Nascent chain, mRNA, and ribosome complexes generated by a pure translation system

Matsuura, Tomoaki; Yanagida, Hayato; Ushioda, Junya; Urabe, Itaru; Yomo, Tetsuya

doi:10.1016/j.bbrc.2006.11.050

Cited by 26 publications

(17 citation statements)

References 20 publications

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“…We used these reconstituted ribosomes, labeled with the FRET donor dye Cy3 or Cy3B, to prepare stalled RNCs by translating different length N-terminal portions of leader peptidase (lepB), an in vivo-validated SRP substrate with an N-terminal signal sequence (29). To this end, we used 3Ј-truncated mRNAs lacking a stop codon and, in agreement with previous work (11,14,30), found that ribosomes reaching the truncated end remained stably associated with the mRNA and peptidyl-tRNA, producing stalled RNCs (Fig. 1D).…”

Section: Protein-labeled Fret Assay To Detect Srp-rnc Interactions-mentioning

confidence: 92%

Signal Recognition Particle-ribosome Binding Is Sensitive to Nascent Chain Length

Noriega

Tsai

Elvekrog

et al. 2014

Journal of Biological Chemistry

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Section: Protein-labeled Fret Assay To Detect Srp-rnc Interactions-mentioning

confidence: 92%

Signal Recognition Particle-ribosome Binding Is Sensitive to Nascent Chain Length

Noriega

Tsai

Elvekrog

et al. 2014

Journal of Biological Chemistry

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“…Although we are still at the starting point of investigating the precise roles of the modifier proteins on the reaction, combining the experimental data with the PPI network may provide insight into how these proteins interact with the translation machinery. Moreover our data may be useful not only for technologies utilizing in vitro translation systems (19,48,49) but also for designing in vitro translation systems with significantly improved performance than those available at present and also for protein production in vivo, such as by coexpressing beneficial components together with a protein of interest.…”

Section: Discussionmentioning

confidence: 96%

Comprehensive Analysis of the Effects of Escherichia coli ORFs on Protein Translation Reaction

Kazuta¹,

Adachi²,

Matsuura³

et al. 2008

Molecular & Cellular Proteomics

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Protein synthesis is one of the most important reactions in the cell. Recent experimental studies indicated that this complex reaction can be achieved with a minimum complement of 36 proteins and ribosomes by reconstituting an Escherichia coli-based in vitro translation system with these protein components highly purified on an individual basis. From the protein-protein interaction (PPI) network of E. coli proteins, these minimal protein components are known to interact physically with large numbers of proteins. However, it is unclear what fraction of E. coli proteins are linked functionally with the minimal protein synthesis system. We investigated the effects of each of the 4194 E. coli ORF products on the minimal protein synthesis system; at least 12% of the entire ORF products, a significant fraction of the gene product of E. coli, affect the activity of this system. Furthermore 34% of these functional modifiers present in the PPI network were shown by mapping to be directly linked (i.e. to interact physically) with the minimal components of the PPI network. Topological analysis of the relationships between modifiers and the minimal components in the PPI network indicated clustering of the minimal components. The modifiers showed no such clustering, indicating that the location of functional modifiers is spread across the PPI network rather than clustering close to the minimal protein components. These observations may reflect the evolutionary process of the protein synthesis system. Molecular & Cellular Proteomics 7:1530 -1540, 2008.The protein translation reaction (1), one of the most important regulators of cell behavior, involves the interactions of a large number of components and can thus be seen as an intermolecular interaction network. It has been demonstrated experimentally that 36 enzymes and the ribosomes are sufficient to carry out protein translation (2). These minimal protein components include the ribosomal proteins; initiation, elongation, and release factors; aminoacyl-tRNA synthetases; and enzymes involved in energy regeneration. This was demonstrated by constructing an Escherichia coli-based reconstituted in vitro translation system with these protein components highly purified on an individual basis.Although the genome of E. coli contains more than 4000 genes (3), constituting a very large interaction network (4, 5), the number of protein components constituting the minimal protein synthesis system corresponds to only 2.1% of the genes encoded in the genome. Thus, only small subsets of the protein components are required for protein synthesis. On the other hand, a number of previous studies, including protein-protein interaction (PPI) 1 network analysis in E. coli (4, 5), indicated that protein components constituting the minimal protein synthesis system interact with a large number of other proteins. To gain a deeper understanding of the protein translation system, it is important to identify not only the proteins that interact physically but also those that interact functionally, i.e. t...

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“…Moreover, in vitro translation using the PURE system, [19] in which all the components necessary for translation are highly purified and reconstituted to carry out the protein translation reaction, can provide further advantages with this technology. [20][21][22] This is mainly attributable to the very low RNase activity of this system compared with the E. coli S30 extract, which has been widely used for in vitro selection techniques, and because more than tenfold more mRNA can be recovered after selection when using the PURE system. [23] Therefore, we achieved the rapid generation of covalently bound proteins through five rounds of selection in just one week, primarily because of the in vitro selection by ribosome display with the PURE system.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Selection of Proteins that Undergo Covalent Labeling with Small Molecules by Thiol‐Disulfide Exchange by Using Ribosome Display

et al. 2011

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There is a great deal of interest in proteins that can bind covalently to target molecules, as they allow unambiguous experiments by tight binding to molecules of interest. Here, we report the generation of proteins that undergo covalent labeling with small molecules through in vitro selection by using ribosome display. Selection was performed from a mutant library of the WW domain with a biotinylated peptide as its binding target, in which the biotin and the peptide are connected by a disulfide bond. After five rounds of selection, we identified mutants carrying a particular cysteine mutation. The binding target reacted specifically with the selected mutant, even in the presence of other proteins, and resulted in the generation of biotin- or peptide-labeled WW domains by thiol-disulfide exchange. When the mutant was fused to a protein of interest, the fusion protein was also labeled with biotin. Thus, the characteristics of the selected mutant should be suitable as a tag sequence that can be covalently labeled with small synthetic molecules. These results indicate that the rapid and efficient generation of such proteins is possible by ribosome display.

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Nascent chain, mRNA, and ribosome complexes generated by a pure translation system

Cited by 26 publications

References 20 publications

Signal Recognition Particle-ribosome Binding Is Sensitive to Nascent Chain Length

Signal Recognition Particle-ribosome Binding Is Sensitive to Nascent Chain Length

Comprehensive Analysis of the Effects of Escherichia coli ORFs on Protein Translation Reaction

In Vitro Selection of Proteins that Undergo Covalent Labeling with Small Molecules by Thiol‐Disulfide Exchange by Using Ribosome Display

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