Construction of Escherichia coli amber suppressor tRNA genes

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.

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Shifted positioning of the anticodon nucleotide residues of amber suppressor tRNA species by Escherichia coli arginyl-tRNA synthetase

European Journal of Biochemistry ◽

10.1046/j.0014-2956.2001.02570.x ◽

2001 ◽

Vol 268 (23) ◽

pp. 6207-6213 ◽

Cited By ~ 5

Author(s):

Daisuke Kiga ◽

Kensaku Sakamoto ◽

Saori Sato ◽

Ichiro Hirao ◽

Shigeyuki Yokoyama

Keyword(s):

Escherichia Coli ◽

Trna Synthetase ◽

Suppressor Trna ◽

Amber Suppressor ◽

Trna Species

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Nonsense suppression in archaea

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1501558112 ◽

2015 ◽

Vol 112 (19) ◽

pp. 6015-6020 ◽

Cited By ~ 4

Author(s):

Arpita Bhattacharya ◽

Caroline Köhrer ◽

Debabrata Mandal ◽

Uttam L. RajBhandary

Keyword(s):

Nonsense Suppression ◽

Trna Genes ◽

Bacterial Strains ◽

Nonsense Mutations ◽

Suppressor Trna ◽

Viral Genetics ◽

Stop Codons ◽

Amber Suppressor ◽

Nonsense Suppressor

Bacterial strains carrying nonsense suppressor tRNA genes played a crucial role in early work on bacterial and bacterial viral genetics. In eukaryotes as well, suppressor tRNAs have played important roles in the genetic analysis of yeast and worms. Surprisingly, little is known about genetic suppression in archaea, and there has been no characterization of suppressor tRNAs or identification of nonsense mutations in any of the archaeal genes. Here, we show, using the β-gal gene as a reporter, that amber, ochre, and opal suppressors derived from the serine and tyrosine tRNAs of the archaeonHaloferax volcaniiare active in suppression of their corresponding stop codons. Using a promoter for tRNA expression regulated by tryptophan, we also show inducible and regulatable suppression of all three stop codons inH. volcanii. Additionally, transformation of aΔpyrE2 H. volcaniistrain with plasmids carrying the genes for apyrE2amber mutant and the serine amber suppressor tRNA yielded transformants that grow on agar plates lacking uracil. Thus, an auxotrophic amber mutation in thepyrE2gene can be complemented by expression of the amber suppressor tRNA. These results pave the way for generating archaeal strains carrying inducible suppressor tRNA genes on the chromosome and their use in archaeal and archaeviral genetics. We also provide possible explanations for why suppressor tRNAs have not been identified in archaea.

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.6.9.3059 ◽

1986 ◽

Vol 6 (9) ◽

pp. 3059-3067 ◽

Cited By ~ 35

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.

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Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

Journal of Bacteriology ◽

10.1128/jb.183.17.5187-5197.2001 ◽

2001 ◽

Vol 183 (17) ◽

pp. 5187-5197 ◽

Cited By ~ 302

Author(s):

Vanessa Sperandio ◽

Alfredo G. Torres ◽

Jorge A. Girón ◽

James B. Kaper

Keyword(s):

Escherichia Coli ◽

Quorum Sensing ◽

Type Strain ◽

Wild Type Strain ◽

Regulatory Mechanism ◽

Sos Response ◽

Enterohemorrhagic Escherichia Coli ◽

Trna Genes ◽

Wild Type ◽

E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

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Nucleotides that determine Escherichia coli tRNA(Arg) and tRNA(Lys) acceptor identities revealed by analyses of mutant opal and amber suppressor tRNAs.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.87.23.9260 ◽

1990 ◽

Vol 87 (23) ◽

pp. 9260-9264 ◽

Cited By ~ 59

Author(s):

W. H. McClain ◽

K. Foss ◽

R. A. Jenkins ◽

J. Schneider

Keyword(s):

Escherichia Coli ◽

Amber Suppressor

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An amber mutation in the gene encoding the beta' subunit of Escherichia coli RNA polymerase

Journal of Bacteriology ◽

10.1128/jb.152.2.736-746.1982 ◽

1982 ◽

Vol 152 (2) ◽

pp. 736-746

Author(s):

S P Ridley ◽

M P Oeschger

Keyword(s):

Escherichia Coli ◽

High Temperature ◽

Rna Polymerase ◽

Coli Strain ◽

Beta Subunit ◽

Temperature Sensitive ◽

Amber Mutation ◽

Gene Encoding ◽

Amber Suppressor

An Escherichia coli strain carrying an amber mutation (UAG) in rpoC, the gene encoding the beta prime subunit of RNA polymerase, was isolated after mutagenesis with nitrosoguanidine. The mutation was moved into an unmutagenized strain carrying the supD43,74 allele, which encodes a temperature-sensitive su1 amber suppressor, and sue alleles, which enhance the efficiency of the suppressor. In this background, beta prime is not synthesized at high temperature. Suppression of the mutation by the non-temperature-sensitive amber suppressor su1+ yields a protein which is functional at all temperatures examined (30, 37, and 42 degrees C).

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Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

The EMBO Journal ◽

10.1002/j.1460-2075.1985.tb02338.x ◽

1985 ◽

Vol 4 (1) ◽

pp. 213-221 ◽

Cited By ~ 36

Author(s):

J.P. Capone ◽

P.A. Sharp ◽

U.L. RajBhandary

Keyword(s):

Trna Gene ◽

Trna Genes ◽

Suppressor Trna

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

Biomolecules ◽

10.3390/biom9070255 ◽

2019 ◽

Vol 9 (7) ◽

pp. 255 ◽

Cited By ~ 16

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.

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