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 Potyvirus Genome-linked Protein, VPg, Directly Affects Wheat Germ in Vitro Translation

2007 ◽  
Vol 283 (3) ◽  
pp. 1340-1349 ◽  
Author(s):  
Mateen A. Khan ◽  
Hiroshi Miyoshi ◽  
Daniel R. Gallie ◽  
Dixie J. Goss
Keyword(s):  
Translation Initiation ◽  
Wheat Germ ◽  
Mrna Translation ◽  
Kinetic Studies ◽  
Dissociation Rate ◽  
In Vitro Translation ◽  
Translation Efficiency ◽  
Viral Mrna ◽  
Initiation Factors

Potyvirus genome linked protein, VPg, interacts with translation initiation factors eIF4E and eIFiso4E, but its role in protein synthesis has not been elucidated. We show that addition of VPg to wheat germ extract leads to enhancement of uncapped viral mRNA translation and inhibition of capped viral mRNA translation. This provides a significant competitive advantage to the uncapped viral mRNA. To understand the molecular basis of these effects, we have characterized the interaction of VPg with eIF4F, eIFiso4F, and a structured RNA derived from tobacco etch virus (TEV RNA). When VPg formed a complex with eIF4F, the affinity for TEV RNA increased more than 4-fold compared with eIF4F alone (19.4 and 79.0 nm, respectively). The binding affinity of eIF4F to TEV RNA correlates with translation efficiency. VPg enhanced eIFiso4F binding to TEV RNA 1.6-fold (178 nm compared with 108 nm). Kinetic studies of eIF4F and eIFiso4F with VPg show ∼2.6-fold faster association for eIFiso4F·VPg as compared with eIF4F·VPg. The dissociation rate was ∼2.9-fold slower for eIFiso4F than eIF4F with VPg. These data demonstrate that eIFiso4F can kinetically compete with eIF4F for VPg binding. The quantitative data presented here suggest a model where eIF4F·VPg interaction enhances cap-independent translation by increasing the affinity of eIF4F for TEV RNA. This is the first evidence of direct participation of VPg in translation initiation.

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.

scholarly journals eIF4G-driven translation initiation of downstream ORFs in mammalian cells

2020 ◽  
Vol 48 (18) ◽  
pp. 10441-10455
Author(s):  
Risa Nobuta ◽  
Kodai Machida ◽  
Misaki Sato ◽  
Satoshi Hashimoto ◽  
Yasuhito Toriumi ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Open Reading Frames ◽  
Translation System ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Genome Wide

Abstract Comprehensive genome-wide analysis has revealed the presence of translational elements in the 3′ untranslated regions (UTRs) of human transcripts. However, the mechanisms by which translation is initiated in 3′ UTRs and the physiological function of their products remain unclear. This study showed that eIF4G drives the translation of various downstream open reading frames (dORFs) in 3′ UTRs. The 3′ UTR of GCH1, which encodes GTP cyclohydrolase 1, contains an internal ribosome entry site (IRES) that initiates the translation of dORFs. An in vitro reconstituted translation system showed that the IRES in the 3′ UTR of GCH1 required eIF4G and conventional translation initiation factors, except eIF4E, for AUG-initiated translation of dORFs. The 3′ UTR of GCH1-mediated translation was resistant to the mTOR inhibitor Torin 1, which inhibits cap-dependent initiation by increasing eIF4E-unbound eIF4G. eIF4G was also required for the activity of various elements, including polyU and poliovirus type 2, a short element thought to recruit ribosomes by base-pairing with 18S rRNA. These findings indicate that eIF4G mediates translation initiation of various ORFs in mammalian cells, suggesting that the 3′ UTRs of mRNAs may encode various products.

scholarly journals eIF5B gates the transition from translation initiation to elongation

2019 ◽  
Author(s):  
Jinfan Wang ◽  
Alex G. Johnson ◽  
Christopher P. Lapointe ◽  
Junhong Choi ◽  
Arjun Prabhakar ◽  
...  
Keyword(s):  
Single Molecule ◽  
Translation Initiation ◽  
Protein Product ◽  
Initiation Factor ◽  
Translation System ◽  
80S Ribosome ◽  
Reading Frame ◽  
Initiation Factors ◽  
Translation Initiation Factors

Translation initiation determines both the quantity and identity of the protein product by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors (eIFs) prepare ribosomes for polypeptide elongation, yet the underlying dynamics of this process remain enigmatic1–4. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here, we applied in vitro single-molecule fluorescence microscopy approaches to monitor directly in real time the pathways of late translation initiation and the transition to elongation using a purified yeast Saccharomyces cerevisiae translation system. This transition was remarkably slower in our eukaryotic system than that reported for Escherichia coli5–7. The slow entry to elongation was defined by a long residence time of eIF5B on the 80S ribosome after joining of individual ribosomal subunits, which is catalyzed by this universally conserved initiation factor. Inhibition of eIF5B GTPase activity following subunit joining prevented eIF5B dissociation from the 80S complex, thereby preventing elongation. Our findings illustrate how eIF5B dissociation serves as a kinetic checkpoint for the transition from initiation to elongation, and its release may be governed by a conformation of the ribosome complex that triggers GTP hydrolysis.

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.

Sign in / Sign up

Export Citation Format

Share Document