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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 path of the RNA in polynucleotide phosphorylase (PNPase). (a) A speculative model for the path of the RNA in the bacterial PNPase from the S1 domains to the active site. (i,ii) Two views rotated approximately 120°; for clarity, only two PNPase protomers are shown (green and blue). RNA bound to C. crescentus PNPase is shown as orange cartoon. RNA modelled at the active site is based on the position of RNA bound to the structurally homologous archaeal exosome (3M7N). RNA bound to the S1 domains is based on the position of RNA bound to the S1 domain of E. coli RNase E (2C0B). Predicted links between the RNA segments are shown as a dashed red line. (b) Schematic of the proposed threading mechanism. PNPase core protomers are depicted as blue cylinders, KH domains as black curved lines and single-stranded RNA as orange arrows. (i,ii,iii) The proposed rotary movement of the KH domains threading the RNA substrate to active sites of adjacent protomers, with the dark blue protomer representing the active site currently engaging the substrate. The model is speculative and proposes that the RNA may be bound and cleaved in three different active sites in the PNPase trimer.
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RSOB120028F5: The path of the RNA in polynucleotide phosphorylase (PNPase). (a) A speculative model for the path of the RNA in the bacterial PNPase from the S1 domains to the active site. (i,ii) Two views rotated approximately 120°; for clarity, only two PNPase protomers are shown (green and blue). RNA bound to C. crescentus PNPase is shown as orange cartoon. RNA modelled at the active site is based on the position of RNA bound to the structurally homologous archaeal exosome (3M7N). RNA bound to the S1 domains is based on the position of RNA bound to the S1 domain of E. coli RNase E (2C0B). Predicted links between the RNA segments are shown as a dashed red line. (b) Schematic of the proposed threading mechanism. PNPase core protomers are depicted as blue cylinders, KH domains as black curved lines and single-stranded RNA as orange arrows. (i,ii,iii) The proposed rotary movement of the KH domains threading the RNA substrate to active sites of adjacent protomers, with the dark blue protomer representing the active site currently engaging the substrate. The model is speculative and proposes that the RNA may be bound and cleaved in three different active sites in the PNPase trimer.

Mentions: We have prepared a speculative model for the path of single-stranded RNA by combining the crystal structures of the C. crescentus PNPase and of an archaeal exosome with a short segment of RNA leading from the active site to the proximal aperture (figure 5b). In the RNA-free structure of PNPase, the S1 domains were resolved but were poorly ordered and could not be resolved in the RNA-bound structure, and as such their role in substrate recognition and processing remains to be elucidated. In the model shown in figure 5, we have used the RNA bound to the S1 domain of E. coli RNase E as a guide to speculate on the path of the nucleic acid from one extreme end of the PNPase trimer to the active site.Figure 5.


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 path of the RNA in polynucleotide phosphorylase (PNPase). (a) A speculative model for the path of the RNA in the bacterial PNPase from the S1 domains to the active site. (i,ii) Two views rotated approximately 120°; for clarity, only two PNPase protomers are shown (green and blue). RNA bound to C. crescentus PNPase is shown as orange cartoon. RNA modelled at the active site is based on the position of RNA bound to the structurally homologous archaeal exosome (3M7N). RNA bound to the S1 domains is based on the position of RNA bound to the S1 domain of E. coli RNase E (2C0B). Predicted links between the RNA segments are shown as a dashed red line. (b) Schematic of the proposed threading mechanism. PNPase core protomers are depicted as blue cylinders, KH domains as black curved lines and single-stranded RNA as orange arrows. (i,ii,iii) The proposed rotary movement of the KH domains threading the RNA substrate to active sites of adjacent protomers, with the dark blue protomer representing the active site currently engaging the substrate. The model is speculative and proposes that the RNA may be bound and cleaved in three different active sites in the PNPase trimer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120028F5: The path of the RNA in polynucleotide phosphorylase (PNPase). (a) A speculative model for the path of the RNA in the bacterial PNPase from the S1 domains to the active site. (i,ii) Two views rotated approximately 120°; for clarity, only two PNPase protomers are shown (green and blue). RNA bound to C. crescentus PNPase is shown as orange cartoon. RNA modelled at the active site is based on the position of RNA bound to the structurally homologous archaeal exosome (3M7N). RNA bound to the S1 domains is based on the position of RNA bound to the S1 domain of E. coli RNase E (2C0B). Predicted links between the RNA segments are shown as a dashed red line. (b) Schematic of the proposed threading mechanism. PNPase core protomers are depicted as blue cylinders, KH domains as black curved lines and single-stranded RNA as orange arrows. (i,ii,iii) The proposed rotary movement of the KH domains threading the RNA substrate to active sites of adjacent protomers, with the dark blue protomer representing the active site currently engaging the substrate. The model is speculative and proposes that the RNA may be bound and cleaved in three different active sites in the PNPase trimer.
Mentions: We have prepared a speculative model for the path of single-stranded RNA by combining the crystal structures of the C. crescentus PNPase and of an archaeal exosome with a short segment of RNA leading from the active site to the proximal aperture (figure 5b). In the RNA-free structure of PNPase, the S1 domains were resolved but were poorly ordered and could not be resolved in the RNA-bound structure, and as such their role in substrate recognition and processing remains to be elucidated. In the model shown in figure 5, we have used the RNA bound to the S1 domain of E. coli RNase E as a guide to speculate on the path of the nucleic acid from one extreme end of the PNPase trimer to the active site.Figure 5.

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