검색 상세

패혈증비브리오균 조절RNA를 통한 산소의 혐기성 대사 단백질 발현 조절 규명

초록/요약

FrsA의 pyruvate decarboxylase 활성이 혐기성조건에서만 나타나는 원인을 찾고자 산소유무에 따른 FrsA 단백질의 양을 비교하였다. 혐기성조건에서만 FrsA 단백질이 발현되었지만, frsA mRNA의 양은 산소에 영향을 받지 않았다. 따라서 small regulatory RNA와 같은 전사 후 조절이 관여할 것이라는 추측을 하게 되었고, in silico 분석을 통해 sRNA 후보 서열을 동정하였다. Northern blot을 통해 sRNA의 존재를 확인하고 Rsf(Regulatory sRNA for FrsA expression)라 명명하였다. Rsf에 의해 FrsA단백질의 발현이 조절되는 방식을 확인하기 위해 frsA mRNA의 안정성조절과 번역여부조절에서 Rsf의 영향을 조사하였고, Rsf가 frsA mRNA의 번역 단계를 억제하는 것을 확인할 수 있었다. 또한, 혐기성 조건에서 전사조절자 FNR(Fumarate and nitrate reduction regulatory protein)이 Rsf의 전사를 억제하여 혐기성조건에서 호기성조건에 비해 Rsf의 양이 감소하는 것을 확인하였다. 그러므로 산소에 산소 유무에 따라 FNR이 Rsf의 전사를 조절하고, Rsf는 pyruvate decarboxylase이자 pyruvate의 anaerobic 대사를 촉진하는 역할의 FrsA(Fermentation respiration switch protein) 단백질의 번역 여부를 전사 후 조절 단계에서 결정한다.

more

초록/요약

Fermentation respiration switch (FrsA) is an enzyme catalyzing a conversion of pyruvate to acetaldehyde and carbon dioxide. FrsA protein level was not detectable in Vibrio vulnificus cells grown under oxygen-rich condition, and thus the in vivo activity of pyruvate decarboxylation derived from FrsA was observed in the cells grown under oxygen-limited condition. To investigate the regulatory mechanism(s) for the anaerobic induction of FrsA expression and activity, its transcription was monitored using both frsA-transcription reporter and quantitative RT-PCR assays. However, no significant difference was observed in its transcription and the resultant transcripts in the cells grown under aerobic or anaerobic conditions. This result lead us to consider the specific regulation at the post-transcription level and to examine the involvement of sRNA in FrsA expression. A candidate regulatory sRNA for FrsA expression (Rsf), including the sequences complementary to the 5'-UTR of frsA mRNA, was identified in V. vulnificus genome. A northern blot revealed the presence of 350 nucleotide-long sRNA. Its regulatory role was examined via monitoring FrsA levels in the rsf-deleted mutant V. vulnificus. In the absence of rsf gene, the negative effect of oxygen on the cellular level of FrsA was abolished, and thus the rsf mutant exhibited high activity of pyruvate decarboxylation even under the aerobic condition. It was further determined the regulatory dependency of Rsf on oxygen in repressing FrsA expression. Expression of the rsf gene was repressed by a transcription factor FNR under anaerobic condition, whereas repression of rsf transcription by FNR was relieved in the presence of oxygen. Thus, this study demonstrates that the cellular content of FrsA is minimized during aerobic growth via repression of its expression by Rsf. This repression, however, is relieved under anaerobic condition via repression of the rsf transcription by FNR, resulting in higher levels of the cellular FrsA and the mixed-acid fermentative metabolisms.

more