Volume 20, Issue 3, Pages 427-435 (November 2005) A Protein Component at the Heart of an RNA Machine: The Importance of Protein L27 for the Function of the Bacterial Ribosome  Bruce A. Maguire, Artemy D. Beniaminov, Haripriya Ramu, Alexander S. Mankin, Robert A. Zimmermann  Molecular Cell  Volume 20, Issue 3, Pages 427-435 (November 2005) DOI: 10.1016/j.molcel.2005.09.009 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Incorporation of L27 into 50S Subunits from Wt and Mutant Cells Two-dimensional PAGE of ribosomal proteins isolated from 70S ribosomes was performed as described in the Experimental Procedures. The complete gel of proteins from ribosomes of strain LG90 stained with Coomassie blue is shown on the left. The spot corresponding to protein L27 is indicated. On the right are portions of similar gels of proteins from the ribosomes of cells expressing the indicated L27 mutants. Area of gel shown corresponds to that which is boxed in the gel for LG90. Molecular Cell 2005 20, 427-435DOI: (10.1016/j.molcel.2005.09.009) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Peptidyl Transferase Activity of Wt and Mutant 70S Ribosomes Ribosomes were isolated from cells expressing no L27 (IW312), wt L27 (LG90 and IW312 pL27), or mutant L27 truncated at the N terminus. Peptidyl transferase was measured by the SPARK-Pmn assay. Histogram shows puromycin reactivity of ribosomes containing L27(−3) relative to that of ribosomes containing wt L27 measured by standard puromycin assay. The error bars show the range of results in three independent experiments. Molecular Cell 2005 20, 427-435DOI: (10.1016/j.molcel.2005.09.009) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Crosslinking of 32P Labeled [2N3A76]tRNAPhe to Ribosomal Components (A) SDS-PAGE of crosslinked 50S ribosomal subunit components. (B) SDS-PAGE of protein-AMP adducts from crosslinked 50S subunits. Ribosome samples were digested with S1 nuclease and separated by SDS-PAGE. The strain used as the source of the 50S subunits is indicated above each lane. The identities of the radioactive bands are indicated by arrows at the side of the gel. (C) Two-dimensional PAGE of protein-AMP adducts from crosslinked 50S subunits. Crosslinked subunits were digested with S1 nuclease and mixed with carrier ribosomal proteins from strain MRE600. The carrier protein spots were visualized by staining with Coomassie blue. The positions of radiolabeled protein-AMP adducts were determined by an overlay of a gel phosphorimage. Each gel is labeled with the strain used as the source of the crosslinked 50S subunits. The positions of proteins L27 and L33 stained with Coomassie blue, as well as their radioactive adducts, are indicated. Molecular Cell 2005 20, 427-435DOI: (10.1016/j.molcel.2005.09.009) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Summary of Crosslinking Analysis Crosslinking of tRNA to proteins L27 and L33, and to 23S rRNA, is expressed as a percentage of the total tRNA bound to ribosomes. Values represent the average of four experiments; the standard deviations for the tRNA-23S, tRNA-L27, and tRNA-L33 adducts were 0.21–1.74, 0.13–0.81, and 0.09–0.51, respectively. Molecular Cell 2005 20, 427-435DOI: (10.1016/j.molcel.2005.09.009) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 The Crystal Structure of the 50S Ribosomal Subunit from D. radiodurans Atoms are rendered as space filled; only the Cα coordinates are available for the proteins. The N terminus of protein L27 is colored red, and the C terminus is green. Amino acids 2, 5, 8, and 11, which after removal of fMet residue are N-terminal in wt L27 and in the −3, −6, and −9 derivatives, respectively, are indicated. The −20 truncation lacks the entire segment in red. Nucleotides that correspond to A2451, U2506, and U2585 in the 23S rRNA of E. coli are colored cyan, blue, and magenta, respectively. The rest of the ribosome is rendered in translucent gray. The view in (A) is from the subunit interface side, with the L7/L12 stalk on the right and the central protuberance (CP) in the center. In (B), the subunit is viewed from the side of the L7/L12 stalk, with the nearest section cut away for clarity. Figures were generated from PDB file 1KPJ (Harms et al., 2001) by using Protein Explorer (http://www.proteinexplorer.org). Molecular Cell 2005 20, 427-435DOI: (10.1016/j.molcel.2005.09.009) Copyright © 2005 Elsevier Inc. Terms and Conditions