RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity.
RraA is a protein inhibitor of RNase E (Rne), which catalyzes the endoribonucleolytic cleavage of a large proportion of RNAs in Escherichia coli. The antibiotic-producing bacterium Streptomyces coelicolor also contains homologs of RNase E and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2, respectively. Here, we report that RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity. Analyses of the steady-state level of RNase E substrates indicated that coexpression of RraAS2 in E. coli cells overproducing Rns effectively inhibits the ribonucleolytic activity of full-length RNase ES, but its inhibitory effects were moderate or undetectable on other truncated forms of Rns, in which the N- or/and C-terminal scaffold domain was deleted. In addition, RraAS2 more efficiently inhibited the in vitro ribonucleolytic activity of RNase ES than that of a truncated form containing the catalytic domain only. Coimmunoprecipitation and in vivo cross-linking experiments further showed necessity of both scaffold domains of RNase ES for high-affinity binding of RraAS2 to the enzyme, resulting in decreased RNA-binding capacity of RNase ES. Our results indicate that RraAS2 is a protein inhibitor of RNase ES and provide clues to how this inhibitor affects the ribonucleolytic activity of RNase ES.
Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.
Jihune
J
Heo
Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.
Daeyoung
D
Kim
Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.
Minju
M
Joo
Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.
Boeun
B
Lee
Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.
Sojin
S
Seo
Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.
Jaejin
J
Lee
Department of Agricultural Biotechnology, Seoul National University, Seoul, 08826, Republic of Korea.
Saemee
S
Song
Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.
Ji-Hyun
JH
Yeom
Department of Agricultural Biotechnology, Seoul National University, Seoul, 08826, Republic of Korea.
Nam-Chul
NC
Ha
Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea. kangseok@cau.ac.kr.
Kangseok
K
Lee
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D004722:Endoribonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020871:RNA Stability; D012329:RNA, Bacterial / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; C477942:RraA protein, E coli; D004722:Endoribonucleases; C022537:ribonuclease E
RNA stability; RNase ES; RraAS2; Streptomyces coelicolor
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