[국내논문]Streptomyces coelicolor RraAS1의 Eschechia coli RNase E의 RNA 분해작용에 대한 활성제로서 기능 암시 Implications of Streptomyces coelicolor RraAS1 as an activator of ribonuclease activity of Escherichia coli RNase E원문보기
RNase E는 대장균(Escherichia coli)에서 수많은 RNA의 가공 및 분해에 관여하는 필수적인 효소이다. RNase E의 효소 활성은 RraA와 RraB에 의해 조절된다. 그람양성균인 Streptomyces coelicolor는 RNase ES, RraAS1, RraAS2라고 명명되는 RNase E와 RraA의 동족체를 가지고 있다. 이 연구에서는 S. coelicolor 유래의 RraAS1이 E. coli에서 RNase E의 효소활성을 저해하는지 연구하였다. 대장균에서 RraAS1의 발현은 RNase E의 과발현에 의해 감소된 세포생장을 더욱 저하시켰으며, RNase E의 기질인 rpsO, ftsZ, rnhB mRNA의 양을 감소시키는 것을 확인 하였다. 이러한 RraAS1의 효과는 공동면역침전실험을 수행한 결과에서 유추할 수 있듯이, Rne 단백질과 RraAS1의 결합으로 유도되는 것으로 보인다. 이러한 결과는 RraAS1이 대장균에서 RNase E의 리보핵산 가수분해 활성을 유도함을 시사한다.
RNase E는 대장균(Escherichia coli)에서 수많은 RNA의 가공 및 분해에 관여하는 필수적인 효소이다. RNase E의 효소 활성은 RraA와 RraB에 의해 조절된다. 그람양성균인 Streptomyces coelicolor는 RNase ES, RraAS1, RraAS2라고 명명되는 RNase E와 RraA의 동족체를 가지고 있다. 이 연구에서는 S. coelicolor 유래의 RraAS1이 E. coli에서 RNase E의 효소활성을 저해하는지 연구하였다. 대장균에서 RraAS1의 발현은 RNase E의 과발현에 의해 감소된 세포생장을 더욱 저하시켰으며, RNase E의 기질인 rpsO, ftsZ, rnhB mRNA의 양을 감소시키는 것을 확인 하였다. 이러한 RraAS1의 효과는 공동면역침전실험을 수행한 결과에서 유추할 수 있듯이, Rne 단백질과 RraAS1의 결합으로 유도되는 것으로 보인다. 이러한 결과는 RraAS1이 대장균에서 RNase E의 리보핵산 가수분해 활성을 유도함을 시사한다.
RNase E (Rne) is an essential enzyme involved in the processing and degradation of a large portion of RNAs in Escherichia coli. The enzymatic activity of RNase E is controlled by regulators of ribonuclease activity, namely, RraA and RraB. Gram-positive bacterium Streptomyces coelicolor also contains...
RNase E (Rne) is an essential enzyme involved in the processing and degradation of a large portion of RNAs in Escherichia coli. The enzymatic activity of RNase E is controlled by regulators of ribonuclease activity, namely, RraA and RraB. Gram-positive bacterium Streptomyces coelicolor also contains homologs of Rne and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2. In the present study, we investigated the effect of S. coelicolor RraAS1 on the ribonucleolytic activity of RNase E in E. coli. Coexpression of RraAS1 with Rne resulted in the decreased levels of rpsO, ftsZ, and rnhB mRNAs, which are RNase E substrates, and augmented the toxic effect of Rne overexpression on cell growth. These in vivo effects appeared to be induced by the binding of RraAS1 to Rne, as indicated by the results of co-immunoprecipitation analysis. These results suggested that RraAS1 induces ribonucleolytic activity of RNase E in E. coli.
