1. Molecular Mechanism of Drug-Dependent Ribosome Stalling
Nora Vazquez-Laslop, Celine Thum, Alexander S. Mankin 
Molecular Cell 
Volume 30, Issue 2, Pages (April 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 The Site of Ribosome Stalling on the ermC Leader ORF
(A) Structure of the regulatory region of the inducible ermC gene in the noninduced conformation. Shine-Dalgarno regions and initiator codons of the leader peptide ORF (ermCL) and the resistance methylase cistron (ermC) are shown in bold. The amino acid sequences of the entire ErmCL peptide and the N-terminal sequence of ErmC are indicated above the corresponding codons.
(B) Structure of the regulatory region in the induced conformation. The location of the toe-printing signal is shown by arrows, and the codon positioned in the ribosomal P site in the stalled complex is underlined.
(C) Gel electrophoresis analysis of [35S] radiolabeled protein products accumulating in the cell-free translation system programmed with the monocistronic ermC template (lane 1) or ermCL-ermC bicistronic template (lanes 2 and 3) in the absence (−) or presence (+) of 2 μM erythromycin. Positions of the full-size ErmC protein and the ErmCL leader peptide are indicated by arrows.
(D) Erythromycin concentration dependence of ermC expression in the cell-free system. The data points were obtained by quantifying the amount of radioactivity in the gel band corresponding to the full-size ErmC and normalizing versus the amount of ErmC expressed in the absence of erythromycin. Shown are the average values of two experiments with the dispersion of actual values represented by error bars.
(E) The stalled ribosome complex on ermCL mRNA. Cell-free translation was carried out in the presence of increasing concentrations of erythromycin, and the position of the stalled ribosome was determined by toe-printing analysis. The reaction products were analyzed in a sequencing gel alongside sequencing reactions (C, U, A, G) obtained from the same primer used for toe-printing. Positions of the toe-printing signals are shown by arrows, and the position of the full-size reverse transcriptase product corresponding to the 5′ end of ermCL mRNA transcript is indicated by an open arrow. The sequence of a portion of the ermCL ORF and the encoded amino acids are expanded next to the sequencing lanes, with the Ile9 codon positioned in the P site of the stalled ribosome boxed.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Characterization of Peptidyl-tRNA from the Stalled Complex
(A) Gel electrophoresis analysis of [35S] radiolabeled products accumulating during in vitro translation of ermCL mRNA. Reaction products were analyzed by gel electrophoresis in the Tricine buffer system (see the Supplemental Experimental Procedures). The position of the RNase-sensitive material that accumulates in the presence of erythromycin is indicated by a filled triangle.
(B) Gel electrophoresis analysis in the Bis-Tris buffer system (see the Supplemental Experimental Procedures) of [35S]fMet-tRNA (indicated by an open triangle) and [35S]peptidyl-tRNA (filled triangle) accumulating during cell-free translation of ermCL mRNA in the presence of erythromycin.
(C) Toe-printing analysis of stalled complex formation on I3L/I6L or S5A ermCL mutant mRNAs. Bands corresponding to the stalled complex-specific toe-printing signals are indicated by arrows.
(D) Accumulation of [14C]Ile peptidyl-tRNA during cell-free translation of wild-type (WT) or mutant (I3L/I6L) ermCL mRNA in the presence of erythromycin. The position of peptidyl-tRNA is indicated by a filled triangle. Open triangles (in [D]–[F]) indicate a faster-migrating band, whose nature is unclear, detected in some experiments. However, its presence was not erythromycin-dependent, suggesting that it did not originate from the stalled complex.
(E) [35S]Met or [14C]Ser peptidyl-tRNA accumulating during translation of wild-type (WT) or mutant (S5A) ermCL mRNA.
(F) Puromycin sensitivity of [35S]peptidyl-tRNA in the stalled complex. Leader mRNA templates truncated immediately after Ile9 codon (ermCLΔ) or full-length ermCL were expressed for 15 min in the cell-free system in the absence or presence of erythromycin and then treated for 90 s at 37°C with 1 mM puromycin. Reaction products were phenol extracted and analyzed by gel electrophoresis.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Dependence of Stalled Complex Formation on the Size and Sequence of the Nascent Peptide
(A) Formation of the stalled complex on ermCL mRNAs that carry mutations of individual ermCL codons to alanine codons (left panel) and the mutation of the Ile9 codon AUC to a Leu codon CUC (right panel).
(B) Stalled complex formation on mutant ermCL mRNA with addition or deletion of codons. The following templates were used: −1, deletion of the Gly2 codon; −2, deletion of Gly2 and Ile3 codons; −3, deletion of Gly2, Ile3, and Phe4 codons; +1, +2, and +3 indicate addition of one, two, or three GCG (Ala) codons after the initiator AUG codon. In (A) and (B), the toe-printing signal corresponding to the stalled complex is indicated by arrows. (B) Efficiency of stalled complex formation on ermCL mRNA with addition or deletion of codons.
(C) Quantification of the bands on the gel shown in (B). The plot was obtained by calculating relative intensity of the two stalled complex-specific toe-printing bands versus the sum of these bands and the full (ribosome-free) mRNA band in the same gel.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Dependence of Stalled Complex Formation on the Nature of the Macrolide Antibiotic
Toe-printing analysis of the stalled complex on the ermCL mRNA upon in vitro translation in the presence of 50 μM cladinose-containing macrolide antibiotics (C) or ketolides (K). Lane marked “−” corresponds to the reaction carried in the absence of antibiotics. The sequence of a portion of the ermCL ORF and the encoded amino acids are indicated, and the Ile9 codon positioned in the P site of the stalled ribosome is boxed.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Ribosomal Elements Involved in Drug-Dependent Stalling
(A) The effect of A2062 mutations on stalled complex formation.
(B) Effect of a 3 amino acid deletion in the β loop of protein L22 on ribosome stalling. In (A) and (B), translation reactions were carried out in the cell-free translation system devoid of endogenous wild-type ribosomes. Filled arrows indicate the toe-printing signal corresponding to the stalled complex, and open arrows indicate the 5′ end of the mRNA transcript.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
A Model of the Molecular Mechanism of Antibiotic-Dependent Ribosome Stalling
(A) A model of the 9 amino acid-long ErmCL nascent peptide in the exit tunnel of the erythromycin-bound ribosome. The peptide is shown in ball-and-stick representation. The five N-terminal amino acid residues are colored pale green, and the four C-terminal residues, critical for stalling, are shown in bright green. Peptide sequence is indicated. The CCA 3′ end of the P site-bound peptidyl-tRNA is colored wheat. Erythromycin is shown as salmon-colored sticks with the cladinose sugar highlighted in red. The van der Waals surface of the drug is represented by mesh. A2062 of 23S rRNA is shown in hot pink, C2063 and G2061 are pale pink, and A2451 is magenta. The β loop of protein L22 is colored cyan.
(B) Relative location of the nascent peptide, erythromycin, and A2062 in the exit tunnel (viewed from the PTC down the tunnel). Color scheme as in (A).
(C) Relative location of A2062, exposed in the tunnel, linked to nucleotides in the PTC active site of the E. coli ribosome (Schuwirth et al., 2005).
(D) A general model of the drug- and nascent peptide-dependent stalling. The color scheme matches that of (A). Solid arrow marks communication of a signal from A2062 to the PTC active site. Dashed arrows indicate possible contribution of L22 (or other tunnel elements) to establishing inactive conformation of the PTC or stabilizing peptidyl-tRNA in the ribosome.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions