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. 2006 Jan;12(1):177–184. doi: 10.1261/rna.2105506

FIGURE 2.

FIGURE 2.

Analysis of loop–loop interactions with the RNA-hybrid system. (A) Northern blot analysis. For sample m26-11, only one RNA is expressed after cotransformation of yeast strain YBZ-1 with plasmids pIIIa-m26-11 MS2 and pACTII. In the other samples, plasmids derived from pAN MS2-2 and pIIIa-m26 containing the indicated RNA X and RNA Y were cotransformed. As controls, the band corresponding to the 5.8S rRNA after ethidium bromide staining of the gel and the band obtained after hybridization of the blot with a probe targeting the mRNA of the housekeeping gene IPP1 encoding the inorganic pyrophosphatase are shown. The probe used to observe expression of the RNAs X and Y is a PCR product from clone m26-11 containing the RNase P RNA 5′ leader and 3′tail as well as the m26 activation domain and MS2 RNA. This probe hybridizes therefore with both the X and Y RNA molecules. In lane 1 (m26-11), only one RNA is expressed (305 nt). In lanes 3–6, both RNA X and Y are detectable (X1, 283 nt; X2, 285 nt; X50, 331 nt; Y1 and Y2, 248 nt; Y50, 294 nt). (B) Quantitative analysis of β-galactosidase activity. The β-galactosidase activity is expressed in Miller units (logarithmic scale). Individual values are shown above each bar. Error bars indicate standard deviation. 3-Hybrid positive control indicates positive control of the yeast three-hybrid system using the IRE–IRP1 interaction; cells were cotransformed with plasmids pIIIa IRE MS2 and pAD IRP (Bernstein et al. 2002). m26-11 indicates MS2-RNA and m26 activation domain are on the same molecule (Buskirk et al. 2003). The other samples show expression after cotransformation of plasmids derived from pAN MS2-2 and pIIIa-m26 containing the indicated RNA X and RNA Y. When one RNA alone is indicated, the corresponding empty vector (pAN MS2-2 or pIIIa-m26) was cotransformed. (C) Growth of dilution series on selective medium. Dilution series of yeast YBZ-1 cultures were plated onto different synthetic defined media lacking leucine, uracil, and histidine, and supplemented with 1 mM 3-aminotriazole (3-AT) as indicated. 3-hybrid – indicates cells were cotransformed with plasmid pIIIa MS2-2 and pAD IRP (Bernstein et al. 2002); 3-hybrid +, same as 3-hybrid positive control in B. Other samples are as in B.