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

1996 ◽  
Vol 16 (3) ◽  
pp. 907-913 ◽  
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.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 255 ◽  
Author(s):  
Sviatlana Smolskaya ◽  
Yaroslav Andreev
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Nonsense Suppression ◽  
Expression Vectors ◽  
Site Specific ◽  
Suppressor Trna ◽  
E Coli ◽  
Orthogonal Pair

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.

scholarly journals Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli

Genes ◽  
2018 ◽  
Vol 9 (10) ◽  
pp. 473 ◽  
Author(s):  
Takuya Umehara ◽  
Saori Kosono ◽  
Dieter Söll ◽  
Koji Tamura
Keyword(s):  
Escherichia Coli ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Lysine Acetylation ◽  
Trna Synthetases ◽  
E Coli ◽  
Domains Of Life ◽  
The Impact

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.

scholarly journals Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

1998 ◽  
Vol 18 (8) ◽  
pp. 4418-4425 ◽  
Author(s):  
Ho-Jin Park ◽  
Uttam L. RajBhandary
Keyword(s):  
Mammalian Cells ◽  
Trna Synthetase ◽  
Nonsense Mutations ◽  
Suppressor Trna ◽  
E Coli ◽  
Tetracycline Transactivator ◽  
Hela Cell Lines ◽  
Amber Codon ◽  
Amber Suppressor ◽  
Chloramphenicol Acetyltransferase Gene

ABSTRACT As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.

Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells

1986 ◽  
Vol 6 (9) ◽  
pp. 3059-3067
Author(s):  
J P Capone ◽  
J M Sedivy ◽  
P A Sharp ◽  
U L RajBhandary
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Mammalian Cells ◽  
Chloramphenicol Acetyltransferase ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Nonsense Mutations ◽  
Suppressor Trna ◽  
Nonsense Codons

We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse NIH3T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors.

scholarly journals Adenosine triphosphate consumption by bacterial arginyl-transfer ribonucleic acid synthetases

1979 ◽  
Vol 179 (2) ◽  
pp. 407-412 ◽  
Author(s):  
J M Godeau ◽  
J Charlier
Keyword(s):  
Escherichia Coli ◽  
Adenosine Triphosphate ◽  
Ribonucleic Acid ◽  
Adenosine Triphosphatase ◽  
Bacillus Stearothermophilus ◽  
Trna Synthetase ◽  
Luciferase Assay ◽  
Trna Synthetases ◽  
E Coli ◽  
Firefly Luciferin

ATP consumption by arginyl-tRNA synthetases from Escherichia coli and Bacillus stearothermophilus has been investigated by the firefly luciferin–luciferase assay. Arginyl-tRNA synthetase from E. coli utilizes ATP only for aminocylation of tRNA with a 1:1 stoicheiometry. In contrast, we have shown an adenosine triphosphatase activity of arginyl-tRNA synthetase from B. stearothermophilus in the absence of tRNAArg. Dowex chromatography revealed the formation of ADP by the thermophile enzyme; under aminoacylation conditions, AMP was also formed in amounts stoicheiometric with arginyl-tRNA formation.

scholarly journals Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells.

1986 ◽  
Vol 6 (9) ◽  
pp. 3059-3067 ◽  
Author(s):  
J P Capone ◽  
J M Sedivy ◽  
P A Sharp ◽  
U L RajBhandary
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Mammalian Cells ◽  
Chloramphenicol Acetyltransferase ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Nonsense Mutations ◽  
Suppressor Trna ◽  
Nonsense Codons

We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse NIH3T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors.

Escherichia coli and colorectal cancer: Unfolding the enigmatic relationship

2021 ◽  
Vol 22 ◽  
Author(s):  
Rogayeh Nouri ◽  
Alka Hasani ◽  
Kourosh Masnadi Shirazi ◽  
Mohammad Reza Aliand ◽  
Bita Sepehri ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Escherichia Coli ◽  
Mammalian Cells ◽  
Chromosomal Rearrangements ◽  
Current Evidence ◽  
Dna Breaks ◽  
Colon Cells ◽  
E Coli ◽  
Dna Mismatch ◽  
Double Strand Dna Breaks