RNase E (Rne) is an essential enzyme involved in the processing and degradation of a large portion of RNAs in Escherichia coli. The enzymatic activity of RNase E is controlled by regulators of ribonuclease activity, namely, RraA and RraB. Gram-positive bacterium Streptomyces coelicolor also contains homologs of Rne and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2. In the present study, we investigated the effect of S. coelicolor RraAS1 on the ribonucleolytic activity of RNase E in E. coli. Coexpression of RraAS1 with Rne resulted in the decreased levels of rpsO, ftsZ, and rnhB mRNAs, which are RNase E substrates, and augmented the toxic effect of Rne overexpression on cell growth. These in vivo effects appeared to be induced by the binding of RraAS1 to Rne, as indicated by the results of co-immunoprecipitation analysis. These results suggested that RraAS1 induces ribonucleolytic activity of RNase E in E. coli.
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문제 정의
그람양성균인 Streptomyces coelicolor는 RNase ES, RraAS1, RraAS2라고 명명되는 RNase E와 RraA의 동족체를 가지고 있다. 이 연구에서는 S. coelicolor 유래의 RraAS1이 E. coli에서 RNase E의 효소활성을 저해하는지 연구하였다. 대장균에서 RraAS1의 발현은 RNase E의 과발현에 의해 감소된 세포생장을 더욱 저하시켰으며, RNase E의 기질인 rpsO, ftsZ, rnhB mRNA의 양을 감소시키는 것을 확인 하였다.
가설 설정
To test whether Rne interacts with RraAS1, C-terminally Myc-tagged RraAS1 (RraAS1-Myc) was overexpressed in KSL2002 and KSL2003 cells, and immunoprecipitated using an antibody against the Myc-tag. Coexpression of RraAS1-Myc in KSL2003 cells overexpressing Rne in the presence of 1 mM IPTG showed a growth pattern similar to that of KSL2003 cells coexpressing untagged RraAS1 and Rne (Fig.
제안 방법
To investigate whether RraAS1 can regulate the ribonucleolytic activity of RNase E in vivo, we analyzed steady-state levels of three RNase E substrates, rpsO, ftsZ, and rnhB mRNAs, in KSL2003 cells. Induced overexpression of Rne in KSL2003 cells resulted in decreased abundance of these mRNAs by approximately 30–50% than that in KSL2003 cells expressing Rne in the presence of 10 μM IPTG (Fig.
성능/효과
coli RNase E activity, we introduced a compatible kanamycin resistance (Kmr) plasmid expressing RraAS1 under the control of an arabinose-inducible promoter (pKAN6B-RraAS1) into KSL2003 cells. Our results showed that growth of KSL2003 cells overexpressing both Rne and RraAS1 in the presence of 1 mM IPTG and 0.2% arabinose was more inhibited than that of KSL2003 cells harboring an empty vector (pKAN6B). Propagation of toxic effect of RraAS1 on the growth of KSL2003 cells overexpressing Rne is not likely to result from RraAS1 overexpression itself because the growth of KSL2002 cells overexpressing both N-Rne and RraAS1 was similar to that of KSL2002 cells harboring the empty vector (pKAN6B) (Fig.
The experiment was repeated at least three times, and standard errors of mean were designated as “± numbers”.
coli. Our results indicated that, unlike other RraA homologs including RraAS2 (Ahn et al., 2008) and RraAV1 (Lee et al., 2009), a Vibrio vulnificus RraA homolog, which exerts similar inhibitory effects on RNase E as RraA, RraAS1 appeared to activate RNase E activity in E. coli. This mode of RraAS1 action on RNase E activity is likely to be mediated by its interaction with the scaffold domain of Rne and probably explains why the toxic effect of Rne overexpression on the growth of KSL2003 cells was propagated when RraAS1 was coexpressed.
coli에서 RNase E의 효소활성을 저해하는지 연구하였다. 대장균에서 RraAS1의 발현은 RNase E의 과발현에 의해 감소된 세포생장을 더욱 저하시켰으며, RNase E의 기질인 rpsO, ftsZ, rnhB mRNA의 양을 감소시키는 것을 확인 하였다. 이러한 RraAS1의 효과는 공동면역침전실험을 수행한 결과에서 유추할 수 있듯이, Rne 단백질과 RraAS1의 결합으로 유도되는 것으로 보인다.
후속연구
, 2003, 2009). Therefore, further studies should be performed to determine molecular mechanisms underlying this unique effect of RraAS1 on Rne and RraAS1-mediated RNA metabolism in S. coelicolor.
참고문헌 (20)
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