Codon assignments and fidelity of translation in a cell-free protein-synthesizing system from an extremely halophilic bacterium

1968 ◽  
Vol 46 (8) ◽  
pp. 937-944 ◽  
Author(s):  
S. T. Bayley ◽  
E. Griffiths
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Halophilic Bacterium ◽  
Quantitative Comparison ◽  
Free System ◽  
Free Protein ◽  
Cell Free System ◽  
Polyuridylic Acid ◽  
A Cell

The incorporation of 14C-labelled amino acids into material insoluble in hot TCA under the direction of synthetic polyribonucleotides has been studied in a preincubated, cell-free system from Halobacterium cutirubrum. In the presence of 3.8 M KCl and 1.1 M NH4 salts, polyuridylic acid directed the incorporation of phenylalanine and also of leucine to the extent of 8% miscoding. This miscoding for leucine was reduced to 1% in the presence of 3.8 M KCl, 1.0 M NaCl, and 0.4 M NH4Cl. Poly C directed the incorporation of proline. The random heteropolyribonucleotides poly CA, poly CU, and poly UG directed the incorporation of only those amino acids expected on the basis of the established genetic code, together with methionine with poly UG. Quantitative comparison of the incorporation with the calculated frequencies of triplets in these polyribonucleotides suggests that the codon assignments investigated in H. cutirubrum could be identical with those in non-halophilic organisms.

Studies on the Incorporation of Amino Acids by a Cell-Free System Obtained from Beef Retina

1972 ◽  
Vol 50 (5) ◽  
pp. 581-587 ◽  
Author(s):  
Y. Matuk
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Incubation Medium ◽  
Optimum Concentration ◽  
Free System ◽  
Cell Free System ◽  
A Cell

The incorporation of 14C-leucine into proteins by a cell-free system from beef retina was studied. It was found that the optimum concentration of ATP depended on the concentration of ribosomes in the incubation medium. Very little incorporation of 14C-leucine was observed in the absence of K+. The optimum concentration of phosphocreatine required for incorporation of radioactive leucine depended on the concentration of Mg2+ in the incubation medium, and the optimum concentration of K+ appears to be independent of the concentrations of Mg2+ and phosphocreatine used.Retinol and retinal had no effect, but ethanol markedly inhibited protein synthesis at concentrations higher than 2%.Puromycin (10−4 M) inhibited incorporation of 14C-leucine by about 80%. The degree of inhibition by cycloheximide depended on the concentration of pH 5 fraction in the incubation medium.

scholarly journals Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins

2019 ◽  
Vol 20 (3) ◽  
pp. 492 ◽  
Author(s):  
Jiro Adachi ◽  
Kazushige Katsura ◽  
Eiko Seki ◽  
Chie Takemoto ◽  
Mikako Shirouzu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Trna Synthetase ◽  
Translation Efficiency ◽  
Free System ◽  
Free Protein ◽  
Cell Free System ◽  
Natural Amino Acids ◽  
Cell Free Protein Synthesis

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

CELL-FREE INCORPORATION OF AMINO ACIDS BY A TROUT-LIVER SYSTEM. HEAT-LABILE AMINOACYL TRANSFERASE ACTIVITY

1967 ◽  
Vol 45 (12) ◽  
pp. 2005-2014 ◽  
Author(s):  
Lawrence Rosen ◽  
E. L. Murray ◽  
G. David Novelli
Keyword(s):  
Amino Acids ◽  
Rat Liver ◽  
Transferase Activity ◽  
Free System ◽  
Cell Free System ◽  
Heat Labile ◽  
A Cell ◽  
Aminoacyl Transferase

A cell-free system from trout liver that incorporates amino acids into protein has been isolated. The system requires both soluble and particulate fractions, and is similar to those isolated from other organisms.The incorporation of amino acids is extremely heat-labile, and this lability has been localized in the aminoacyl transferase activity of the cell sap. The heat lability of the microsomes may also be related to transferase activity.Ribosomal fractions from trout liver and rat liver incorporate amino acids when incubated with cell sap from rat liver and trout liver, respectively.

scholarly journals In vitro synthesis of pp60v-src: myristylation in a cell-free system.

1988 ◽  
Vol 8 (10) ◽  
pp. 4295-4301 ◽  
Author(s):  
I Deichaite ◽  
L P Casson ◽  
H P Ling ◽  
M D Resh
Keyword(s):  
Amino Acids ◽  
Synthetic Peptide ◽  
Covalent Attachment ◽  
Free System ◽  
In Vitro Synthesis ◽  
Cell Free System ◽  
Reticulocyte Lysates ◽  
A Cell

Covalent attachment of myristic acid to pp60v-src, the transforming protein of Rous sarcoma virus, was studied in a cell-free system. Using a synthetic peptide containing the first 11 amino acids of the mature pp60v-src polypeptide sequence as a substrate, we probed lysates from a variety of cells and tissues for N-myristyl transferase (NMT) activity. Nearly every eucaryotic cell type tested contained NMT, including avian, mammalian, insect, and plant cells. Since NMT activity was detected in rabbit reticulocyte lysates, we took advantage of the translational capability of these lysates to determine the precise point during translation at which myristate is attached to pp60v-src. src mRNA, transcribed from cloned v-src DNA, was translated in reticulocyte lysates which had been depleted of endogenous myristate. Addition of [3H]myristate to lysates 10 min after the start of synchronized translation resulted in a dramatic decrease in the incorporation of radiolabeled myristate into pp60v-src polypeptide chains. These results imply that although myristate can be attached posttranslationally to synthetic peptide substrates, myristylation in vivo is apparently a very early cotranslational event which occurs before the first 100 amino acids of the nascent polypeptide chain are polymerized.

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