Dataset Information

Protein synthesis in tomato-fruit locule tissue. Incorporation of amino acids into protein by aseptic cell-free systems.

ABSTRACT: 1. Osmotically disrupted protoplasts and isolated plastids from tomato-fruit locule tissue were found capable of incorporating (14)C-labelled amino acids under aseptic conditions into an exhaustively washed trichloroacetic acid-insoluble protein fraction. 2. The disrupted protoplast system incorporated 20-45mumumoles of amino acid/mg. of protein in 10min. The isolated plastid system incorporated 10-20mumumoles of amino acid/mg. of protein; 40-150mumug. of carbon/mg. of protein was incorporated in 10min. from (14)C-labelled amino acid mixture. 3. Incorporation is stimulated by added ATP in the dark, but no added ATP is required when the system is illuminated. The cell-free plastid system is to some extent self-sufficient and does not normally require an added supernatant fraction or unlabelled amino acids. 4. Amino acid incorporation by plastids is inhibited by chloramphenicol, puromycin, actinomycin D, ribonuclease and deoxyribonuclease. It is suggested that the mechanism of protein synthesis in the cell-free plastids, and in the tissue generally, is basically the same as established for bacteria. Ribosomes and highspeed supernatant from this tissue were to some extent interchangeable with Escherichia coli ribosomes and supernatant in cell-free incubations. 5. Incorporation of amino acids by isolated plastids was stimulated by indol-3-ylacetic acid and kinetin, and, whereas incorporation normally proceeds for only 10-20min., the time-course was extended in the presence of these growth substances. It is suggested that hormones may be involved in the regulation of protein synthesis in plants.

SUBMITTER: Davies JW

PROVIDER: S-EPMC1270542 | biostudies-other | 1967 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Synthetic biology holds promise to revolutionize the life sciences and biomedicine via expansion of macromolecular diversity outside the natural chemical space. Use of non-canonical amino acids (ncAAs) via codon reassignment has found diverse applications in protein structure and interaction analysis, introduction of post-translational modifications, production of constrained peptides, antibody-drug conjugates, and novel enzymes. However, simultaneously encoding multiple ncAAs in vivo requires complex engineering and is sometimes restricted by the cell's poor uptake of ncAAs. In contrast the open nature of cell-free protein synthesis systems offers much greater freedom for manipulation and repurposing of the biosynthetic machinery by controlling the level and identity of translational components and reagents, and allows simultaneous incorporation of multiple ncAAs with non-canonical side chains and even backbones (N-methyl, D-, ?-amino acids, ?-hydroxy acids etc.). This review focuses on the two most used Escherichia coli-based cell-free protein synthesis systems; cell extract- and PURE-based systems. The former is a biological mixture with >500 proteins, while the latter consists of 38 individually purified biomolecules. We delineate compositions of these two systems and discuss their respective advantages and applications. Also, we dissect the translational components required for ncAA incorporation and compile lists of ncAAs that can be incorporated into polypeptides via different acylation approaches. We highlight the recent progress in using unnatural nucleobase pairs to increase the repertoire of orthogonal codons, as well as using tRNA-specific ribozymes for in situ acylation. We summarize advances in engineering of translational machinery such as tRNAs, aminoacyl-tRNA synthetases, elongation factors, and ribosomes to achieve efficient incorporation of structurally challenging ncAAs. We note that, many engineered components of biosynthetic machinery are developed for the use in vivo but are equally applicable to the in vitro systems. These are included in the review to provide a comprehensive overview for ncAA incorporation and offer new insights for the future development in cell-free systems. Finally, we highlight the exciting progress in the genomic engineering, resulting in E. coli strains free of amber and some redundant sense codons. These strains can be used for preparation of cell extracts offering multiple reassignment options.

Dataset Information

Protein synthesis in tomato-fruit locule tissue. Incorporation of amino acids into protein by aseptic cell-free systems.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets