A temperature-sensitive glycyl-transfer ribonucleic acid synthetase mutant of Escherichia coli

1973 ◽  
Vol 19 (4) ◽  
pp. 421-426 ◽  
Author(s):  
E. R. Roback ◽  
J. D. Friesen ◽  
N. P. Fill
Keyword(s):  
Escherichia Coli ◽  
Ribonucleic Acid ◽  
Bacteriophage T4 ◽  
Temperature Shift ◽  
Transfer Rna ◽  
In Vitro Assays ◽  
Temperature Sensitive ◽  
Heat Sensitivity ◽  
Synthetase Activity

A method of selecting temperature-sensitive mutants of Escherichia coli which makes use of a double temperature shift combined with suicide as a result of incorporated 3H-uridine is described. One mutant selected in this way shows restricted growth at 42 °C resulting from early inhibition of protein and ribonucleic acid (RNA) synthesis. In vitro assays of aminoacyl-transfer RNA synthetase activity indicated a complete absence of active glycyl-transfer RNA synthetase in the mutant. This enzyme activity in the mutant was distinguishable from the wild type by its rapid inactivation at 28 °C in cell-free extracts. Infection of the temperature-sensitive mutant with bacteriophage T4 did not alter the heat-sensitivity of the mutant enzyme. The mutation is 72% cotransducible with the xyl locus.

scholarly journals Functional Analysis of an Acyltransferase-Like Domain from Polyunsaturated Fatty Acid Synthase in Thraustochytrium

2021 ◽  
Vol 9 (3) ◽  
pp. 626
Author(s):  
Carla Almendáriz-Palacios ◽  
Dauenpen Meesapyodsuk ◽  
Xiao Qiu
Keyword(s):  
Fatty Acids ◽  
Polyunsaturated Fatty Acids ◽  
Free Fatty Acids ◽  
Fatty Acid Synthase ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Complementation Test ◽  
Temperature Sensitive ◽  
Synthetase Activity

Biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFA) such as docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) and docosapentaenoic acid (DPA, 22:5-4,7,10,13,16) in protist Thraustochytrium is catalyzed by a polyunsaturated fatty acids (PUFA) synthase comprising three large subunits, each with multiple catalytic domains. This study used complementation test, in vitro assays, and functional expression to characterize an acyltransferase (AT)-like domain in Subunit-B of a PUFA synthase from Thraustochytrium. Complementation test in Escherichia coli showed that the AT-like domain could not restore the growth phenotype of a temperature-sensitive mutant (∆fabDts) defective in malonyl-CoA:ACP transacylase activity. In vitro assays showed that the AT-like domain possessed thioesterase activity towards a few acyl-CoAs tested where docosahexaenoyl-CoA (DHA-CoA) was the preferred substrate. Expression of this domain in an E. coli mutant (∆fadD) defective in acyl-CoA synthetase activity resulted in the increased accumulation of free fatty acids. Site-directed mutagenesis showed that the substitution of two putative active site residues, serine at 96 (S96) and histidine at 220 (H220), in the AT-like domain significantly reduced its activity towards DHA-CoA and accumulation of free fatty acids in the ∆fadD mutant. These results indicate that the AT-like domain of the PUFA synthase does not function as a malonyl-CoA:ACP transacylase, rather it functions as a thioesterase. It might catalyze the last step of the VLCPUFA biosynthesis by releasing freshly synthesized VLCPUFAs attached to ACP domains of the PUFA synthase in Thraustochytrium.

In vitro biosynthesis of functional Escherichia coli su3+tyrosine transfer RNA

Biochemistry ◽  
1977 ◽  
Vol 16 (16) ◽  
pp. 3608-3618 ◽  
Author(s):  
Maurille J. Fournier ◽  
Elizabeth Webb ◽  
Shirley Tang
Keyword(s):  
Escherichia Coli ◽  
Transfer Rna ◽  
In Vitro Biosynthesis

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 Cnu, a Novel oriC-Binding Protein of Escherichia coli

2005 ◽  
Vol 187 (20) ◽  
pp. 6998-7008 ◽  
Author(s):  
Myung Suk Kim ◽  
Sung-Hun Bae ◽  
Sang Hoon Yun ◽  
Hee Jung Lee ◽  
Sang Chun Ji ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid Identity ◽  
Replication Initiation ◽  
In Vitro Assays ◽  
Wild Type ◽  
Genetic Method ◽  
Dna Replication Initiation ◽  
Pair Sequence

ABSTRACT We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

scholarly journals Lowering S-Adenosylmethionine Levels inEscherichia coli Modulates C-to-T Transition Mutations

2001 ◽  
Vol 183 (3) ◽  
pp. 921-927 ◽  
Author(s):  
Georgina Macintyre ◽  
C. Victoria Atwood ◽  
Claire G. Cupples
Keyword(s):  
Escherichia Coli ◽  
Dna Methylation ◽  
Genomic Dna ◽  
Synthetase Activity ◽  
Inescherichia Coli ◽  
Genome Methylation ◽  
Sam Synthetase ◽  
Methyl Cytosine

ABSTRACT Deoxycytosine methylase (Dcm) enzyme activity causes mutagenesis in vitro either directly by enzyme-induced deamination of cytosine to uracil in the absence of the methyl donor,S-adenosylmethionine (SAM), or indirectly through spontaneous deamination of [5-methyl]cytosine to thymine. Using a Lac reversion assay, we investigated the contribution of the first mechanism to Dcm mutagenesis in vivo by lowering the levels of SAM.Escherichia coli SAM levels were lowered by reducing SAM synthetase activity via the introduction of a metK84 allele or by hydrolyzing SAM using the bacteriophage T3 SAM hydrolase. ThemetK84 strains exhibited increased C-to-T mutagenesis. Expression of the T3 SAM hydrolase gene, under the control of the arabinose-inducible PBAD promoter, effectively reduced Dcm-mediated genomic DNA methylation. However, increased mutagenesis was not observed until extremely high arabinose concentrations were used, and genome methylation at Dcm sites was negligible.

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