1. Ribosomal Protein L3: Gatekeeper to the A Site
Arturas Meskauskas, Jonathan D. Dinman 
Molecular Cell 
Volume 25, Issue 6, Pages (March 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
Putative Interaction between the Tip of the L3 W Finger and A2940 of 25S rRNA Is Critical for Cell Viability
(A) Predicted interactions between amino acid substitutions at position 255 with large subunit rRNA. Images were modeled using data for yeast proteins obtained from cryo-EM studies threaded into the H. marismortui X-ray crystal structures (Spahn et al., 2004). U2607 of the H. marismortui 23S rRNA (shown here) is the structural equivalent of E. coli A2572, and of A2940 in the S. cerevisiae 25S rRNA.
(B) Viability of substitution mutants at position 255 of L3 as monitored by 10-fold dilution spot assays on −Trp 5-FOA plates. Apparently viable W255E and W255A colonies were proved to be revertants to tryptophan by DNA sequence analysis.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2 Intrinsic but Limited Flexibility of the L3 W Finger
(A) Wild-type L3, W255C, and C251S are viable as the sole forms of L3, but the W255C/C251S double mutant is not.
(B) The W255C mutant is hypersensitive to the reducing agent dithiothreitol.
(C) H259 is absolutely required for viability in combination with W255C, and H256 is partially required, suggesting that the putative interactions normally made between W255 and A2940 (E. coli A2572) are compensated for by a combination of H259 and H256 in the W255C mutant.
(D) Insertions or deletions in the W finger, resulting in shifting of W255 toward the N-terminal side of the central extension (green up arrow) are mostly viable (except iV260 and ΔV249), while those that shift W255 toward the C-terminal side (red down arrow) are lethal.
(E) Model describing these observations. Modeling was based on the cryo-EM map of the yeast 80S ribosome threaded onto the X-ray crystal structure of H. marismortui (Spahn et al., 2004). (Top panel) Mutated cysteine at position 255 presumably forms a disulfide bridge with C251 (red dots), repositioning H256 and H259 to interact with A2940 (shown as H. marismortui U2607). (Bottom panel) In general, mutants that allow movement of the tip of the W finger toward the N-terminal side of the L3 W finger (green arrow) reposition histidines on the C-terminal side to interact with A2940, thus helping to restabilize the A site region of the PTC. In contrast, mutations that move the tip in the other direction (red arrow) are lethal because there are no basic or aromatic amino acid residues on the N-terminal side of W255 to interact with A2940.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Structure Probing of Wild-Type and Mutant Ribosomes
(A–C) Autoradiograms of reverse transcriptase primer extension reactions spanning sequence in helices 89–93 (A), helix 73 (B), and the helix 94–95 regions (C). Sequencing reactions (left sides of panels) are labeled corresponding to the rRNA sense strand. Mutants are indicated at top, and bases with altered chemical protection patterns are indicated to the left. Below each panel, “U” stands for untreated, “D” is DMS, “C” denotes CMCT, and “K” indicates kethoxal.
(D) Localization of bases in yeast 25S rRNA whose modification patterns were affected by the L3 mutants. Protected and deprotected bases are indicated by open and filled arrows, respectively; bases of 25S rRNA located within 4 Å of W255 are boxed in gray.
(E) rRNA protection data mapped onto the H. marismortui large subunit crystal structure. Base numbering follows the S. cerevisiae sequence shown in (D), and E. coli numbering is shown in parentheses. The mutants described above result in conformational changes of helices 90–92 (orange and yellow), helix 95 (light blue), and helix 73 (gray). Nucleotides with altered chemical protection patterns are shown in pale blue. Bases forming the two A site gates are in yellow. Bases interacting with anisomycin are in salmon. A2819 (E. coli A2451) is shown in red. The path taken by the 3′ end of the aa-tRNA from the SRL to the PTC (Sanbonmatsu et al., 2005) is shown as an orange arrow.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Biochemical Characterization of Ribosomes Extracted from Cells Expressing L3 W Finger Mutants
(A) Single site isotherms of eEF-1A stimulated binding of [14C]Phe-tRNA to ribosomal A sites.
(B) eEF2 binding isotherms for wild-type and mutant ribosomes.
(C) Binding isotherms of Ac-[14C]Phe-tRNA to ribosomal P sites.
(D) Characterization of peptidyltransferase activities of wild-type and mutant ribosomes by first order time plots of Ac-[14C]Phe-puromycin formation. Error bars denote standard deviations.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
Model for the Role of L3 in Coordinating Functions of the Elongation Factor-Binding Site: L3 as the Gatekeeper to the A Site
(Left) Positioning of the L3 W finger (green) away from the PTC (red oval) positions the helix 90–92 side of the aa-tRNA accommodation corridor so as to open the accommodation gates (red circles), allowing accommodation (dotted gray line) of the 3′ end of aa-tRNA (aa). “pep” denotes the 3′ end of the peptidyl-tRNA. This W finger conformation also positions helix 95 to form the eEF-1A•aa-tRNA•GTP ternary complex binding site and promotes interaction between L3 H259 and 25S rRNA A2940, denoted by “A” (E. coli A2572). (Right) During/after accommodation, repositioning of the W finger toward the PTC closes the gates, repositions helices 95 and 90–92 to form the eEF2 binding site, and promotes the W255-A2940 stacking interaction.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions