scholarly journals Multiple prebiotic metals mediate translation

2018 ◽  
Vol 115 (48) ◽  
pp. 12164-12169 ◽  
Author(s):  
Marcus S. Bray ◽  
Timothy K. Lenz ◽  
Jay William Haynes ◽  
Jessica C. Bowman ◽  
Anton S. Petrov ◽  
...  
Keyword(s):  
Metal Content ◽  
Protein Production ◽  
Tertiary Structure ◽  
In Vitro Translation ◽  
Translation System ◽  
Ancestral State ◽  
Functional Protein ◽  
Functional Roles ◽  
Shape Selective

Today, Mg2+is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O2, high Fe2+, and high Mn2+) can revert the ribosome to a functional ancestral state. First, SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) was used to compare the effect of Mg2+, Fe2+, and Mn2+on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe2+or Mn2+could mediate protein production, and quantified ribosomal metal content. We found that (i) Mg2+, Fe2+, and Mn2+had strikingly similar effects on rRNA folding; (ii) Fe2+and Mn2+can replace Mg2+as the dominant divalent cation during translation of mRNA to functional protein; and (iii) Fe and Mn associate extensively with the ribosome. Given that the translation system originated and matured when Fe2+and Mn2+were abundant, these findings suggest that Fe2+and Mn2+played a role in early ribosomal evolution.

scholarly journals Multiple prebiotic metals mediate translation

2018 ◽  
Author(s):  
Marcus S. Bray ◽  
Timothy K. Lenz ◽  
Jay William Haynes ◽  
Jessica C. Bowman ◽  
Anton S. Petrov ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Tertiary Structure ◽  
Cell Function ◽  
Living Organism ◽  
Translation System ◽  
Ancestral State ◽  
Functional Protein ◽  
Functional Roles ◽  
And Function

ABSTRACTToday, Mg2+is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O2, high Fe2+, high Mn2+) can revert the ribosome to a functional ancestral state. First, SHAPE (Selective 2’HydroxylAcylation analyzed byPrimerExtension) was used to compare the effect of Mg2+, Fe2+, and Mn2+on the tertiary structure of rRNA. Then, we usedin vitrotranslation reactions to test whether Fe2+or Mn2+could mediate protein production, and quantified ribosomal metal content. We found that: (i) Mg2+, Fe2+, and Mn2+had strikingly similar effects on rRNA folding; (ii) Fe2+and Mn2+can replace Mg2+as the dominant divalent cation during translation of mRNA to functional protein; (iii) Fe and Mn associate extensively with the ribosome. Given that the translation system originated and matured when Fe2+and Mn2+were abundant, these findings suggest that Fe2+and Mn2+played a role in early ribosomal evolution.SIGNIFICANCERibosomes are found in every living organism where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ~4 billion years ago when ecosystems were anoxic and metal-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions (low O2, high Fe2+, high Mn2+). Our results expand the roles of Fe2+and Mn2+in ancient and extant biochemistry as cofactors for ribosomal structure and function.

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

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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