김완태 (University of Texas, Austin) | 한빛사논문 > 한빛사

한빛사논문

조회391

University of Texas, Austin

CV File 113 kb

Proc. Natl. Acad. Sci. USA, Nov 12 2024, 121 (46) | https://doi.org/10.1073/pnas.2317865121 Selective 8-oxo-rG stalling occurs in the catalytic core of polynucleotide phosphorylase (PNPase) during degradation

Lucas G. Miller^a,1, Wantae Kim^a,1, Shawn Schowe^b,1, Kathleen Taylor^a, Runhua Han^a, Vashita Jain^b, Raeyeon Park^c, Mark Sherman^a, Janssen Fang^d, Haydee Ramirez^b, Andrew Ellington^d, Phanourios Tamamis^c,e, Marino J. E. Resendiz^b,2, Y. Jessie Zhang^d,2, and Lydia Contreras^a,d,2,3

^aMcKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712

^bDepartment of Chemistry, University of Colorado Denver, Denver, CO 80217

^cArtie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX 77843

^dDepartment of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712

^eDepartment of Materials Science & Engineering, Texas A&M University, College Station, TX 77840

¹L.G.M., W.K., and S.S. contributed equally to this work.

²To whom correspondence may be addressed. : Marino J. E. Resendiz, Y. Jessie Zhang, and Lydia Contreras

³L.C. is an organizer of this Special Feature.

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 Escherichia coli 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 E. coli 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 E. coli cell growth experiments under H₂O₂-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.

논문정보

형식Research article
게재일2024년 11월 (BRIC 등록일 2024-11-06)
연구진국외 연구진
분야 생명과학 > 구조생물 및 생물물리학

댓글 작성 로그인

CV 열람하기

연락처 보기