8VAH

E.coli PNPase in complex with single 8-oxoG RNA

Summary for 8VAH

• Entry DOI: 10.2210/pdb8vah/pdb
• EMDB information: 43092
• Descriptor: Polyribonucleotide nucleotidyltransferase, RNA (5'-R(P*AP*AP*AP*AP*AP*AP*A*A*A)-3'), RNA (5'-R(P*AP*CP*AP*(8GM))-3'), ... (4 entities in total)
• Functional Keywords: pnpase, oxidized rna, phosphorolysis, 8-oxo g, rna binding protein
• Biological source: Escherichia coli; More
• Total number of polymer chains: 7
• Total formula weight: 238399.86

Authors

Kim, W.,Zhang, Y.J. (deposition date: 2023-12-11, release date: 2024-11-20)

Primary citation

Miller, L.G.,Kim, W.,Schowe, S.,Taylor, K.,Han, R.,Jain, V.,Park, R.,Sherman, M.,Fang, J.,Ramirez, H.,Ellington, A.,Tamamis, P.,Resendiz, M.J.E.,Zhang, Y.J.,Contreras, L.
Selective 8-oxo-rG stalling occurs in the catalytic core of polynucleotide phosphorylase (PNPase) during degradation.
Proc.Natl.Acad.Sci.USA, 121:e2317865121-e2317865121, 2024

PubMed Abstract: RNA oxidation, predominantly through the accumulation of 8-oxo-7,8-dihydroguanosine (8-oxo-rG), represents an important biomarker for cellular oxidative stress. Polynucleotide phosphorylase (PNPase) is a 3'-5' exoribonuclease that has been shown to preferentially recognize 8-oxo-rG-containing RNA and protect cells from oxidative stress. However, the impact of 8-oxo-rG on PNPase-mediated RNA degradation has not been studied. Here, we show that the presence of 8-oxo-rG in RNA leads to catalytic stalling of PNPase through in vitro RNA degradation experiments and electrophoretic analysis. We also link this stalling to the active site of the enzyme through resolution of single-particle cryo-EM structures for PNPase in complex with singly or doubly oxidized RNA oligonucleotides. Following identification of Arg399 as a key residue in recognition of both single and sequential 8-oxo-rG nucleotides, we perform follow-up in vitro analysis to confirm the importance of this residue in 8-oxo-rG-specific PNPase stalling. Finally, we investigate the effects of mutations to active site residues implicated in 8-oxo-rG binding through cell growth experiments under HO-induced oxidative stress. Specifically, Arg399 mutations show significant effects on cell growth under oxidative stress. Overall, we demonstrate that 8-oxo-rG-specific stalling of PNPase is relevant to bacterial survival under oxidative stress and speculate that this enzyme might associate with other cellular factors to mediate this stress.

PubMed: 39495922
DOI: 10.1073/pnas.2317865121

Experimental method

ELECTRON MICROSCOPY (3.15 Å)