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.     

Escherichia coli poly(A)-binding proteins that interact with components of degradosomes or impede RNA decay mediated by polynucleotide phosphorylase and RNase E

The multifunctional ribonuclease RNase E and the 3'-exonuclease polynucleotide phosphorylase (PNPase) are major components of an Escherichia coli ribonucleolytic "machine" that has been termed the RNA degradosome. Previous work has shown that poly(A) additions to the 3' ends of RNA substrates affect RNA degradation by both of these enzymes. To better understand the mechanism(s) by which poly(A) tails can modulate ribonuclease action, we used selective binding in 1 m salt to identify E. coli proteins that interact at high affinity with poly(A) tracts. We report here that CspE, a member of a family of RNA-binding "cold shock" proteins, and S1, an essential component of the 30 S ribosomal subunit, are poly(A)-binding proteins that interact functionally and physically, respectively, with degradosome ribonucleases. We show that purified CspE impedes poly(A)-mediated 3' to 5' exonucleolytic decay by PNPase by interfering with its digestion through the poly(A) tail and also inhibits both internal cleavage and poly(A) tail removal by RNase E. The ribosomal protein S1, which is known to interact with sequences at the 5' ends of mRNA molecules during the initiation of translation, can bind to both RNase E and PNPase, but in contrast to CspE, did not affect the ribonucleolytic actions of these enzymes. Our findings raise the prospect that E. coli proteins that bind to poly(A) tails may link the functions of degradosomes and ribosomes.     

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.     

Enolase in the RNA degradosome plays a crucial role in the rapid decay of glucose transporter mRNA in the response to phosphosugar stress in Escherichia coli

The ptsG mRNA encoding the major glucose transporter is rapidly degraded in an RNase E-dependent manner in response to the accumulation of glucose 6-P or fructose 6-P when the glycolytic pathway is blocked at its early steps in Escherichia coli. RNase E, a major endonuclease, is associated with polynucleotide phosphorylase (PNPase), RhlB helicase and a glycolytic enzyme, enolase, which bind to its C-terminal scaffold region to form a multienzyme complex called the RNA degradosome. The role of enolase within the RNase E-based degradosome in RNA decay has been totally mysterious. In this article, we demonstrate that the removal of the scaffold region of RNase E suppresses the rapid degradation of ptsG mRNA in response to the metabolic stress without affecting the expression of ptsG mRNA under normal conditions. We also demonstrate that the depletion of enolase but not the disruption of pnp or rhlB eliminates the rapid degradation of ptsG mRNA. Taken together, we conclude that enolase within the degradosome plays a crucial role in the regulation of ptsG mRNA stability in response to a metabolic stress. This is the first instance in which a physiological role for enolase in the RNA degradosome has been demonstrated. In addition, we show that PNPase and RhlB within the degradosome cooperate to eliminate short degradation intermediates of ptsG mRNA.     

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.     

The KH and S1 domains of Escherichia coli polynucleotide phosphorylase are necessary for autoregulation and growth at low temperature

PNPase is a phosphate-dependent exonuclease of Escherichia coli required for growth in the cold. In this work we explored the effect of specific mutations in its two RNA binding domains KH and S1 on RNA binding, enzymatic activities, autoregulation and ability to grow at low temperature. We removed critical motifs that stabilize the hydrophobic core of each domain, as well as made a complete deletion of both (DeltaKHS1) that severely impaired PNPase binding to RNA. Nevertheless, a residual RNA binding activity, possibly imputable to catalytic binding, could be observed even in the DeltaKHS1 PNPase. These mutations also resulted in significant changes in the kinetic behavior of both phosphorolysis and polymerization activities of the enzyme, in particular for the double mutant Pnp-DeltaKHS1-H. Additionally, PNPases with mutations in these RNA binding domains did not autoregulate efficiently and were unable to complement the growth defect of a chromosomal Deltapnp mutation at 18 degrees C. Based on these results it appears that in E. coli the RNA binding domains of PNPase, in particular the KH domain, are vital at low temperature, when the stem-loop structures present in the target mRNAs are more stable and a machinery capable to degrade structured RNA may be essential.     

Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome.

In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the endoribonuclease RNase E, a DEAD-RNA helicase and the glycolytic enzyme enolase are associated with a high molecular weight complex, the degradosome. This complex has an important role in processing and degradation of RNA. Chloroplasts contain an exoribonuclease homologous to E. coli PNPase. Size exclusion chromatography revealed that chloroplast PNPase elutes as a 580-600 kDa complex, suggesting that it can form an enzyme complex similar to the E. coli degradosome. Biochemical and mass-spectrometric analysis showed, however, that PNPase is the only protein associated with the 580-600 kDa complex. Similarly, a purified recombinant chloroplast PNPase also eluted as a 580-600 kDa complex after gel filtration chromatography. These results suggest that chloroplast PNPase exists as a homo-multimer complex. No other chloroplast proteins were found to associate with chloroplast PNPase during affinity chromatography. Database analysis of proteins homologous to E. coli RNase E revealed that chloroplast and cyanobacterial proteins lack the C-terminal domain of the E. coli protein that is involved in assembly of the degradosome. Together, our results suggest that PNPase does not form a degradosome-like complex in the chloroplast. Thus, RNA processing and degradation in this organelle differ in several respects from those in E. coli.     

