Volume 19, Issue 3, Pages 333-343 (August 2005) Features of Ribosome-Peptidyl-tRNA Interactions Essential for Tryptophan Induction of tna Operon Expression  Luis Rogelio Cruz-Vera, Soumitra Rajagopal, Catherine Squires, Charles Yanofsky  Molecular Cell  Volume 19, Issue 3, Pages 333-343 (August 2005) DOI: 10.1016/j.molcel.2005.06.013 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Characterization of (bio)UMP-mRNA-TnaC-tRNAPro-Ribosome Complexes (A) Template organization of the PCR-derived DNA fragments used for preparation of mRNAs for TnaC-tRNAPro-ribosome complex isolation. The template DNA contained a T7 promoter, the tna operon leader region from the transcription start site to tnaA (this region contains a rut site and Rho termination sequences), and the rpoBC terminator sequence. Codon 12 of tnaC was either a Trp(W) codon or an Arg(R) codon. The transcript produced, whether biotinylated or not, is shown as a curved line. (B) Isolation of biotinylated tna mRNA-TnaC-tRNAPro-ribosome complexes using streptavidin beads. Reactions were performed with tnaC mRNAs that were either biotinylated (bio-UMP) (+) or not (−) in the presence (+) or absence (−) of 1 mM L-tryptrophan (Trp). After incubation, a portion of each reaction mixture was mixed with streptavidin beads to isolate biotinylated tna mRNA-TnaC-tRNAPro-ribosome complexes (see Experimental Procedures). Reaction products were analyzed by electrophoresis on Tris-Tricine 10% polyacrylamide gels. TnaC-tRNAPro and TnaC bands are marked. An asterisk marks the principal background-labeled band produced in the cell-free extracts. (C) Detection of the presence of RF2 protein in isolated translation complexes. Top, [35S]-methionine-labeled products were isolated from translation reactions performed with (+) or without (−) L-Trp, using cell-free extracts either treated (+) or not treated (−) with anti-RF2 and different tnaC mRNAs, as shown in (B). Bottom, Western blot assays using the anti-RF2 antibody. The control lane (lane 1) displays an in vitro translation reaction mixture that did not contain biotinylated tnaC mRNA. (D) Evaluation of the stability of [35S]-methionine-labeled TnaC-tRNAPro after washing of beads in the presence or absence of L-tryptophan. Isolated complexes were washed several times with a buffer that did (+) or did not (−) contain 1 mM tryptophan (Trp in wash) and then incubated at 37°C for 5 min. Components were resolved as indicated above. Molecular Cell 2005 19, 333-343DOI: (10.1016/j.molcel.2005.06.013) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Analysis of Features of the Ribosome that Are Altered upon tna Operon Induction (A) Determination of the location of Lys11 of TnaC-tRNAPro. Primer extension analyses were performed on 23S rRNA isolated from mRNA-TnaC-tRNAPro-ribosome complexes. The mRNAs used were: tnaC (K18R) (lanes 1 and 2) or tnaC (W12R,K18R) (lane 3). Complexes containing tnaC (W12R,K18R) mRNA were obtained by using cell-free extracts treated with anti-RF2. The in vitro translation reactions were carried out in the absence (lane 1) or presence (lanes 2 and 3) of (ANB-NOS)-Lysyl-tRNALys. The labeled products were resolved in 6% polyacrylamide-7 M urea gels. The arrow at the right of the figure indicates the specific bands created as a consequence of the presumed presence of a crosslink between the indicated nucleotide (marked by an asterisk) and the ANB-NOS molecule on Lys11 of the TnaC peptidyl-tRNA. The specific sequencing lanes obtained by primer extension are shown. The very dark band observed corresponds to G745, which is methylated in vivo, stopping the retro-transcriptase reaction. (B) Footprinting analysis of nucleotides in domain II of 23S rRNA in the presence of wt TnaC-tRNAPro or mutant TnaC(W12R)-tRNAPro. The 23S rRNA treated (see Experimental Procedures) was from isolated, stalled mRNA-ribosome complexes (lanes 3–6) or free ribosomes (Ribo, lane 2). As a control, free ribosomes were treated with ethanol (lane 1) or with DMS (lane 2). The lines and arrows designate the specific bands that were changed upon methylation. Arrows identify nucleotides A788 and A792, which were protected from methylation by TnaC-tRNAPro. An asterisk identifies each nucleotide in a sequence that was affected by the presence of W12. Molecular Cell 2005 19, 333-343DOI: (10.1016/j.molcel.2005.06.013) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Detecting TnaC-tRNAPro and TnaC In Vitro by Using Ribosomes Containing Different L22 Alterations Ribosomes were removed from a cell-free extract by centrifugation and replaced by ribosomes obtained from strain SVS1144 that had been transformed with plasmids expressing L22 wt (lane 1), L22 K90H (lane 2), L22 G91W (lane 3), or L22 K90W (lane 4). In vitro translation assays were performed by using [3H]-proline in the presence of 1 mM L-Trp. The [3H]-proline-labeled components in the samples were resolved by electrophoresis, as described in Figure 1B. Molecular Cell 2005 19, 333-343DOI: (10.1016/j.molcel.2005.06.013) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Diagram of a Portion of the Exit Tunnel of the 50S Ribosomal Subunit Showing the Locations of the Nucleotides and Amino Acids at the Tunnel Gate that Appear to Be Involved in or Altered by Ribosome Stalling Induced by TnaC-tRNAPro and L-tryptophan The crystal structure shown is that of a region of the Haloarcula marismortui 50 S ribosomal subunit with a pretranslocation peptidyl-tRNA (Schmeing et al., 2002). Nucleotides and amino acids identified in this study are labeled. 23 rRNA nucleotides in the exit tunnel are in gray; those not in the exit tunnel are in white. Yellow lines indicate the locations of segments of ribosomal proteins, especially L4 and L22. A peptidyl-tRNA is located in the A site: it consists of cytosine (in green) -puromycin (pink spheres) -phenylalanine-caproic acid-biotin (pink line) (end point marked with a star). It is followed by a curved line and arrow indicating the location of the nascent peptide and its presumed direction of movement in the exit tunnel away from the peptidyl-transferase center (PTC). The ribosomal P site is filled by the CCA sequence (in orange). Circles on the peptide arrow colored green and yellow mark the putative positions of W12 and K11, respectively, of TnaC-tRNAPro. K11 is ∼15 Å (extension of the photo-reactive chemical group plus the lysine radical) from nucleotide A750 and ∼35 Å (extension of a peptide with 13 amino acids) from the PTC. The compound puromycin-phenylalanine-caproic acid-biotin would correspond to a peptide containing about five to seven amino acid residues. Molecular Cell 2005 19, 333-343DOI: (10.1016/j.molcel.2005.06.013) Copyright © 2005 Elsevier Inc. Terms and Conditions