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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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Recognition of RNase E by Caulobacter crescentus PNPase. (a) (i) Interaction of C. crescentus PNPase with RNase E recognition peptide. (ii) Interaction of E. coli PNPase with the recognition site in RNase E (pdb entry 3GME). The GWW motif, conserved in the α-proteobacterial RNase E, is bound at the opposite face of the PNPase ring from the surface exposing the SI and KH domains (at the top, not shown). The GWW motif peptide is shown in red. The inset is an expanded view showing the GWW peptide and an omit map for this peptide. (b) Mutation of the GWW motif in vitro. GST fusions of the PNPase recognition peptide were expressed with alanine mutations of the conserved tryptophan residues, and the ability to bind to PNPase was assessed by an electrophoretic mobility shift assay. (c) Deletion of the PNPase-binding site from RNase E in vivo. The PNPase recognition peptide was deleted from chromosomal RNase E, and the resulting pull-down of degradosome components was assessed by SDS–PAGE.
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RSOB120028F4: Recognition of RNase E by Caulobacter crescentus PNPase. (a) (i) Interaction of C. crescentus PNPase with RNase E recognition peptide. (ii) Interaction of E. coli PNPase with the recognition site in RNase E (pdb entry 3GME). The GWW motif, conserved in the α-proteobacterial RNase E, is bound at the opposite face of the PNPase ring from the surface exposing the SI and KH domains (at the top, not shown). The GWW motif peptide is shown in red. The inset is an expanded view showing the GWW peptide and an omit map for this peptide. (b) Mutation of the GWW motif in vitro. GST fusions of the PNPase recognition peptide were expressed with alanine mutations of the conserved tryptophan residues, and the ability to bind to PNPase was assessed by an electrophoretic mobility shift assay. (c) Deletion of the PNPase-binding site from RNase E in vivo. The PNPase recognition peptide was deleted from chromosomal RNase E, and the resulting pull-down of degradosome components was assessed by SDS–PAGE.

Mentions: It has previously been shown that C. crescentus PNPase interacts with the endoribonuclease RNase E to form part of an RNA–degradosome complex [26]. This interaction is mediated by a short segment at the C-terminus of RNase E containing a GWW motif that is strikingly conserved in RNase E of the α-proteobacteria. To examine further the importance of the conserved GWW motif in the PNPase recognition peptide, we substituted the tryptophan residues individually and in combination with alanine, and assessed the impact on PNPase binding using a native electrophoretic mobility shift assay (figure 4b). GST fused to the PNPase recognition peptide (TAPPEKPRRGWWRR) was mixed at an approximate fourfold molar excess with PNPase, and the mixture was then resolved on a native gel. The band corresponding to free PNPase was completely shifted to a slower migrating species in the presence of the wild-type GST–GWW peptide. However, when either tryptophan was mutated to alanine the formation of the complex with PNPase was severely disrupted.Figure 4.


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)

Recognition of RNase E by Caulobacter crescentus PNPase. (a) (i) Interaction of C. crescentus PNPase with RNase E recognition peptide. (ii) Interaction of E. coli PNPase with the recognition site in RNase E (pdb entry 3GME). The GWW motif, conserved in the α-proteobacterial RNase E, is bound at the opposite face of the PNPase ring from the surface exposing the SI and KH domains (at the top, not shown). The GWW motif peptide is shown in red. The inset is an expanded view showing the GWW peptide and an omit map for this peptide. (b) Mutation of the GWW motif in vitro. GST fusions of the PNPase recognition peptide were expressed with alanine mutations of the conserved tryptophan residues, and the ability to bind to PNPase was assessed by an electrophoretic mobility shift assay. (c) Deletion of the PNPase-binding site from RNase E in vivo. The PNPase recognition peptide was deleted from chromosomal RNase E, and the resulting pull-down of degradosome components was assessed by SDS–PAGE.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120028F4: Recognition of RNase E by Caulobacter crescentus PNPase. (a) (i) Interaction of C. crescentus PNPase with RNase E recognition peptide. (ii) Interaction of E. coli PNPase with the recognition site in RNase E (pdb entry 3GME). The GWW motif, conserved in the α-proteobacterial RNase E, is bound at the opposite face of the PNPase ring from the surface exposing the SI and KH domains (at the top, not shown). The GWW motif peptide is shown in red. The inset is an expanded view showing the GWW peptide and an omit map for this peptide. (b) Mutation of the GWW motif in vitro. GST fusions of the PNPase recognition peptide were expressed with alanine mutations of the conserved tryptophan residues, and the ability to bind to PNPase was assessed by an electrophoretic mobility shift assay. (c) Deletion of the PNPase-binding site from RNase E in vivo. The PNPase recognition peptide was deleted from chromosomal RNase E, and the resulting pull-down of degradosome components was assessed by SDS–PAGE.
Mentions: It has previously been shown that C. crescentus PNPase interacts with the endoribonuclease RNase E to form part of an RNA–degradosome complex [26]. This interaction is mediated by a short segment at the C-terminus of RNase E containing a GWW motif that is strikingly conserved in RNase E of the α-proteobacteria. To examine further the importance of the conserved GWW motif in the PNPase recognition peptide, we substituted the tryptophan residues individually and in combination with alanine, and assessed the impact on PNPase binding using a native electrophoretic mobility shift assay (figure 4b). GST fused to the PNPase recognition peptide (TAPPEKPRRGWWRR) was mixed at an approximate fourfold molar excess with PNPase, and the mixture was then resolved on a native gel. The band corresponding to free PNPase was completely shifted to a slower migrating species in the presence of the wild-type GST–GWW peptide. However, when either tryptophan was mutated to alanine the formation of the complex with PNPase was severely disrupted.Figure 4.

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