The Protein Interaction of RNA Helicase B (RhlB) and Polynucleotide Phosphorylase (PNPase) Contributes to the Homeostatic Control of Cysteine in Escherichia coli.
PNPase, one of the major enzymes with 3' to 5' single-stranded RNA degradation and processing activities, can interact with the RNA helicase RhlB independently of RNA degradosome formation in Escherichia coli. Here, we report that loss of interaction between RhlB and PNPase impacts cysteine homeostasis in E. coli. By random mutagenesis, we identified a mutant RhlB(P238L) that loses 75% of its ability to interact with PNPase but retains normal interaction with RNase E and RNA, in addition to exhibiting normal helicase activity. Applying microarray analyses to an E. coli strain with impaired RNA degradosome formation, we investigated the biological consequences of a weakened interaction between RhlB and PNPase. We found significant increases in 11 of 14 genes involved in cysteine biosynthesis. Subsequent Northern blot analyses showed that the up-regulated transcripts were the result of stabilization of the cysB transcript encoding a transcriptional activator for the cys operons. Furthermore, Northern blots of PNPase or RhlB mutants showed that RhlB-PNPase plays both a catalytic and structural role in regulating cysB degradation. Cells expressing the RhlB(P238L) mutant exhibited an increase in intracellular cysteine and an enhanced anti-oxidative response. Collectively, this study suggests a mechanism by which bacteria use the PNPase-RhlB exosome-like complex to combat oxidative stress by modulating cysB mRNA degradation.
From the Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, the Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 10617, Taiwan.
Yi-Ting
YT
Tseng
From the Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, the Institute of Biochemistry & Molecular Biology, National Yang-Ming University, Taipei 11221, Taiwan.
Ni-Ting
NT
Chiou
From the Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan.
Rajinikanth
R
Gogiraju
From the Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, mbsue@gate.sinica.edu.tw.
Sue
S
Lin-Chao
D000255:Adenosine Triphosphate / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006706:Homeostasis; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism*; D011485:Protein Binding
D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D011117:Polyribonucleotide Nucleotidyltransferase; C502431:RhlB protein, E coli; D053487:DEAD-box RNA Helicases; D003545:Cysteine
Cysteine homeostasis; Escherichia coli (E. coli); PNPase; RNA degradation; RhlB; bacterial metabolism; exosome complex; oxidative stress; ribonuclease