Structure-Function Analysis of Rny1 in tRNA Cleavage and Growth Inhibition
Figure 5
Multiple loops regulate RNA cleavage.
(A) Cleavage of tRNA Met(CAT) by over-expressed full-length (WT), full-length catalytically inactive (ci), vector control (v), or RNY1 containing catalytic histidines but mutated in cis at indicated loops (L#). Strains deleted for RNY1 were grown as described to induce plasmid over-expression of GAL-RNY1 in the plasmid mutant or control indicated, and equivalent amounts of RNA were resolved and transferred to gels for Northern blots using oRP1401, all performed as indicated in the Materials and Methods. All samples are from the same image of the same blot, and the image was cropped after scaling the image to show only relevant samples (for A and B). Bracket reveals where expected bands accumulate with over-expression of the full-length (WT) RNY1. Migration of oligonucleotide standards is shown in base pairs (bp). (B) Western blot (performed as indicated in Materials and Methods) of strains expressing constructs as shown in (A). Migration of molecular weight standards is indicated.