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. Author manuscript; available in PMC: 2012 Nov 29.
Published in final edited form as: Biochemistry. 2011 Nov 2;50(47):10203–10214. doi: 10.1021/bi200964p

Figure 3.

Figure 3

Effect of the R562E/Y563S mutation in Rna14 on yeast growth and mRNA processing. A. Viability of strains at various temperatures when the RNA14 shuffle strain is plated on 5FOA-containing media to force loss of the wild-type covering plasmid and leave only plasmid containing no insert (Empty vector), or the RNA14 or rna14- R562E/Y563S genes. B. In vitro processing assays using the indicated yeast extracts. Coupled cleavage and poly(A) addition assays were performed with precursor containing sequences upstream and downstream of the GAL7 poly(A) site (left panel) and a cleavage only assay (middle panel). Poly(A) addition in the absence of cleavage was assayed using substrate that ends at the GAL7 poly(A) site (right panel). The positions of precursors and products are shown on the right. C. Western blot to test for expression levels of Rna14, rna14-ES, and Rna15 in yeast grown at 30°C and 39°C.