glutaminase domain at Cys1135. The N-terminal (PurS-like) domain is hypothesized to form the putative channel through which ammonia passes from the glutaminase domain to the FGAM synthetase domain.     

The archaeal exosome core is a hexameric ring structure with three catalytic subunits

The exosome is a 3' --> 5' exoribonuclease complex involved in RNA processing. We report the crystal structure of the RNase PH core complex of the Sulfolobus solfataricus exosome determined at a resolution of 2.8 A. The structure reveals a hexameric ring-like arrangement of three Rrp41-Rrp42 heterodimers, where both subunits adopt the RNase PH fold common to phosphorolytic exoribonucleases. Structure-guided mutagenesis reveals that the activity of the complex resides within the active sites of the Rrp41 subunits, all three of which face the same side of the hexameric structure. The Rrp42 subunit is inactive but contributes to the structuring of the Rrp41 active site. The high sequence similarity of this archaeal exosome to eukaryotic exosomes and its high structural similarity to the bacterial mRNA-degrading PNPase support a common basis for RNA-degrading machineries in all three domains of life.     

Activation of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) in Streptomyces antibioticus.

The activity of the ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase I [GPSI]) from Streptomyces antibioticus is stimulated maximally by methanol at 20% (vol/vol) in assay mixtures. Although the enzyme is not activated by ribosomes, its activity is stimulated by tRNA (uncharged or charged) and by synthetic mRNA [e.g., poly(U)]. The level of stimulation is greater in the presence of tRNA and poly(U) together than with either RNA alone. Incubation of GPSI with low levels of trypsin also leads to activation of the enzyme. Analysis of the products of mild trypsin digestion revealed the presence of two intermediates whose M(r)s are identical to those of species produced by incubation of purified GPSI with crude extracts of S. antibioticus mycelium. GPSI can be activated by incubation with crude mycelial extracts, and this activation is partially inhibited by the inclusion of trypsin inhibitor in reaction mixtures.     

Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase

The dihydrolipoamide succinyltransferase (E2o) component of the alpha-ketoglutarate dehydrogenase complex catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A. E2o is normally a 24-mer, but is found as a trimer when E2o is expressed with a C-terminal [His]6 tag. The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 A resolution using the Molecular Replacement method. The refined model contains an intact trimer in the asymmetric unit and has an R-factor of 0.257 (Rfree = 0.286) for 18,699 reflections between 10.0 and 3.0 A resolution. The core of tE2oCD (residues 187-396) superimposes onto that of the cubic E2oCD with an RMS difference of 0.4 A for all main-chain atoms. The C-terminal end of tE2oCD (residues 397-404) rotates by an average of 37 degrees compared to cubic E2oCD, disrupting the normal twofold interface. Despite the alteration of quaternary structure, the active site of tE2oCD shows no significant differences from that of the cubic E2oCD, although several side chains in the active site are more ordered in the trimeric form of E2oCD. Analysis of the available sequence data suggests that the majority of E2 components have active sites that resemble that of E. coli E2oCD. The remaining E2 components can be divided into three groups based on active-site sequence similarity. Analysis of the surface properties of both crystal forms of E. coli E2oCD suggests key residues that may be involved in the protein-protein contacts that occur between the catalytic and lipoyl domains of E2o.     

The translational regulation of threonyl-tRNA synthetase. Functional relationship between the enzyme, the cognate tRNA and the ribosome

The E. coli threonyl-tRNA synthetase gene is negatively autoregulated at the translational level by a direct binding of the enzyme to the leader region of the thrS mRNA. This region folds in four well-defined domains. The enzyme binds to the leader at two major sites: the first is a stem-loop structure located in domain II upstream of the translational initiation site (domain I) which shares structural analogies with the anticodon arm of several tRNA(Thr) isoacceptors. The second site corresponds to a stable stem-loop structure located in domain IV. Both sites are separated by a large unpaired region (domain III). In vivo and in vitro experiments show that the structural integrity of both sites is required for the regulatory process. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. tRNA(Thr) suppresses this inhibitory effect by displacing the mRNA from the enzyme at both the upstream stem-loop structure and the tRNA-like anticodon arm.     

Polynucleotide phosphorylase interacts with ribonuclease E through a betabetaalphabetabetaalpha domain

In the present work we have used a double-hybrid assay in bacteria to identify a putative domain in E. coli PNPase required for in vivo interaction with RNase E. We used a 202 aa fragment of RNase E previously reported as the PNPase binding domain in this enzyme and a collection of 13 different fragments of 105 aa, spanning the entire sequence of 734 aa PNPase (GenBank Accession number NP_417633). Our results indicate that two clones of PNPase including residues 158-262 and residues 473-577 contain interaction sites for RNase E within a betabetaalphabetabetaalpha domain configuration. Three-dimensional modeling of the E. coli PNPase based on the S. antibioticus protein structure indicates that the putative binding domain is located on the monomer surface, facing outward from the trimeric tertiary structure. Since a copy of the betabetaalphabetabetaalpha domain is also found in RNase PH, we investigated and found an interaction with RNase E in a pull-down assay. We suggest this interaction takes place through the similar betabetaalphabetabetaalpha domain present in the tertiary structure of this enzyme. Based on these results, we propose that RNase PH and RNase E could form functional assemblies in E. coli.