1. Spb1p-Directed Formation of Gm2922 in the Ribosome Catalytic Center Occurs at a Late Processing Stage 
Bruno Lapeyre, Suresh K. Purushothaman 
Molecular Cell 
Volume 16, Issue 4, Pages (November 2004)
DOI: /j.molcel

2. Figure 1 Characterization of the snr52-0 and spb1-D52A Mutant Strains
(A) Northern blot analysis of pre-rRNA prepared from wild-type and snR52-0 strains using probes for the internal transcribed spacers. Lanes 1 and 2, oligo 1; lanes 3 and 4, oligo 2 (Supplemental Figure S1). Major pre-rRNA species are indicated on the left. The 23S is designated by a dot.
(B) Secondary structure of the MTase domain of Spb1p. The six α helices and the seven β strands are represented by ovals and triangles, respectively. The sequence of motif I of the AdoMet binding domain has been aligned with Trm7p, Mrm2p, and RrmJ to show the aspartate residue at position 52. The arrowheads point to two residues that were proposed to make contact with AdoMet.
(C) Serial dilutions of yeast cells (1/10) were spotted onto YPD plates that were incubated at 30°C, 36°C, or 17°C, as indicated. 1, wild-type strain (BMA64); 2, snr52-0 (YBL4564); 3, spb1-D52A (YBL4637); 4, snr52-0 spb1-D52A (YBL4646).
(D) Cellular extracts from strains YBL4637 (spb1-D52A) and YBL4646 (snr52-0 spb1-D52A) were fractionated onto 10%–50% sucrose gradients. Continuous A254nm records are presented with the top of the gradients on the left.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Secondary Structure Representation of a Region of the Yeast LSU Comprising the PTC
A portion of the 25S LSU (nucleotides 2365–2423 and 2607–2994) is represented, according to Cannone et al. (2002). Modified nucleotides have been represented as follows: pseudouridine (Ψ), black triangle flag; 2′-O-methylriboses, black circle flag; methylbases, black arrow. The uncertain m5U at position 2924 is depicted with a question mark. The position of the three primers described in the text is shown with a black thick line and an arrow indicating their 5′ to 3′ direction. OBL277 is biotinylated at its 5′ end, as shown. The 29-mer fragment that is protected from RNase T1 digestion by OBL277 is shown with the two nearest sites of digestion indicated by white arrowheads. The putative tRNA docking sites have been boxed: the A site with nucleotide Gm2922 and the P site with nucleotides Gm2619G2620. Two nucleotides (A2820 and G2816) potentially involved in catalyzing the peptidyl transfer reaction are shown with white arrows. Three nucleotides (U2873, U2954, and A2971) that might be important for peptide release have been circled and designated with shaded arrows. Bottom right: the A loops from the LSU of yeast mitochondria and E. coli are represented, with their unique modified nucleotide (Um2791 and Um2552) that is catalyzed by Mrm2p and RrmJ, respectively.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 TLC Analyses of Modified Nucleotides from the A Loop
(A) The 32P-labeled RNA fragment protected by OBL277 (Figure 2) was captured using streptavidin coupled to magnetic particles and was washed under stringent conditions before being eluted and fractionated onto 15%-7M urea-PAGE. Lanes 1 and 2, protected RNA prepared from the wild-type (BMA64) and the snr52-0 strain (YBL4564), respectively. The 29-mer fragment was then identified and eluted from the gel. Lanes 3 and 4, analysis of the gel-purified fragment (strains BMA64 and YBL4564, respectively).
(B) The purified 29-mer fragment was digested with nuclease P1, and then the resulting mononucleotides were analyzed on 2D-TLC plates using the NI/RII solvent system. Approximately 2 × 104 dpm was spotted on each plate, which were then exposed overnight using a phosphorimager screen. Identification of the spots was carried out by comparison with standard maps (Keith, 1995). Free phosphate generates a double stain on the right (p). The four panels correspond to the wild-type strain BMA64 (upper left) and to strains YBL4564 (snr52-0, upper right), YBL4637 (spb1-D52A, lower left), and YBL4646 (snr52-0 spb1-D52A, lower right). The positions of the three modified nucleotides of interest are shown with arrows.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 Primer Extension Analyses on Pre-rRNA and Mature rRNA
Primer extension with AMV reverse transcriptase was performed using primer LSU-R2746 to map the methylriboses at position 2724 and 2729, and OBL213 to map position 2921 or Extension was performed with 1 mM dNTP (odd numbered lanes) and 4 μM dNTP (even numbered lanes). Each primer was tested using total RNA (i.e., mostly mature rRNA) or purified 35S pre-rRNA, as indicated at the top of the figure. These RNA were prepared either from the wild-type (BMA64, denoted wt) or the snr52-0 strain (YBL4564, denoted here 52-0). The position of the modified nucleotides, which have been determined accurately from running side by side a sequencing reaction of the same fragment (data not shown), is given on the sides of the gels, and the bands of interest are shown with arrows.
Molecular Cell  , DOI: ( /j.molcel )