The Ribosomal Exit Tunnel Functions as a Discriminating Gate

Nakatogawa, Hitoshi; Ito, Koreaki

doi:10.1016/s0092-8674(02)00649-9

Cited by 514 publications

(668 citation statements)

References 26 publications

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“…SecM arrest sequence, upon its translation interacts with the ribosomal proteins L4 and L22 as well as 23S rRNA in the ribosomal tunnel 7,8 . A number of critical residues, constituting the SecM arrest motif, FXXXXWIXXXXGIRAGP 7 (in bold type) are of immense importance, ensuring the efficiency of the translational arrest.…”

Section: Discussionmentioning

confidence: 99%

“…A number of critical residues, constituting the SecM arrest motif, FXXXXWIXXXXGIRAGP 7 (in bold type) are of immense importance, ensuring the efficiency of the translational arrest. Mutations, or deletions of these critical residues may lead to the relief of the translation elongation arrest 7,8 . Thus, maintaining the sequence of the SecM arrest motif FXXXXWIXXXXGIRAGP 7 intact is absolutely critical to ensure that the protein under study would remain stably bound to the ribosome.…”

Section: Discussionmentioning

confidence: 99%

“…SP6 promoter based plasmid. To obtain RNCs of interest, C-terminus of the target polypeptide is extended by adding arrest inducing sequence of SecM FXXXXWIXXXXGIRAGP 7 . In order to ensure that the polypeptide fragment of interest will extrude out of the ribosomal tunnel, the C-terminal part of the target protein has to be extended by at least 30 amino acids [12][13][14] .…”

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Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Jha

Komar

2012

JoVE

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Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process [1][2][3][4][5] . However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon 6 . Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site 7-9 . More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes 7 . Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains 10,11 . Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system. Video LinkThe video component of this article can be found at http://www.jove.com/video/4027/ Protocol DNA Template Preparation and in vitro Transcription In vitro TranslationFor in vitro translation using the RTS 100 E. coli HY Kit (5 Prime, Gaithersburg, MD) follow the steps below: In order to ensure that the polypeptide fragment of interest will extrude out of the ribosomal tunnel, the C-terminal part of the target protein has to be extended by at least 30 amino acids [12][13][14] . A flexible Glycine-Serine rich linker can be introduced between the protein and the SecM arrest sequence to avoid any possible conformational constraints. 2. For in vitro transcription, template DNA should be linearized with restriction enzyme cutting downstream of the ORF. One needs to verify complete linearization of the plasmid DNA by running the restriction digestion product on agarose gel electrophoresis. 3. The linearized plasmid is further used for in vitro transcription reaction. Different concentration of template DNA can be tested to identify optimum DNA concentration required for in vitro transcript...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Jha

Komar

2012

JoVE

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“…A number of nascent peptides interact with the exit tunnel and stall elongation at specific sites within their peptide chain. The tunnel constriction has also been implicated in peptide-mediated pausing (Nakatogawa and Ito, 2002;Cruz-Vera et al, 2005). Previous research had indicated that in bacteria, mutations in RPL22 and RPL4 mediate erythromycin resistance by perturbing the conformation of rRNA, and a variety of changes in these proteins could mediate macrolide resistance (Gregory and Dahlberg, 1999;Gabashvili et al, 2001;Zaman et al, 2007;Caldwell et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

“…RPL22 and RPL4 form a constriction that results in the narrowest passage in the tunnel. This exit tunnel interacts with nascent translation products and functions as a discriminating gate and may control the nascent chain elongation (Nakatogawa and Ito, 2002;Berisio et al, 2003). But the correlation between the function of RPL22 and insecticide resistance has not been reported.…”

Section: Introductionmentioning

confidence: 99%

Cloning and characterization of 60S ribosomal protein L22 (RPL22) from Culex pipiens pallens

Sun

Zhang

et al. 2009

Comparative Biochemistry and Physiology Part B: Biochemistry an

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The 60S ribosomal protein L22 (GenBank accession no. EF990190) was cloned from Culex pipiens pallens. An open reading frame (ORF) of 447 bps was found to encode a putative 148 amino acids protein which shares 90% and 80% identity with RPL22 genes from Aedes aegypti and Anopheles gambiae respectively. Real-time quantitative PCR analysis demonstrated that the transcription level of RPL22 in deltamethrin-resistant strain was 2.57 folds higher than in deltamethrin-susceptible strain of Cx. pipiens pallens. Overexpression of RPL22 in C6/36 cells showed that the deltamethrin-resistance was decreased in C6/36-RPL22 cell compared to the control. The mRNA level of cytochrome P450 6A1 (CYP6A1, GenBank accession no. FJ423553) showed that CYP6A1 was down-regulated in the C6/36 transfected with RPL22 (C6/36-RPL22) cells, suggesting that CYP6A1 was repressed by RPL22. Our study provides the first evidence that RPL22 may play some role in the regulation of deltamethrin-resistance in Cx. pipiens pallens.

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Ribosomal RNA

2004

Encyclopedic Dictionary of Genetics, Genomics and Proteomics

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The Ribosomal Exit Tunnel Functions as a Discriminating Gate

Cited by 514 publications

References 26 publications

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Cloning and characterization of 60S ribosomal protein L22 (RPL22) from Culex pipiens pallens

Ribosomal RNA

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