The 3′ Codon Context Effect on UAG Suppressor tRNA is Different in Escherichia coli and Human Cells

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

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Replicative Bypass of Abasic Site in Escherichia coli and Human Cells: Similarities and Differences

PLoS ONE ◽

10.1371/journal.pone.0107915 ◽

2014 ◽

Vol 9 (9) ◽

pp. e107915 ◽

Cited By ~ 21

Author(s):

Savithri Weerasooriya ◽

Vijay P. Jasti ◽

Ashis K. Basu

Keyword(s):

Escherichia Coli ◽

Human Cells ◽

Abasic Site ◽

Similarities And Differences

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A highly conserved endonuclease activity present in Escherichia coli, bovine, and human cells recognizes oxidative DNA damage at sites of pyrimidines.

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.26 ◽

1987 ◽

Vol 7 (1) ◽

pp. 26-32 ◽

Cited By ~ 82

Author(s):

P W Doetsch ◽

W D Henner ◽

R P Cunningham ◽

J H Toney ◽

D E Helland

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Oxidative Dna Damage ◽

Uv Light ◽

Human Fibroblasts ◽

Human Cells ◽

Sequence Specificity ◽

Endonuclease Activity ◽

E Coli ◽

Endonuclease Iii

We have compared the sites of nucleotide incision on DNA damaged by oxidizing agents when cleavage is mediated by either Escherichia coli endonuclease III or an endonuclease present in bovine and human cells. E. coli endonuclease III, the bovine endonuclease isolated from calf thymus, and the human endonuclease partially purified from HeLa and CEM-C1 lymphoblastoid cells incised DNA damaged with osmium tetroxide, ionizing radiation, or high doses of UV light at sites of pyrimidines. For each damaging agent studied, regardless of whether the E. coli, bovine, or human endonuclease was used, the same sequence specificity of cleavage was observed. We detected this endonuclease activity in a variety of human fibroblasts derived from normal individuals as well as individuals with the DNA repair deficiency diseases ataxia telangiectasia and xeroderma pigmentosum. The highly conserved nature of such a DNA damage-specific endonuclease suggests that a common pathway exists in bacteria, humans, and other mammals for the reversal of certain types of oxidative DNA damage.

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Codon context effect in virus translational readthrough A study in vitro of the determinants of TMV and Mo-MuLV amber suppression

FEBS Letters ◽

10.1016/0014-5793(92)80984-o ◽

1992 ◽

Vol 306 (2-3) ◽

pp. 133-139 ◽

Cited By ~ 19

Author(s):

Rosaura P.C. Valle ◽

Gabrièle Drugeon ◽

Marie-Dominique Devignes-Morch ◽

Andrzej B. Legocki ◽

Anne-Lise Haenni

Keyword(s):

Context Effect ◽

Translational Readthrough ◽

Codon Context ◽

Amber Suppression

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Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene.

Molecular and Cellular Biology ◽

10.1128/mcb.16.3.907 ◽

1996 ◽

Vol 16 (3) ◽

pp. 907-913 ◽

Cited By ~ 52

Author(s):

H J Drabkin ◽

H J Park ◽

U L RajBhandary

Keyword(s):

Escherichia Coli ◽

Mammalian Cells ◽

Trna Gene ◽

Trna Synthetase ◽

Developmentally Regulated ◽

Coding Sequence ◽

Suppressor Trna ◽

Trna Synthetases ◽

E Coli ◽

Nonsense Codons

As an approach to inducible suppression of nonsense mutations in mammalian and in higher eukaryotic cells, we have analyzed the expression of an Escherichia coli glutamine-inserting amber suppressor tRNA gene in COS-1 and CV-1 monkey kidney cells. The tRNA gene used has the suppressor tRNA coding sequence flanked by sequences derived from a human initiator methionine tRNA gene and has two changes in the coding sequence. This tRNA gene is transcribed, and the transcript is processed to yield the mature tRNA in COS-1 and CV-1 cells. We show that the tRNA is not aminoacylated in COS-1 cells by any of the endogenous aminoacyl-tRNA synthetases and is therefore not functional as a suppressor. Concomitant expression of the E. coli glutaminyl-tRNA synthetase gene results in aminoacylation of the suppressor tRNA and its functioning as a suppressor. These results open up the possibility of attempts at regulated suppression of nonsense codons in mammalian cells by regulating expression of the E. coli glutaminyl-tRNA synthetase gene in an inducible, cell-type specific, or developmentally regulated manner.

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BK virus-plasmid expression vector that persists episomally in human cells and shuttles into Escherichia coli.

Molecular and Cellular Biology ◽

10.1128/mcb.4.8.1551 ◽

1984 ◽

Vol 4 (8) ◽

pp. 1551-1560 ◽

Cited By ~ 35

Author(s):

G Milanesi ◽

G Barbanti-Brodano ◽

M Negrini ◽

D Lee ◽

A Corallini ◽

...

Keyword(s):

Escherichia Coli ◽

Expression Vector ◽

Selective Pressure ◽

T Antigen ◽

Bk Virus ◽

Human Cells ◽

Transformed Cells ◽

Low Molecular Weight ◽

Simplex Virus

We describe a novel expression vector, pBK TK-1, that persists episomally in human cells that can be shuttled into bacteria. This vector includes sequences from BK virus (BKV), the thymidine kinase (TK) gene of herpes simplex virus type 1, and plasmid pML-1. TK+-transformed HeLa and 143 B cells contained predominantly full-length episomes. There were typically 20 to 40 (HeLa) and 75 to 120 143 B vector copies per cell, although some 143 B transformants contained hundreds. Low-molecular-weight DNA from TK+-transformed cells introduced into Escherichia coli were recovered as plasmids that were indistinguishable from the input vector. Removal of selective pressure had no apparent effect upon the episomal status of pBK TK-1 molecules in TK+-transformed cells. BKV T antigen may play a role in episomal replication of pBK TK-1 since this viral protein was expressed in TK+ transformants and since a plasmid that contained only the BKV origin of replication was highly amplified in BKV-transformed human cells that synthesize BKV T antigen.

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