Incorporation of Nonnatural Amino Acids into Proteins by Using Various Four-Base Codons in anEscherichia coliin Vitro Translation System

Aminoacylation of tRNA is an essential event in the translation system. Although in the modern system protein enzymes play the sole role in tRNA aminoacylation, in the primitive translation system RNA molecules could have catalysed aminoacylation onto tRNA or tRNA-like molecules. Even though such RNA enzymes so far are not identified from known organisms, in vitro selection has generated such RNA catalysts from a pool of random RNA sequences. Among them, a set of RNA sequences, referred to as flexizymes (Fxs), discovered in our laboratory are able to charge amino acids onto tRNAs. Significantly, Fxs allow us to charge a wide variety of amino acids, including those that are non-proteinogenic, onto tRNAs bearing any desired anticodons, and thus enable us to reprogramme the genetic code at our will. This article summarizes the evolutionary history of Fxs and also the most recent advances in manipulating a translation system by integration with Fxs.

Download Full-text

Chemical aminoacylation of tRNAs with fluorinated amino acids for in vitro protein mutagenesis

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.6.40 ◽

2010 ◽

Vol 6 ◽

Cited By ~ 8

Author(s):

Shijie Ye ◽

Allison Ann Berger ◽

Dominique Petzold ◽

Oliver Reimann ◽

Benjamin Matt ◽

...

Keyword(s):

Amino Acids ◽

Translation System ◽

Biologically Relevant ◽

Fluorinated Amino Acids ◽

Free Translation ◽

Trna Species ◽

A Cell ◽

Protein Environment ◽

Vitro Protein

This article describes the chemical aminoacylation of the yeast phenylalanine suppressor tRNA with a series of amino acids bearing fluorinated side chains via the hybrid dinucleotide pdCpA and ligation to the corresponding truncated tRNA species. Aminoacyl-tRNAs can be used to synthesize biologically relevant proteins which contain fluorinated amino acids at specific sites by means of a cell-free translation system. Such engineered proteins are expected to contribute to our understanding of discrete fluorines’ interaction with canonical amino acids in a native protein environment and to enable the design of fluorinated proteins with arbitrary desired properties.

Download Full-text

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs

RNA ◽

10.1261/rna.2024810 ◽

2010 ◽

Vol 16 (8) ◽

pp. 1660-1672 ◽

Cited By ~ 16

Author(s):

J. Gubbens ◽

S. J. Kim ◽

Z. Yang ◽

A. E. Johnson ◽

W. R. Skach

Keyword(s):

Amino Acids ◽

Nonnatural Amino Acids ◽

Amber Suppressor

Download Full-text

Identification of a talin binding site in the cytoskeletal protein vinculin.

The Journal of Cell Biology ◽

10.1083/jcb.109.6.2917 ◽

1989 ◽

Vol 109 (6) ◽

pp. 2917-2927 ◽

Cited By ~ 42

Author(s):

P Jones ◽

P Jackson ◽

G J Price ◽

B Patel ◽

V Ohanion ◽

...

Keyword(s):

Amino Acids ◽

In Vitro Transcription ◽

Binding Activity ◽

Cytoskeletal Protein ◽

Translation System ◽

Cos Cells ◽

Cell Matrix ◽

Do So

Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746-867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.

Download Full-text