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

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Title
A novel fluorescent reporter system for monitoring and identifying RNase III activity and its target RNAs
Author(s)
Kwang-Sun Kim; Sunyoung Park; Soohyun Lee; S B Kang; J Lee; Seung Goo LeeChoong-Min Ryu
Bibliographic Citation
RNA Biology, vol. 9, no. 9, pp. 1167-1176
Publication Year
2012
Abstract
Bacteriophage 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.
Keyword
Bacteriophage vectorCleavage targetFluorescenceRibonuclease III (RNase III)Translational fusion reporter system
ISSN
1547-6286
Publisher
T&F (Taylor & Francis)
DOI
http://dx.doi.org/10.4161/rna.21499
Type
Article
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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