Molecular evolution: aminoacyl-tRNA synthetases on the loose. (25/1309)

Modified versions - paralogs - of the catalytic domain of at least three different aminoacyl-tRNA synthetases have been found to serve catalytic or regulatory roles in other reactions. These findings suggest that the first modern tRNA-synthetases could have been derived from amino-acid biosynthetic enzymes.  (+info)

Nuclear tRNA aminoacylation and its role in nuclear export of endogenous tRNAs in Saccharomyces cerevisiae. (26/1309)

Nuclear tRNA aminoacylation was proposed to provide a proofreading step in Xenopus oocytes, ensuring nuclear export of functional tRNAs [Lund, E. & Dahlberg, J. E. (1998) Science 282, 2082-2085]. Herein, it is documented that tRNA aminoacylation also occurs in yeast nuclei and is important for tRNA export. We propose that tRNA aminoacylation functions in one of at least two parallel paths of tRNA export in yeast. Alteration of one aminoacyl-tRNA synthetase affects export of only cognate tRNA, whereas alterations of two other aminoacyl-tRNA synthetases affect export of both cognate and noncognate tRNAs. Saturation of tRNA export pathway is a possible explanation of this phenomenon.  (+info)

Global transposon mutagenesis and a minimal Mycoplasma genome. (27/1309)

Mycoplasma genitalium with 517 genes has the smallest gene complement of any independently replicating cell so far identified. Global transposon mutagenesis was used to identify nonessential genes in an effort to learn whether the naturally occurring gene complement is a true minimal genome under laboratory growth conditions. The positions of 2209 transposon insertions in the completely sequenced genomes of M. genitalium and its close relative M. pneumoniae were determined by sequencing across the junction of the transposon and the genomic DNA. These junctions defined 1354 distinct sites of insertion that were not lethal. The analysis suggests that 265 to 350 of the 480 protein-coding genes of M. genitalium are essential under laboratory growth conditions, including about 100 genes of unknown function.  (+info)

Aminoacyl-tRNA synthetases database Y2K. (28/1309)

The aminoacyl-tRNA synthetases (AARS) are a diverse group of enzymes that ensure the fidelity of transfer of genetic information from DNA into protein. They catalyse the attachment of amino acids to transfer RNAs and thereby establish the rules of the genetic code by virtue of matching the nucleotide triplet of the anticodon with its cognate amino acid. Currently, 818 AARS primary structures have been reported from archaebacteria, eubacteria, mitochondria, chloro-plasts and eukaryotic cells. The database is a compilation of the amino acid sequences of all AARSs, known to date, which are available as separate entries or alignments of related proteins via the WWW at http://rose.man.poznan.pl/aars/index.html  (+info)

A single base substitution in the variable pocket of yeast tRNA(Arg) eliminates species-specific aminoacylation. (29/1309)

Early biochemical data showed that aminoacyl-tRNA synthetases often displayed species-specific recognition of tRNA. We compared the ability of purified Saccharomyces cerevisiae and Escherichia coli arginyl-tRNA synthetases to aminoacylate native and transcribed yeast tRNA(Arg) as well as E. coli tRNA(Arg). The kinetic data revealed that yeast ArgRS could charge E. coli tRNA(Arg), but at a lower efficiency than it charged either the transcribed or native yeast tRNA(Arg). E. coli ArgRS can acylate only its cognate E. coli tRNA. Strikingly, a single base change from C to A at position 20 in yeast tRNA(3)(Arg) altered the species specificity. The transcript of yeast tRNA(3)(Arg)CA20 mutant was aminoacylated by E. coli ArgRS with a 10(6) increase in k(cat)/K(m) over that for aminoacylation of yeast tRNA(3)(Arg) transcript. This indicates that A20 is not only an important identity of E. coli tRNA(Arg), but is also the key to altering species-specific aminoacylation of yeast tRNA(Arg).  (+info)

Direct experimental evidence for kinetic proofreading in amino acylation of tRNAIle. (30/1309)

Kinetic proofreading is a reaction scheme with a structure more complicated than that of Michaelis kinetics, which leads to a proofreading for errors in the recognition of a correct substrate by an enzyme. We have measured the stoichiometry between ATP hydrolysis and tRNAIle charging, using the enzyme isoleucyl-tRNA synthetase [L-isoleucine:tRNAIle ligase (AMP-forming), EC 6.1.1.5] and the amino acids isoleucine (correct) and valine (incorrect). The enzymatic deacylation of charged tRNA, which would normally prevent meaningful stoichiometry studies, was eliminated by the use of transfer factor Tu-GTP, (which binds strongly to charged tRNA) in the reaction mixture. For isoleucine, 1.5 ATP molecules are hydrolyzed per tRNA charged, but for valine, 270. These stoichiometry ratios are fundamental to kinetic proofreading, for the energy coupling is essential and proofreading is obtained only by departing from 1:1 stoichiometry between energy coupling and product formation. Within the known reaction pathway, these ratios demonstrate that kinetic proofreading induces a reduction in errors by a factor of 1/180. An overall error rate of about 10(-4) for tRNA charging is obtained by a kinetic proofreading using a fundamental discrimination level of about 10(-2), and is compatible with the low in vivo error rate of protein synthesis.  (+info)

Drug target validation: lethal infection blocked by inducible peptide. (31/1309)

Genome projects are generating large numbers of potential new targets for drug discovery. One challenge is target validation, proving the usefulness of a specific target in an animal model. In this paper, we demonstrate a new approach to validation and assay development. We selected in vitro specific peptide binders to a potential pathogen target. By inducing the expression of a selected peptide in pathogen cells causing a lethal infection in mice, the animals were rescued. Thus, by combining in vitro selection methods for peptide binders with inducible expression in animals, the target's validity was rigorously tested and demonstrated. This approach to validation can be generalized and has the potential to become a valuable tool in the drug discovery process.  (+info)

One polypeptide with two aminoacyl-tRNA synthetase activities. (32/1309)

The genome sequences of certain archaea do not contain recognizable cysteinyl-transfer RNA (tRNA) synthetases, which are essential for messenger RNA-encoded protein synthesis. However, a single cysteinyl-tRNA synthetase activity was detected and purified from one such organism, Methanococcus jannaschii. The amino-terminal sequence of this protein corresponded to the predicted sequence of prolyl-tRNA synthetase. Biochemical and genetic analyses indicated that this archaeal form of prolyl-tRNA synthetase can synthesize both cysteinyl-tRNA(Cys) and prolyl-tRNA(Pro). The ability of one enzyme to provide two aminoacyl-tRNAs for protein synthesis raises questions about concepts of substrate specificity in protein synthesis and may provide insights into the evolutionary origins of this process.  (+info)