The heterotrimeric Thermus thermophilus Asp-tRNAAsn amidotransferase can also generate Gln-tRNAGln

AbstractThermus thermophilus strain HB8 is known to have a heterodimeric aspartyl-tRNAAsn amidotransferase (Asp-AdT) capable of forming Asn-tRNAAsn [Becker, H.D. and Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832–12837]. Here we show that, like other bacteria, T. thermophilus possesses the canonical set of amidotransferase (AdT) genes (gatA, gatB and gatC). We cloned and sequenced these genes, and constructed an artificial operon for overexpression in Escherichia coli of the thermophilic holoenzyme. The overproduced T. thermophilus AdT can generate Gln-tRNAGln as well as Asn-tRNAAsn. Thus, the T. thermophilus tRNA-dependent AdT is a dual-specific Asp/Glu-AdT resembling other bacterial AdTs. In addition, we observed that removal of the 44 carboxy-terminal amino acids of the GatA subunit only inhibits the Asp-AdT activity, leaving the Glu-AdT activity of the mutant AdT unaltered; this shows that Asp-AdT and Glu-AdT activities can be mechanistically separated

The COMBREX Project: Design, Methodology, and Initial Results

© 2013 Brian P. et al.Prior to the “genomic era,” when the acquisition of DNA sequence involved significant labor and expense, the sequencing of genes was strongly linked to the experimental characterization of their products. Sequencing at that time directly resulted from the need to understand an experimentally determined phenotype or biochemical activity. Now that DNA sequencing has become orders of magnitude faster and less expensive, focus has shifted to sequencing entire genomes. Since biochemistry and genetics have not, by and large, enjoyed the same improvement of scale, public sequence repositories now predominantly contain putative protein sequences for which there is no direct experimental evidence of function. Computational approaches attempt to leverage evidence associated with the ever-smaller fraction of experimentally analyzed proteins to predict function for these putative proteins. Maximizing our understanding of function over the universe of proteins in toto requires not only robust computational methods of inference but also a judicious allocation of experimental resources, focusing on proteins whose experimental characterization will maximize the number and accuracy of follow-on predictions.COMBREX is funded by a GO grant from the National Institute of General Medical Sciences (NIGMS) (1RC2GM092602-01).Peer Reviewe

The amino acid substitution and some chemical properties of a variant human erythrocyte carbonic anhydrase: Carbonic anhydrase Id Michigan

Carbonic anhydrase Id Michigan , an electrophoretic variant of human red cell carbonic anhydrase I, was purified from erythrocytes obtained from an individual heterozygous for the trait. Primary structural analysis indicates that a lysine residue has exchanged for a threonine residue in the variant enzyme. After isolation, there was approximately 1.8 times as much normal as variant enzyme. Thermostability studies demonstrated that carbonic anhydrase Id was more thermolabile than the normal enzyme. The normal and variant enzymes showed no differences in specific carboxylesterase activity or CO 2 hydratase activity. Utilizing the carboxylesterase activity toward β-naphthyl acetate, the normal and variant enzymes had similar Michaelis constants, p H profiles, and rates of inhibition by acetazolamide. Immunochemical studies did not demonstrate an antigenic difference for the variant enzyme.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44117/1/10528_2004_Article_BF00486517.pd

The developmental pattern of homologous and heterologous tRNA methylation in rat brain differential effect of spermidine

Using S -adenosyl- L -[Me- 14 C] methionine, rat cerebral cortex methyltransferase activity was determined during the early postnatal period in the absence of added Escherichia coli tRNA and in its presence. [Me- 14 C] tRNA was purified from both systems and its [Me- 14 C] base composition determined. The endogenous formation of [Me- 14 C] tRNA (homologous tRNA methylation) was totally abolished in the presence of 2.5 mM spermidine, whereas E. coli B tRNA methylation (heterologous methylation) was markedly stimulated. Only [Me- 14 C] 1-methyl guanine and [Me- 14 C] N 2 -methyl guanine were formed by homologous methylation, there being an inverse shift in their relative proportions with age. Heterologous tRNA methylation led, additionally, to the formation of [Me- 14 C] N 2 2 -dimethyl guanine, 5-methyl cytosine, 1-methyl adenine, 5-methyl uracil, 2-methyl adenine, and 1-methyl hypoxanthine. A comparison of heterologous tRNA methylation between the whole brain cortex (containing nerve and glial cells) and bulk-isolated nerve cell bodies revealed markedly lower proportions of [Me- 14 C] N 2 -methyl and N 2 2 -dimethyl guanine and significantly higher proportions of [Me- 14 C] 1-methyl adenine in the neurons. The present findings suggest (1) that homologous tRNA methylation may provide developing brain cells with continuously changing populations of tRNA and (2) that neurons are enriched in adenine residue-specific tRNA methyltransferases that are highly sensitive to spermidine.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45399/1/11064_2004_Article_BF00966229.pd

Analogs of methionyl-tRNA synthetase substrates containing photolabile groups.

Three photolabile analogs of substrates of methionyl-tRNA synthetase were synthesized. In one, the 4-thiouridine at the 8 position of E. coli tRNAfMet was alkylated with [14C]p-azidobromoacetanilide. In the second, [14C]p-azidobenzoic acid hydrazide was condensed with the 3'-terminal dialdehyde of periodate-oxidized Escherichia coli tRNAfMet. The modified tRNAs could be purified by chromatography on benzoylated DEAE-cellulose. The third photolabile compound was [3H]methioninyl-8-azido-adenosine 5'-phosphate, an analog of the methionyl adenylate intermediate in the aminoacylation reaction. Irradiation of each of these compounds in the presence of equimolar amounts of E. coli methionyl-tRNA synthetase of micrometer concentrations gave 5-15% crosslinking