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

scholarly journals The translocation, folding, assembly and redox-dependent degradation of secretory and membrane proteins in semi-permeabilized mammalian cells

1995 ◽  
Vol 307 (3) ◽  
pp. 679-687 ◽  
Author(s):  
R Wilson ◽  
A J Allen ◽  
J Oliver ◽  
J L Brookman ◽  
S High ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Intact Cell ◽  
Cell System ◽  
In Vitro Translation ◽  
Tissue Type ◽  
Translation System ◽  
Secretory Proteins ◽  
Permeabilized Cell ◽  
Permeabilized Cells

We describe here a semi-permeabilized cell-system which reconstitutes the efficient synthesis, translocation, folding, assembly and degradation of membrane and secretory proteins. Cells grown in culture were treated with the detergent digitonin which selectively permeabilized the plasma membrane leaving the cellular organelles, such as the endoplasmic reticulum (ER) and trans-Golgi network intact. These permeabilized cells were added to an in vitro translation system, either wheatgerm or reticulocyte lysate, supplemented with RNA coding for either membrane or secretory proteins. Efficient translocation and modification of proteins by these cells was demonstrated by protease protection, photocross-linking of nascent chains to components of the translocation apparatus and by post-translational modifications such as glycosylation or hydroxylation. A comparison was made between the ability of semi-permeabilized cells and microsomal vesicles to fold and assemble proteins. The results show that the intact ER within these cells can assemble proteins much more efficiently than vesicularized ER. Furthermore, the semi-permeabilized cells carried out the redox-dependent degradation of tissue-type plasminogen activator. This system has all the advantages of conventional cell-free systems, including speed and, importantly, the ability to manipulate the components of the assay, while retaining intracellular organelles and, therefore, allowing cellular processes to occur as they would in the intact cell.

Structural and functional similarities between the central eukaryotic initiation factor (eIF)4A-binding domain of mammalian eIF4G and the eIF4A-binding domain of yeast eIF4G

2001 ◽  
Vol 355 (1) ◽  
pp. 223-230 ◽  
Author(s):  
Diana DOMINGUEZ ◽  
Elisabeth KISLIG ◽  
Michael ALTMANN ◽  
Hans TRACHSEL
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Mammalian Cells ◽  
Amino Acid Level ◽  
Binding Domain ◽  
In Vitro Translation ◽  
Initiation Factor ◽  
Translation System ◽  
Eukaryotic Initiation Factor

The translation eukaryotic initiation factor (eIF)4G of the yeast Saccharomyces cerevisiae interacts with the RNA helicase eIF4A (a member of the DEAD-box protein family; where DEAD corresponds to Asp-Glu-Ala-Asp) through a C-terminal domain in eIF4G (amino acids 542–883). Mammalian eIF4G has two interaction domains for eIF4A, a central domain and a domain close to the C-terminus. This raises the question of whether eIF4A binding to eIF4G is conserved between yeast and mammalian cells or whether it is different. We isolated eIF4G1 mutants defective in eIF4A binding and showed that these mutants are strongly impaired in translation and growth. Extracts from mutants displaying a temperature-sensitive phenotype for growth have low in vitro translation activity, which can be restored by addition of the purified eIF4G1–eIF4E complex, but not by eIF4E alone. Analysis of mutant eIF4G542–883 proteins defective in eIF4A binding shows that the interaction of yeast eIF4A with eIF4G1 depends on amino acid motifs that are conserved between the yeast eIF4A-binding site and the central eIF4A-binding domain of mammalian eIF4G. We show that mammalian eIF4A binds tightly to yeast eIF4G1 and, furthermore, that mutant yeast eIF4G542–883 proteins, which do not bind yeast eIF4A, do not interact with mammalian eIF4A. Despite the conservation of the eIF4A-binding site in eIF4G and the strong sequence conservation between yeast and mammalian eIF4A (66% identity; 82% similarity at the amino acid level) mammalian eIF4A does not substitute for the yeast factor in vivo and is not functional in a yeast in vitro translation system.

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

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

scholarly journals CTELS: A Cell-Free System for the Analysis of Translation Termination Rate

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 911 ◽  
Author(s):  
Kseniya A. Lashkevich ◽  
Valeriya I. Shlyk ◽  
Artem S. Kushchenko ◽  
Vadim N. Gladyshev ◽  
Elena Z. Alkalaeva ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Protein Biosynthesis ◽  
Inhibitory Effect ◽  
In Vitro Translation ◽  
Nonsense Suppression ◽  
Translation System ◽  
Free System ◽  
Termination Rate

Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression.

Fibrinogen Biosynthesis: In Vitro Translation. Glycosylation And Translocation Of Fibrinogen Peptide Chains

1981 ◽  
Author(s):  
G M Fuller ◽  
J M Nickerson
Keyword(s):  
Signal Peptide ◽  
Hepatoma Cell ◽  
Temporal Analysis ◽  
In Vitro Translation ◽  
Translation System ◽  
Hepatoma Cell Line ◽  
Amino Terminal ◽  
Cellular Processes ◽  
Microsomal Membranes

Fibrinogen is a hepatically derived plasma glycoprotein that is composed of three pairs of nonidentical chains linked together by complex sets of disulfide bridges. In an effort to understand the molecular and cellular processes of translating and assembling this important multichained protein we have utilized an in vitro translating system using mRNA’s for rat fibrinogen. Highly specific antibodies to fibrinogen and to each chain have been developed and used to immunoprecipitate the nascent Aα, Bβ, and γ polypeptides. We have also used a rat hepatoma cell line which synthesizes and secretes fibrinogen to prepare nonglycosylated but processed fibrinogen subunits. SDS/PAGE analysis of the translation products clearly show that each polypeptide has a “signal” peptide located at its amino terminal end. The size of the signal peptide is different for each chain. These results demonstrate that separate mRNA’s exist for each of the fibrinogen subunits. Temporal analysis of the glycosylation of the Bβ and γ chain reveal that the γ chain receives its Asn-linked carbohydrate as an early cotranslational event. The Bβ chain’s core carbohydrate moiety is near the end of the polypeptide and our evidence shows that the glycosylation event likely occurs posttranslationally. When microsomal membranes are added to an on-going translation system, all three of fibrinogen's polypeptides translocate into the cisternal space, with an apparent equal stiochiometry. Additional experiments suggest that fibrinogen assembly occurs as a cotranslational process.These studies have been supported in part by NIH HL - 16445 and HL 00162.

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