A novel fluorescent reporter system for monitoring and identifying RNase III activity and its target RNAs

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dc.contributor.authorKwang-Sun Kim-
dc.contributor.authorSunyoung Park-
dc.contributor.authorSoohyun Lee-
dc.contributor.authorS B Kang-
dc.contributor.authorJ Lee-
dc.contributor.authorSeung Goo Lee-
dc.contributor.authorChoong-Min Ryu-
dc.date.accessioned2017-04-19T09:33:27Z-
dc.date.available2017-04-19T09:33:27Z-
dc.date.issued2012-
dc.identifier.issn15476286-
dc.identifier.uri10.4161/rna.21499ko
dc.identifier.urihttps://oak.kribb.re.kr/handle/201005/10904-
dc.description.abstractBacteriophage vectors for achieving single-copy gene expression linked to a colorigenic reporter assay have been used successfully for genetic screening applications. However, the limited number of cloning sites in these vectors, combined with the requirement for lac-strains and the time-and/or media-dependence of the chemical-based colorimetric reaction, have limited the range of applications for these vectors. An alternative approach using a fluorescent reporter gene such as green fluorescent protein (GFP) or GFP derivatives could overcome some of these technical issues and facilitate real-time monitoring of promoter and/or protein activity. Here, we report the development of a novel translational bacteriophage fusion vector encoding enhanced GFP (eGFP) that can be incorporated into the chromosome as a single-copy gene. We identified a Bacillus promoter (BP) that is stably expressed in Escherichia coli and drives ~6-fold more expression of eGFP than the T7 promoter in the absence of inducer. Incorporating this BP and RNase III target signals into a single system enabled clear detection of the absence or downregulation of RNase III activity in vivo, thereby establishing a system for screening and identifying novel RNase II targets in a matter of days. An RNase III target signal identified in this manner was confirmed by post-transcriptional analysis. We anticipate that this novel translational fusion vector will be used extensively to study activity of both interesting RNases and related complex or to identify or validate targets of RNases that are otherwise difficult to study due to their sensitivity to environmental stresses and/or autoregulatory processes.-
dc.publisherT&F (Taylor & Francis)-
dc.titleA novel fluorescent reporter system for monitoring and identifying RNase III activity and its target RNAs-
dc.title.alternativeA novel fluorescent reporter system for monitoring and identifying RNase III activity and its target RNAs-
dc.typeArticle-
dc.citation.titleRNA Biology-
dc.citation.number9-
dc.citation.endPage1176-
dc.citation.startPage1167-
dc.citation.volume9-
dc.contributor.affiliatedAuthorSoohyun Lee-
dc.contributor.affiliatedAuthorSeung Goo Lee-
dc.contributor.affiliatedAuthorChoong-Min Ryu-
dc.contributor.alternativeName김광선-
dc.contributor.alternativeName박선영-
dc.contributor.alternativeName이수현-
dc.contributor.alternativeName강선빈-
dc.contributor.alternativeName이정민-
dc.contributor.alternativeName이승구-
dc.contributor.alternativeName류충민-
dc.identifier.bibliographicCitationRNA Biology, vol. 9, no. 9, pp. 1167-1176-
dc.identifier.doi10.4161/rna.21499-
dc.subject.keywordBacteriophage vector-
dc.subject.keywordCleavage target-
dc.subject.keywordFluorescence-
dc.subject.keywordRibonuclease III (RNase III)-
dc.subject.keywordTranslational fusion reporter system-
dc.subject.localBacteriophage vector-
dc.subject.localCleavage target-
dc.subject.localFluorescence-
dc.subject.localRibonuclease III (RNase III)-
dc.subject.localTranslational fusion reporter system-
dc.description.journalClassY-
Appears in Collections:
Division of Research on National Challenges > Infectious Disease Research Center > 1. Journal Articles
Division of Biomaterials Research > Synthetic Biology and Bioengineering Research Center > 1. Journal Articles
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