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.
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.
Steven W
SW
Hardwick
Tobias
T
Gubbey
Isabelle
I
Hug
Urs
U
Jenal
Ben F
BF
Luisi
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain; D016935:Caulobacter crescentus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005095:Exoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D011117:Polyribonucleotide Nucleotidyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D020365:RNA Helicases / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; C111244:degradosome; C056502:ribonuclease PH; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; D005095:Exoribonucleases; C022537:ribonuclease E; D020365:RNA Helicases