Figure 1.
25S rRNA in the peptiptidyl transferase center of yeast.
(A) Secondary structure of yeast 25S rRNA in the PTC. snoRNAs targeted for this study are indicated along with the residues they modify. Ψ – pseudouridylated residue; Nm – 2′-O-ribose methylated residue. Helices are numbered in black. (B) Three dimensional representation of the E. coli PTC [34]. Modified residues are labeled by the colors indicated in panel A. Left: view into the PTC from the top of the LSU, right: 90° rotation of Left. Helices and tRNAs are labeled.
Figure 2.
Sensitivity of rRNA base modification mutants to Translational Inhibitors.
Mutant and isogenic wild-type yeast strains were spotted as ten fold dilutions from 105 to 101 CFU onto YPAD media containing 20 µg/ml anisomycin or sparsomycin. Cells were incubated for 3 days at 30°C, and growth was monitored as compared to growth on plates in the absence of drug. Each strain and drug was assayed at least twice.
Table 1.
Summary of rRNA modification mutant phenotypes.
Figure 3.
Many of the rRNA base modification mutants have M1 virus propagation defects.
Yeast rRNA modification mutants were tested for their ability to maintain the L-A and M1 viruses. (A) Mutant and isogenic wild-type yeast strains were spotted onto YPAD plates, and allowed to grow at 30°C, and then replica plated to a seeded lawn of 5X47 indicator cells. Plates were incubated at room temperature for 3–5 days until a zone of inhibition was clearly visible for wild-type cells. (B) Total RNAs were extracted from mutant and isogenic wild-type yeast strains and digested with RNase A under high salt conditions. The resulting double-stranded RNA was separated on a 1% agarose gel and visualized with ethidium bromide. L-A and M1 dsRNAs are indicated. The image was inverted for clarity.
Figure 4.
Mutant strain snr46 Δ and the isogenic wild-type are shown in the top row, and mutant strain spb1DA/snr52 Δ and the isogenic wild-type are show in the bottom row. Error bars represent standard error for all graphs. A. [14C]Phe-tRNA binding to the A-site of the ribosome. One site binding curves of bound tRNA as analyzed using GraphPad Prism software. Data are reported as a percentage of the total tRNA bound. B. Ac-[14C]Phe-tRNA binding to the P-site of the ribosome. One site binding curves of bound tRNA as analyzed using GraphPad Prism software. Data are reported as a percentage of the total tRNA bound. C. Peptidyltransfer. Timecourse assays of peptidyltransferase activities as measured by the puromycin reaction.
Figure 5.
25S rRNA structure probing analysis in spb1DA/snr52 Δ mutant ribosomes.
(A) Puromycin treated ribosomes isolated from isogenic wild-type and spb1DA/snr52 Δ 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.