Most regulation of small RNAs (sRNAs) occurs at the posttranscriptional level. We used primer extension, northern blotting, quantitative reverse transcription (RT)–PCR, β-galactosidase reporter fusion (LacZ), and other techniques to screen candidate targets that interact directly with sRNAs of interest. We used a low-copy-number green fluorescent protein (GFP) reporter gene fusion vector (pXG-10-SF) to determine the direct target genes of specific small RNAs in a simple target gene screening system for Yersinia pestis. Reporter gene fusion systems based on LacZ or GFP are usually used to validate sRNA-mediated target regulation in vivo. A GFP-based reporter fusion method used to identify the sRNA targets in Y. pestis is described here. pXG10-SF is a modified GFP translation vector that contains the PLtetO promoter. GFP is translated from the fusion vector and its fluorescence detected, and the fluorescence intensity reflects the expression of the target gene itself. When the GFP fusion construct is coexpressed with an sRNA of interest, the effects of the sRNA on the regulation of the target gene is reflected in the relative level of GFP fluorescence.
Yersinia pestisLacZ GFP Reporter gene fusion
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
Vogel J, Bartels V, Tang TH, Churakov G, Slagter-Jager JG, Huttenhofer A, Wagner EG (2003) RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria. Nucleic Acids Res 31(22):6435–6443CrossRefPubMedPubMedCentralGoogle Scholar
Wassarman KM, Repoila F, Rosenow C, Storz G, Gottesman S (2001) Identification of novel small RNAs using comparative genomics and microarrays. Genes Dev 15(13):1637–1651CrossRefPubMedPubMedCentralGoogle Scholar
Argaman L, Hershberg R, Vogel J, Bejerano G, Wagner EG, Margalit H, Altuvia S (2001) Novel small RNA-encoding genes in the intergenic regions of Escherichia coli. Curr Biol 11(12):941–950CrossRefPubMedGoogle Scholar
Zhang A, Wassarman KM, Rosenow C, Tjaden BC, Storz G, Gottesman S (2003) Global analysis of small RNA and mRNA targets of Hfq. Mol Microbiol 50(4):1111–1124CrossRefPubMedGoogle Scholar
Rivas E, Klein RJ, Jones TA, Eddy SR (2001) Computational identification of noncoding RNAs in E. Coli by comparative genomics. Curr Biol 11(17):1369–1373CrossRefPubMedGoogle Scholar
Kawano M, Reynolds AA, Miranda-Rios J, Storz G (2005) Detection of 5′- and 3′-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli. Nucleic Acids Res 33(3):1040–1050CrossRefPubMedPubMedCentralGoogle Scholar
Delihas N (1997) Antisense micF RNA and 5′-UTR of the target ompF RNA: phylogenetic conservation of primary and secondary structures. Nucleic Acids Symp Ser 36:33–35Google Scholar
Corcoran CP, Podkaminski D, Papenfort K, Urban JH, Hinton JC, Vogel J (2012) Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA. Mol Microbiol 84(3):428–445CrossRefPubMedGoogle Scholar