Cell-free translation reconstituted with purified components

Shimizu, Yoshihiro; Inoue, Akio; Tomari, Yukihide; Suzuki, Tsutomu; Yokogawa, Takashi; Nishikawa, Kazuya; Ueda, Takuya

doi:10.1038/90802

Cited by 1,733 publications

(1,679 citation statements)

References 25 publications

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“…To date, several replication systems have been constructed either from various sets of RNA and proteins 19,20 or from RNA ribozymes only 21,22 , although these replication systems lack the process by which the genetic information is translated into proteins. To construct an artificial system that replicates in the same manner as natural organisms, through the translation of a replication enzyme, we combined an artificial genomic RNA that encodes an RNA-dependent RNA polymerase, the Qb replicase, with a reconstituted translation system 23,24 . In this translationcoupled RNA replication (TcRR) system, the genomic plus-strand RNA (2,125 nucleotides) replicates using an RNA replicase translated from its own sequence via the synthesis of the complementary minus-strand (Genomic RNA replication, Fig.…”

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confidence: 99%

Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment

et al. 2013

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The ability to evolve is a key characteristic that distinguishes living things from non-living chemical compounds. The construction of an evolvable cell-like system entirely from non-living molecules has been a major challenge. Here we construct an evolvable artificial cell model from an assembly of biochemical molecules. The artificial cell model contains artificial genomic RNA that replicates through the translation of its encoded RNA replicase. We perform a long-term (600-generation) replication experiment using this system, in which mutations are spontaneously introduced into the RNA by replication error, and highly replicable mutants dominate the population according to Darwinian principles. During evolution, the genomic RNA gradually reinforces its interaction with the translated replicase, thereby acquiring competitiveness against selfish (parasitic) RNAs. This study provides the first experimental evidence that replicating systems can be developed through Darwinian evolution in a cell-like compartment, even in the presence of parasitic replicators.

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confidence: 99%

Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment

et al. 2013

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“…Ideally, since one would like to investigate the activity just as a function of the relative composition and concentration of the components, a more detailed knowledge of the protein synthesis kit would be advisable. In 2001, the group of Takuya Ueda at the Tokyo University has formulated a ''reconstituted'' cell-free transcription and translation system from purified E. coli components, named PURE system (Protein synthesis Using Recombinant Elements) (Shimizu et al, 2001). The composition of this kit is shown in Table 2.…”

Section: The Technology For Building Minimal Cellsmentioning

confidence: 99%

Advances in Minimal Cell Models: a New Approach to Synthetic Biology and Origin of Life

Stano¹

2011

Progress in Molecular and Environmental Bioengineering - From Analysis and Modeling to Technology Applications

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“…The genes for the Cys 7 3 Ser (⌬cys) and the Cys 7 3 Ser/Ser 42 3 Cys/Asn 49 3 Cys (SCC) mutants of pCoxIV-DHFR were constructed as described previously for the 7/188/189 and 7/42/49 mutants of pCoxIV-DHFR (30); the ⌬cys mutant lacks Gln 188 and Cys 189 of the 7/188/189 mutant, and the SCC mutant is the same as the 7/42/49 mutant. The radiolabeled precursor proteins were synthesized in a cell-free translation system with rabbit reticulocyte lysate or PURESYSTEM (Post Genome Institute Co., Ltd.) (31) in the presence of [ 35 S]methionine.…”

Section: Methodsmentioning

confidence: 99%

Reinvestigation of the Requirement of Cytosolic ATP for Mitochondrial Protein Import

Asai

Takahashi

Esaki

et al. 2004

Journal of Biological Chemistry

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Protein import into mitochondria requires the energy of ATP hydrolysis inside and/or outside mitochondria. Although the role of ATP in the mitochondrial matrix in mitochondrial protein import has been extensively studied, the role of ATP outside mitochondria (external ATP) remains only poorly characterized. Here we developed a protocol for depletion of external ATP without significantly reducing the import competence of precursor proteins synthesized in vitro with reticulocyte lysate. We tested the effects of external ATP on the import of various precursor proteins into isolated yeast mitochondria. We found that external ATP is required for maintenance of the import competence of mitochondrial precursor proteins but that, once they bind to mitochondria, the subsequent translocation of presequencecontaining proteins, but not the ADP/ATP carrier, proceeds independently of external ATP. Because depletion of cytosolic Hsp70 led to a decrease in the import competence of mitochondrial precursor proteins, external ATP is likely utilized by cytosolic Hsp70. In contrast, the ADP/ATP carrier requires external ATP for efficient import into mitochondria even after binding to mitochondria, a situation that is only partly attributed to cytosolic Hsp70.Many eukaryotic proteins have to move across one or more biological membrane(s) to reach their destinations after synthesis (1). Protein translocation across the organellar membranes is mediated by translocators or translocases, which are membrane-protein complexes and provide a protein-conducting channel through which organellar proteins thread, in most cases, in unfolded conformations. Several translocation systems can actively unfold precursor proteins and achieve their vectorial movement across the membranes by using various forms of energy (2, 3).Mitochondria consist of the outer and inner membranes and two aqueous compartments, the intermembrane space and matrix. The vast majority of mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. Precursors of most matrix proteins and many inner membrane proteins have a positively charged presequence that contains targeting information for mitochondria and is removed by the processing peptidase in the matrix. These proteins utilize the translocase of the outer mitochondrial membrane (TOM) 1 to cross the outer membrane and the translocase of the inner mitochondrial membrane, the TIM23 complex, for translocation across the inner membrane (4, 5). The targeting information in the presequence is recognized by Tom20, a general presequence receptor subunit of the TOM complex (6). Polytopic inner membrane proteins, including members of the carrier protein family such as the ADP/ATP carrier (AAC), are not synthesized with cleavable presequences but instead contain internal mitochondrial targeting signals. Members of the carrier protein family utilize the TOM complex to cross the outer membrane and another translocase of the inner membrane, the TIM22 complex, for integration into the inner membrane (7,8). These...

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Cell-free translation reconstituted with purified components

Cited by 1,733 publications

References 25 publications

Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment

Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment

Advances in Minimal Cell Models: a New Approach to Synthetic Biology and Origin of Life

Reinvestigation of the Requirement of Cytosolic ATP for Mitochondrial Protein Import

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