The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo

RNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay. While the C-terminal half of this 1061-residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into a 'degradosome', only the N-terminal half, which carries the catalytic activity, is required for growth. We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E. This mutation resembles the N-terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected. Another mutation (rne105 ) removing the last 469 residues behaves similarly. Thus, the C-terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA. A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants. All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization. Artificial mRNAs generated by E. coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth.     

Nucleic acid and protein factors involved in Escherichia coli polynucleotide phosphorylase function on RNA

It has been reported that polynucleotide phosphorylase (PNPase) binds to RNA via KH and S1 domains, and at least two main complexes (I and II) have been observed in RNA-binding assays. Here we describe PNPase binding to RNA, the factors involved in this activity and the nature of the interactions observed in vitro. Our results show that RNA length and composition affect PNPase binding, and that PNPase interacts primarily with the 3′ end of RNA, forming the complex I-RNA, which contains trimeric units of PNPase. When the 5′ end of RNA is blocked by a hybridizing oligonucleotide, the formation of complex II-RNA is inhibited. In addition, PNPase was found to form high molecular weight (>440 kDa) aggregates in vitro in the absence of RNA, which may correspond to the hexameric form of the enzyme. We confirmed that PNPase in vitro RNA binding, degradation and polyadenylation activities depend on the integrity of KH and S1 domains. These results can explain the defective in vivo autoregulation of PNPase71, a KH point substitution mutant. As previously reported, optimal growth of a cold-sensitive strain at 18 °C requires a fully active PNPase, however, we show that overexpression of a novel PNPaseΔS1 partially compensated the growth impairment of this strain, while PNPase71 showed a minor compensation effect. Finally, we propose a mechanism of PNPase interactions and discuss their implications in PNPase function.     

Regulation of Escherichia coli polynucleotide phosphorylase by ATP

Polynucleotide phosphorylase (PNPase), an enzyme conserved in bacteria and eukaryotic organelles, processively catalyzes the phosphorolysis of RNA, releasing nucleotide diphosphates, and the reverse polymerization reaction. In Escherichia coli, both reactions are implicated in RNA decay, as addition of either poly(A) or heteropolymeric tails targets RNA to degradation. PNPase may also be associated with the RNA degradosome, a heteromultimeric protein machine that can degrade highly structured RNA. Here, we report that ATP binds to PNPase and allosterically inhibits both its phosphorolytic and polymerization activities. Our data suggest that PNPase-dependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge. These findings connect RNA turnover with the energy charge of the cell and highlight unforeseen metabolic roles of PNPase.     

Preferential degradation of polyadenylated and polyuridinylated RNAs by the bacterial exoribonuclease polynucleotide phosphorylase.

Polyadenylation of mRNA has been shown to target the RNA molecule for rapid exonucleolytic degradation in bacteria. To elucidate the molecular mechanism governing this effect, we determined whether the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated PNPase degraded polyadenylated and non-polyadenylated RNAs at similar rates. However, when the two molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. The same phenomenon was observed with polyuridinylated RNA. The poly(A) tail has to be located at the 3' end of the RNA, as the addition of several other nucleotides at the 3' end prevented competition for polyadenylated RNA. In RNA-binding experiments, E. coli PNPase bound to poly(A) and poly(U) sequences with much higher affinity than to poly(C) and poly(G). This high binding affinity defines poly(A) and poly(U) RNAs as preferential substrates for this enzyme. The high affinity of PNPase for polyadenylated RNA molecules may be part of the molecular mechanism by which polyadenylated RNA is preferentially degraded in bacterial cells.     

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.     

KH domains with impaired nucleic acid binding as a tool for functional analysis

In eukaryotes, RNA-binding proteins that contain multiple K homology (KH) domains play a key role in coordinating the different steps of RNA synthesis, metabolism and localization. Understanding how the different KH modules participate in the recognition of the RNA targets is necessary to dissect the way these proteins operate. We have designed a KH mutant with impaired RNA-binding capability for general use in exploring the role of individual KH domains in the combinatorial functional recognition of RNA targets. A double mutation in the hallmark GxxG loop (GxxG-to-GDDG) impairs nucleic acid binding without compromising the stability of the domain. We analysed the impact of the GDDG mutations in individual KH domains on the functional properties of KSRP as a prototype of multiple KH domain-containing proteins. We show how the GDDG mutant can be used to directly link biophysical information on the sequence specificity of the different KH domains of KSRP and their role in mRNA recognition and decay. This work defines a general molecular biology tool for the investigation of the function of individual KH domains in nucleic acid binding proteins.