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Crystal Structure of Escherichia coli Polynucleotide Phosphorylase Core Bound to RNase E, RNA and Manganese: Implications for Catalytic Mechanism and RNA Degradosome Assembly

DOI: 10.1016/j.jmb.2009.03.051 DOI Help
PMID: 19327365 PMID Help

Authors: Salima Nurmohamed (University of Cambridge) , Bhamini Vaidialingam (University of Cambridge) , Anastasia J. Callaghan (University of Portsmouth) , Ben F Luisi (Department of Biochemistry, University of Cambridge)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Molecular Biology , VOL 389 (1) , PAGES 17 - 33

State: Published (Approved)
Published: May 2009

Open Access Open Access

Abstract: Polynucleotide phosphorylase (PNPase) is a processive exoribonuclease that contributes to messenger RNA turnover and quality control of ribosomal RNA precursors in many bacterial species. In Escherichia coli, a proportion of the PNPase is recruited into a multi-enzyme assembly, known as the RNA degradosome, through an interaction with the scaffolding domain of the endoribonuclease RNase E. Here, we report crystal structures of E. coli PNPase complexed with the recognition site from RNase E and with manganese in the presence or in the absence of modified RNA. The homotrimeric PNPase engages RNase E on the periphery of its ring-like architecture through a pseudo-continuous anti-parallel β-sheet. A similar interaction pattern occurs in the structurally homologous human exosome between the Rrp45 and Rrp46 subunits. At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites. Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state. We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.

Journal Keywords: Biocatalysis; Calorimetry; Catalytic; Crystallography; X-Ray; Endoribonucleases; Escherichia; Manganese; Models; Molecular; Multienzyme; Polyribonucleotide; Protein; Quaternary; Protein; Tertiary; RNA; RNA; Bacterial

Subject Areas: Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography

Other Facilities: ESRF

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