1. Genetically Encoded but Nonpolypeptide Prolyl-tRNA Functions in the A Site for SecM- Mediated Ribosomal Stall 
Hiroki Muto, Hitoshi Nakatogawa, Koreaki Ito 
Molecular Cell 
Volume 22, Issue 4, Pages (May 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
In Vitro Reproduction of the SecM Elongation Arrest in PURE SYSTEM Translation
(A) Time course of SecM synthesis. Coupled in vitro transcription translation was directed by the secM template described in Experimental Procedures in the presence of 35[S]methionine at 37°C for the indicated periods. Radioactive products were separated by the NuPAGE version of SDS-PAGE and visualized by Fuji BAS1800 phosphor imager. Numbers “30” and “12.3” indicate positions of 30 kDa and 12.3 kDa molecular mass markers. Asterisk indicates a nonspecific material that is produced even in the absence of added template.
(B) Products from different templates. Transcription/translation was directed by the indicated templates for 60 min. Δ indicates a 3′ truncation. Lanes 1–5, detection by 35[S]methionine labeling; lanes 6–10, detection by anti-SecM immunoblotting.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2
Determination of the Ribosome Stall Position on mRNA Induced by the SecM Arrest Sequence
(A) Toeprint analysis. Coupled in vitro transcription translation was directed by the secM (lane 1), secM(P166A) (lane 2), secM(amber167) (lane 3), secM(P166A, amber167) (lane 4), and secM(amber166) (lane 5) templates. Reactions for lanes 1 and 2 were by regular PURE SYSTEM reaction mixture. Reactions for lanes 3–5 were by PURE SYSTEM reaction mixture that lacked release factors (RF−) to enable ribosome stall at the amber nonsense codon. Translation products were then used as templates for reverse transcriptase reactions that were primed by the 32P-labeled oligonucleotide described in Experimental Procedures. Radioactive products were separated by 8% polyacrylamide gel electrophoresis and exposed to X-ray films. Lanes A, T, G, and C received respective dideoxy nucleotide-terminated products of DNA polymerase reactions using the same primer and pAN1 (secM) template. The secM mRNA bases shown on the right are numbered from the first base of the Gly165 codon.
(B) Cartoon representation of the PTC positioning on each mRNA. PTC is shown only conceptually. tRNAs are shown by stars and nascent polypeptides by dotted lines. The numbers correspond to the lane numbers in (A).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3
Isolation of the Polypeptide Part of the Arrested SecM Peptidyl-tRNA
(A and B) Two-dimensional separation by SDS-PAGE under low and high pH conditions. PURE SYSTEM 1 hr reaction products directed by the secM template (left) or without template (right) were separated first by NuPAGE electrophoresis. Lanes were cut out, equilibrated with sample buffer, and layered on the top of the second dimension gel of Laemmli system. When samples entered into the stacking and separation gels, electrophoresis was halted for 90 min and then continued. Gels were subjected to anti-SecM immunoblotting (A) and silver staining (B). Arrow indicates the spot of the SecM arrested peptide without tRNA.
(C) Mass spectroscopic determination of the C-terminal end. The separation principle shown above was applied for larger scale isolation using wider sample slot for the first dimension gel. The position of peptidyl-tRNA was cut out and subjected to the second dimension electrophoresis. The arrested SecM fragment formed a horizontal band, which was stained with Commassie brilliant blue, cut out, and then subjected to lysyl endopeptidase digestion and mass spectroscopic analyses (nano ESI-MS and nano ESI-MS/MS, custom services of APRO Life Science Institute).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. Figure 4
Detection of the tRNA Components of Peptidyl-tRNAs by Northern Hybridization
(A) Products directed by full-length secM templates. PURE SYSTEM transcription/translation was directed by indicated templates and subjected to NuPAGE electrophoresis and Northern hybridization using probes (see Experimental Procedures) indicated on the top.
(B) Products directed by truncated templates as well as by full-length template containing amber mutations. PURE SYSTEM (lanes 1–5, normal system; lanes 6–9, release-factor-deficient system) transcription/translation was directed by indicated templates and subjected to NuPAGE electrophoresis and Northern hybridization using indicated probes.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5
Puromycin Action against Various Nascent Chain-Ribosome Complexes
(A) Northern hybridization assay of tRNA loss. PURE SYSTEM transcription/translation was directed by indicated templates at 37°C for 60 min, followed by incubation with puromycin (1 mg/ml; Puro) at 37°C for 0, 0.5, and 3 min, as indicated. Samples were then analyzed as in Figure 4.
(B) Immunoblotting assay of electrophoretic mobility changes. NuPAGE patterns were visualized by anti-SecM immunoblotting. SecM-Puro indicates released peptidyl-puromycin.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions