scholarly journals In Vitro Reconstitution of Yeast Translation System Capable of Synthesizing Long Polypeptide and Recapitulating Programmed Ribosome Stalling

2021 ◽  
Vol 4 (3) ◽  
pp. 45
Author(s):  
Riku Nagai ◽  
Yichen Xu ◽  
Chang Liu ◽  
Ayaka Shimabukuro ◽  
Nono Takeuchi-Tomita
Keyword(s):  
Unnatural Amino Acids ◽  
Molecular Mechanisms ◽  
In Vitro Translation ◽  
Critical Parameters ◽  
Translation System ◽  
Reaction Conditions ◽  
Translation Elongation ◽  
Functional Studies ◽  
Ribosome Stalling

The rates of translation elongation or termination in eukaryotes are modulated through cooperative molecular interactions involving mRNA, the ribosome, aminoacyl- and nascent polypeptidyl-tRNAs, and translation factors. To investigate the molecular mechanisms underlying these processes, we developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors, utilizing CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing not only short oligopeptides but also long reporter proteins such as nanoluciferase. By setting appropriate translation reaction conditions, such as the Mg2+/polyamine concentration, the arrest of translation elongation by known ribosome-stalling sequences (e.g., polyproline and CGA codon repeats) is properly recapitulated in this system. We describe protocols for the preparation of the system components, manipulation of the system, and detection of the translation products. We also mention critical parameters for setting up the translation reaction conditions. This reconstituted translation system not only facilitates biochemical analyses of translation but is also useful for various applications, such as structural and functional studies with the aim of designing drugs that act on eukaryotic ribosomes, and the development of systems for producing novel functional proteins by incorporating unnatural amino acids by eukaryotic ribosomes.

Reconstitution of yeast translation elongation and termination in vitro utilizing CrPV IRES-containing mRNA

2020 ◽  
Vol 167 (5) ◽  
pp. 441-450
Author(s):  
Taisho Abe ◽  
Riku Nagai ◽  
Hiroaki Imataka ◽  
Nono Takeuchi-Tomita
Keyword(s):  
Molecular Weight ◽  
Translation Initiation ◽  
In Vitro Translation ◽  
Translation System ◽  
Reporter Protein ◽  
Translation Elongation ◽  
Initiation Factors ◽  
Translation Factors ◽  
Internal Translation

Abstract We developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors and programmed by CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing the active reporter protein, nanoLuciferase, with a molecular weight of 19 kDa. The protein synthesis by the system is appropriately regulated by controlling its composition, including translation factors, amino acids and antibiotics. We found that a high eEF1A concentration relative to the ribosome concentration is critically required for efficient IRES-mediated translation initiation, to ensure its dominance over IRES-independent random internal translation initiation.

scholarly journals Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

2019 ◽  
Author(s):  
Petr Tesina ◽  
Laura N. Lessen ◽  
Robert Buschauer ◽  
Jingdong Cheng ◽  
Colin Chih-Chien Wu ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
Ribosome Profiling ◽  
Translation System ◽  
Structural Explanation ◽  
Ribosome Stalling ◽  
A Site ◽  
Codon Pairs ◽  
Protein Output

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination ofin vitrobiochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use anin vitroreconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

scholarly journals Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA

2020 ◽  
Author(s):  
Muhoon Lee ◽  
Noriko Matsunaga ◽  
Shiori Akabane ◽  
Ippei Yasuda ◽  
Takuya Ueda ◽  
...  
Keyword(s):  
Liver Mitochondria ◽  
In Vitro Translation ◽  
Translation System ◽  
Mitochondrial Translation ◽  
Ribosome Stalling ◽  
Leaderless Mrna ◽  
Mammalian Mitochondria ◽  
Mitochondrial Ribosome ◽  
Nascent Chain

Abstract Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.

scholarly journals Development of a tRNA-dependent in vitro translation system

RNA ◽  
2001 ◽  
Vol 7 (5) ◽  
pp. 765-773 ◽  
Author(s):  
RICHARD J. JACKSON ◽  
SAWSAN NAPTHINE ◽  
IAN BRIERLEY
Keyword(s):  
In Vitro Translation ◽  
Translation System

scholarly journals Arabidopsis Cell-free Extract, ACE, a New In Vitro Translation System Derived from Arabidopsis Callus Cultures

2012 ◽  
Vol 53 (3) ◽  
pp. 602-602
Author(s):  
K. Murota ◽  
Y. Hagiwara-Komoda ◽  
K. Komoda ◽  
H. Onouchi ◽  
M. Ishikawa ◽  
...  
Keyword(s):  
Callus Cultures ◽  
In Vitro Translation ◽  
Translation System ◽  
Cell Free Extract ◽  
Arabidopsis Callus

scholarly journals An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

RNA ◽  
2008 ◽  
Vol 14 (3) ◽  
pp. 593-602 ◽  
Author(s):  
V. V. Zeenko ◽  
C. Wang ◽  
M. Majumder ◽  
A. A. Komar ◽  
M. D. Snider ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
In Vitro Translation ◽  
Translation System ◽  
Translational Inhibition ◽  
Eif2 Phosphorylation

four-jointed is required for intermediate growth in the proximal-distal axis in Drosophila

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2767-2777 ◽  
Author(s):  
J.L. Villano ◽  
F.N. Katz
Keyword(s):  
Optic Lobe ◽  
Regional Growth ◽  
Positional Information ◽  
In Vitro Translation ◽  
Translation System ◽  
Regional Pattern ◽  
Microsomal Membranes ◽  
Position Sensitive ◽  
Coordination Function

Genes capable of translating positional information into regulated growth lie at the heart of morphogenesis, yet few genes with this function have been identified. Mutants in the Drosophila four-jointed (fj) gene show reduced growth and altered differentiation only within restricted sectors of the proximal-distal (PD) axis in the leg and wing, thus fj is a candidate for a gene with this coordination function. Consistent with a position-sensitive role, we show that fj is expressed in a regional pattern in the developing leg, wing, eye and optic lobe. The fj gene encodes a novel type II membrane glycoprotein. When the cDNA is translated in an in vitro translation system in the presence of exogenous microsomal membranes, the intralumenal portion of some of the molecules is cleaved, yielding a secreted C-terminal fragment. We propose that fj encodes a secreted signal that functions as a positive regulator of regional growth and differentiation along the PD axis of the imaginal discs.

scholarly journals Oxidative Stress Can Affect the Gene Silencing Effect of DOTAP Liposome in an In Vitro Translation System

2011 ◽  
Vol 7 (3) ◽  
pp. 253-260 ◽  
Author(s):  
Hiroshi Umakoshi ◽  
Tomoyuki Tanabe ◽  
Keishi Suga ◽  
Huong Thi Bui ◽  
Toshinori Shimanouchi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Silencing ◽  
In Vitro Translation ◽  
Translation System
Sign in / Sign up

Export Citation Format

Share Document