25S rRNA structure probing analysis in spb1DA/snr52Δ mutant ribosomes.

(A) Puromycin treated ribosomes isolated from isogenic wild-type and spb1DA/snr52Δ mutant strains were used for in vitro chemical probing of the structure around the peptidyltransferase center of the ribosome, specifically helices 89-93. Reactions were performed in triplicate, representative autoradiographs are shown. U - untreated; C - CMCT; D – DMS; K – Kethoxal. Residues with changes in banding pattern labeled. Strongly modified bases at positions U2923, C2843, and C2851 are indicated, as are the more weakly deprotected A2932 and A2933. The increased intensity corresponding to U2845 (marked by *) is not DMS-specific. (B–D). Locations of residues demonstrating strong changes in protection patterns mapped to LSU rRNA structures. Um2920 and Gm2921 are indicated and color coded. Bases with altered protection patterns are circled in orange. aa-tRNA accommodation ‘gate’ bases [36] are indicated with purple (gate 1) and green (gate 2). The “catalytic” A base (equivalent to E. coli 23S rRNA A2451) is incicated with red. Individual LSU helices are numbered and color coded. (B) Secondary structure of yeast 25S rRNA around the PTC. (C) Three dimensional representation of rRNA bases of interest mapped onto the E. coli PTC [34]. Arrow represents the path the 3′ end of the aa-tRNA travels when entering the A-site. (D) Rotation and zoom in of Panel C. Shows the path into the A-site from the aa-tRNA perspective. Residues demonstrating changes in protection patterns are labeled in orange. Locations of modified bases and A-site ‘gate’ residues are labeled.