: Colorectal cancer (CRC) is one of the deadliest cancers in the world. Specific strains of intestinal Escherichia coli (E. coli) may influence the initiation and development of CRC by exploiting virulence factors and inflammatory pathways. Mucosa-associated E. coli strains are more prevalent in CRC biopsies in comparison to healthy controls. Moreover, these strains can survive and replicate within macrophages and induce a pro-inflammatory response. Chronic exposure to inflammatory mediators can lead to increased cell proliferation and cancer. Production of colobactin toxin by the majority of mucosa-associated E. coli isolated from CRC patients is another notable finding. Colibactin-producing E. coli strains, in particular, induce double-strand DNA breaks, stop the cell cycle, involve in chromosomal rearrangements of mammalian cells and are implicated in carcinogenic effects in animal models. Moreover, some enteropathogenic E. coli (EPEC) strains are able to survive and replicate in colon cells as chronic intracellular pathogens and may promote susceptibility to CRC by downregulation of DNA Mismatch Repair (MMR) proteins. In this review, we discuss current evidence and focus on the mechanisms by which E. coli can influence the development of CRC.

scholarly journals E1A 13S and 12S mRNA products made in Escherichia coli both function as nucleus-localized transcription activators but do not directly bind DNA.

1985 ◽  
Vol 5 (10) ◽  
pp. 2653-2661 ◽  
Author(s):  
B Ferguson ◽  
B Krippl ◽  
O Andrisani ◽  
N Jones ◽  
H Westphal ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Biochemical Properties ◽  
Protein Product ◽  
Gene Products ◽  
E Coli ◽  
Adenovirus E1a ◽  
Myc Gene ◽  
E1a Gene ◽  
Transcription Activators

We previously purified and characterized functionally the Escherichia coli-expressed product of the human subgroup C adenovirus E1A 13S mRNA (B. Ferguson, N. Jones, J. Richter, and M. Rosenberg, Science 224:1343-1346, 1984; B. Krippl, B. Ferguson, M. Rosenberg, and H. Westphal, Proc. Natl. Acad. Sci. USA 81:6988-6992, 1984). We have now expressed in E. coli and purified the protein product encoded by the human subgroup C adenovirus E1A 12S mRNA and have compared the functional properties of this protein with those of the E1A 13S mRNA product. Using microinjection techniques to introduce these proteins into mammalian cells, we found that the E1A 12S mRNA product, like the 13S mRNA product, localized rapidly to the cell nucleus and induced adenovirus gene expression. Although both E1A gene products localized to the nucleus and stimulated adenovirus gene transcription, these proteins did not directly bind to DNA under conditions in which a known DNA-binding protein, the human c-myc gene product, bound DNA efficiently. Thus, the E1A and myc gene products, which have been related both structurally and functionally, exhibit distinctly different biochemical properties.

scholarly journals Novel Roles for the AIDA Adhesin from Diarrheagenic Escherichia coli: Cell Aggregation and Biofilm Formation

2004 ◽  
Vol 186 (23) ◽  
pp. 8058-8065 ◽  
Author(s):  
Orla Sherlock ◽  
Mark A. Schembri ◽  
Andreas Reisner ◽  
Per Klemm
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Mammalian Cells ◽  
Gastrointestinal Disease ◽  
Cell Aggregation ◽  
Bacterial Attachment ◽  
E Coli ◽  
Abiotic Surfaces ◽  
Broad Variety ◽  
Self Association

ABSTRACT Diarrhea-causing Escherichia coli strains are responsible for numerous cases of gastrointestinal disease and constitute a serious health problem throughout the world. The ability to recognize and attach to host intestinal surfaces is an essential step in the pathogenesis of such strains. AIDA is a potent bacterial adhesin associated with some diarrheagenic E. coli strains. AIDA mediates bacterial attachment to a broad variety of human and other mammalian cells. It is a surface-displayed autotransporter protein and belongs to the selected group of bacterial glycoproteins; only the glycosylated form binds to mammalian cells. Here, we show that AIDA possesses self-association characteristics and can mediate autoaggregation of E. coli cells. We demonstrate that intercellular AIDA-AIDA interaction is responsible for bacterial autoaggregation. Interestingly, AIDA-expressing cells can interact with antigen 43 (Ag43)-expressing cells, which is indicative of an intercellular AIDA-Ag43 interaction. Additionally, AIDA expression dramatically enhances biofilm formation by E. coli on abiotic surfaces in flow chambers.

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