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

Hardwick SW, Gubbey T, Hug I, Jenal U, Luisi BF - Open Biol (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.

ABSTRACT
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.

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The structure of Caulobacter crescentus polynucleotide phosphorylase. (a) Linear schematic of domain organization, showing the two RNase PH domains, the helical domain, the KH and S1 RNA-binding domains. (b) A schematic of the structure of the trimeric C. crescentus PNPase. (i) Side view and (ii) perpendicular view along the threefold axis. For one of the protomers, the individual subdomains are colour coded according to the scheme in figure 1a. The other two protomers are grey.
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RSOB120028F1: The structure of Caulobacter crescentus polynucleotide phosphorylase. (a) Linear schematic of domain organization, showing the two RNase PH domains, the helical domain, the KH and S1 RNA-binding domains. (b) A schematic of the structure of the trimeric C. crescentus PNPase. (i) Side view and (ii) perpendicular view along the threefold axis. For one of the protomers, the individual subdomains are colour coded according to the scheme in figure 1a. The other two protomers are grey.

Mentions: All known PNPase homologues share the same modular organization of conserved structural domains, depicted schematically in figure 1a. Two domains, both resembling closely the phosphorolytic exoribonuclease RNase PH, almost certainly have originated from duplication and fusion of an ancestral gene. While the C-terminal RNase PH-like domain catalyses phosphorolytic attack of RNA, the N-terminal domain has lost this capacity; instead, it contributes to the ring-like quaternary structure of the trimeric PNPase assembly [17]. Two recurrent structural motifs that are found in many RNA-binding proteins, the S1 and K-homology (KH) domains, are appended at the C-terminus of PNPase [18]. Finally, an all-helical domain is situated between the two RNase PH-like domains. The crystal structures of Streptomyces antibioticus, E. coli and human PNPase reveal how these domains are spatially organized [17,19]. Foremost, the structures reveal that the trimer is stabilized mainly by interactions of neighbouring RNase PH-like domains, and the quaternary structure generates a central channel coincident with the molecular threefold axis. The exosome assemblies of eukaryotes and archaea share molecular ancestry with PNPase, and have a similar ring-like architecture of RNase PH, S1 and KH subunits [20].Figure 1.


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

Hardwick SW, Gubbey T, Hug I, Jenal U, Luisi BF - Open Biol (2012)

The structure of Caulobacter crescentus polynucleotide phosphorylase. (a) Linear schematic of domain organization, showing the two RNase PH domains, the helical domain, the KH and S1 RNA-binding domains. (b) A schematic of the structure of the trimeric C. crescentus PNPase. (i) Side view and (ii) perpendicular view along the threefold axis. For one of the protomers, the individual subdomains are colour coded according to the scheme in figure 1a. The other two protomers are grey.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376730&req=5

RSOB120028F1: The structure of Caulobacter crescentus polynucleotide phosphorylase. (a) Linear schematic of domain organization, showing the two RNase PH domains, the helical domain, the KH and S1 RNA-binding domains. (b) A schematic of the structure of the trimeric C. crescentus PNPase. (i) Side view and (ii) perpendicular view along the threefold axis. For one of the protomers, the individual subdomains are colour coded according to the scheme in figure 1a. The other two protomers are grey.
Mentions: All known PNPase homologues share the same modular organization of conserved structural domains, depicted schematically in figure 1a. Two domains, both resembling closely the phosphorolytic exoribonuclease RNase PH, almost certainly have originated from duplication and fusion of an ancestral gene. While the C-terminal RNase PH-like domain catalyses phosphorolytic attack of RNA, the N-terminal domain has lost this capacity; instead, it contributes to the ring-like quaternary structure of the trimeric PNPase assembly [17]. Two recurrent structural motifs that are found in many RNA-binding proteins, the S1 and K-homology (KH) domains, are appended at the C-terminus of PNPase [18]. Finally, an all-helical domain is situated between the two RNase PH-like domains. The crystal structures of Streptomyces antibioticus, E. coli and human PNPase reveal how these domains are spatially organized [17,19]. Foremost, the structures reveal that the trimer is stabilized mainly by interactions of neighbouring RNase PH-like domains, and the quaternary structure generates a central channel coincident with the molecular threefold axis. The exosome assemblies of eukaryotes and archaea share molecular ancestry with PNPase, and have a similar ring-like architecture of RNase PH, S1 and KH subunits [20].Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.

ABSTRACT
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.

Show MeSH