Polynucleotide phosphorylase protects Escherichia coli against oxidative stress.
Escherichia coli polynucleotide phosphorylase (PNPase) primarily functions in RNA degradation. It is an exoribonuclease and integral component of the multienzyme RNA degradosome complex [Carpousis et al. (1994) Cell 76, 889]. PNPase was previously shown to specifically bind a synthetic RNA containing the oxidative lesion 8-hydroxyguanine (8-oxoG) [Hayakawa et al. (2001) Biochemistry 40, 9977], suggesting a possible role in removing oxidatively damaged RNA. Here we show that PNPase binds to RNA molecules of natural sequence that were oxidatively damaged by treatment with hydrogen peroxide (H(2)O(2)) postsynthetically. PNPase bound oxidized RNA with higher affinity than untreated RNA of the same sequence, raising the possibility that it may act against a wide variety of lesions. The importance of such a protective role is illustrated by the observation that, under conditions known to cause oxidative damage to cytoplasmic components, PNPase-deficient cells are less viable than wild-type cells. Further, when challenged with H(2)O(2), PNPase-deficient cells accumulate 8-oxoG in cellular RNA to a greater extent than wild-type cells, suggesting that this RNase functions in minimizing oxidized RNA in vivo. Introducing the pnp gene encoding PNPase rescues defects in growth and RNA quality of the pnp mutant cells. Our results also suggest that protection against oxidative stress is an intrinsic function of PNPase because association with the RNA degradosome or with RNA helicase B (RhlB) is not required.
Wu
Jinhua
J
College of Biomedical Science, Florida Atlantic University, 777 Glades Road, Boca Raton, Florida 33431, USA.
Jiang
Zhe
Z
Liu
Min
M
Gong
Xin
X
Wu
Shaohui
S
Burns
Christopher M
CM
Li
Zhongwei
Z
D000080242:8-Hydroxy-2'-Deoxyguanosine; D001483:Base Sequence; D001667:Binding, Competitive; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D050296:Microbial Viability / Q000187:drug effects; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010269:Paraquat / Q000494:pharmacology; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012329:RNA, Bacterial / Q000378:metabolism
D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D012329:RNA, Bacterial; C024829:8-hydroxyguanine; D006147:Guanine; D000080242:8-Hydroxy-2'-Deoxyguanosine; D006861:Hydrogen Peroxide; D011117:Polyribonucleotide Nucleotidyltransferase; D003849:Deoxyguanosine; D010269:Paraquat