期刊
NUCLEIC ACIDS RESEARCH
卷 46, 期 2, 页码 -出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkx1086
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资金
- National Institutes of Health [GM044025, FGM102999]
The interaction of RNA molecules with proteins is a critical aspect of gene regulation across all domains of life. Here, we report the development of a bacterial three-hybrid (B3H) assay to genetically detect RNA-protein interactions. The basis for this three-hybrid assay is a transcription-based bacterial two-hybrid assay that has been used widely to detect and dissect protein-protein interactions. In the three-hybrid assay, a DNA-bound protein with a fused RNA-binding moiety (the coat protein of bacteriophage MS2 (MS2(CP))) is used to recruit a hybrid RNA upstream of a test promoter. The hybrid RNA consists of a constant region that binds the tethered MS2CP and a variable region. Interaction between the variable region of the hybrid RNA and a target RNA-binding protein that is fused to a subunit of Escherichia coli RNA polymerase (RNAP) stabilizes the binding of RNAP to the test promoter, thereby activating transcription of a reporter gene. We demonstrate that this three-hybrid assay detects interaction between non-coding small RNAs (sRNAs) and the hexameric RNA chaperone Hfq from E. coli and enables the identification of Hfqmutants with sRNA-binding defects. Our findings suggest that this B3H assay will be broadly applicable for the study of RNA-protein interactions.
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