tRNA synthetase mutants of Escherichia coli K-12 are resistant to the gyrase inhibitor novobiocin. (1/81)

In previous studies we demonstrated that mutations in the genes cysB, cysE, and cls (nov) affect resistance of Escherichia coli to novobiocin (J. Rakonjac, M. Milic, and D. J. Savic, Mol. Gen. Genet. 228:307-311, 1991; R. Ivanisevic, M. Milic, D. Ajdic, J. Rakonjac, and D. J. Savic, J. Bacteriol. 177:1766-1771, 1995). In this work we expand this list with mutations in rpoN (the gene for RNA polymerase subunit sigma54) and the tRNA synthetase genes alaS, argS, ileS, and leuS. Similarly to resistance to the penicillin antibiotic mecillinam, resistance to novobiocin of tRNA synthetase mutants appears to depend upon the RelA-mediated stringent response. However, at this point the overlapping pathways of mecillinam and novobiocin resistance diverge. Under conditions of stringent response induction, either by the presence of tRNA synthetase mutations or by constitutive production of RelA protein, inactivation of the cls gene diminishes resistance to novobiocin but not to mecillinam.  (+info)

SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of SsrA (tmRNA). (2/81)

In bacteria, SsrA RNA recognizes ribosomes stalled on defective messages and acts as a tRNA and mRNA to mediate the addition of a short peptide tag to the C-terminus of the partially synthesized nascent polypeptide chain. The SsrA-tagged protein is then degraded by C-terminal-specific proteases. SmpB, a unique RNA-binding protein that is conserved throughout the bacterial kingdom, is shown here to be an essential component of the SsrA quality-control system. Deletion of the smpB gene in Escherichia coli results in the same phenotypes observed in ssrA-defective cells, including a variety of phage development defects and the failure to tag proteins translated from defective mRNAs. Purified SmpB binds specifically and with high affinity to SsrA RNA and is required for stable association of SsrA with ribosomes in vivo. Formation of an SmpB-SsrA complex appears to be critical in mediating SsrA activity after aminoacylation with alanine but prior to the transpeptidation reaction that couples this alanine to the nascent chain. SsrA RNA is present at wild-type levels in the smpB mutant arguing against a model of SsrA action that involves direct competition for transcription factors.  (+info)

Single-nucleotide polymorphisms can cause different structural folds of mRNA. (3/81)

Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation in man. Genes containing one or more SNPs can give rise to two or more allelic forms of mRNAs. These mRNA variants may possess different biological functions as a result of differences in primary or higher order structures that interact with other cellular components. Here we report the observation of marked differences in mRNA secondary structure associated with SNPs in the coding regions of two human mRNAs: alanyl tRNA synthetase and replication protein A, 70-kDa subunit (RPA70). Enzymatic probing of SNP-containing allelic fragments of the mRNAs revealed pronounced allelic differences in cleavage pattern at sites 14 or 18 nt away from the SNP, suggesting that a single-nucleotide variation can give rise to different mRNA folds. By using phosphorothioate oligodeoxyribonucleotides complementary to the region of different allelic structures in the RPA70 mRNA, but not extending to the SNP itself, we find that the SNP exerts an allele-specific effect on the accessibility of its flanking site in the endogenous human RPA70 mRNA. This further supports the allele-specific structural features identified by enzymatic probing. These results demonstrate the contribution of common genetic variation to structural diversity of mRNA and suggest a broader role than previously thought for the effects of SNPs on mRNA structure and, ultimately, biological function.  (+info)

CDC64 encodes cytoplasmic alanyl-tRNA synthetase, Ala1p, of Saccharomyces cerevisiae. (4/81)

The cdc64-1 mutation causes G(1) arrest in Saccharomyces cerevisiae corresponding to a type II Start phenotype. We report that CDC64 encodes Ala1p, an alanyl-tRNA synthetase. Thus, cdc64-1 might affect charging of tRNA(Ala) and thereby initiation of cell division.  (+info)

Identification of discriminator base atomic groups that modulate the alanine aminoacylation reaction. (5/81)

Specific aminoacylation of tRNAs involves activation of an amino acid with ATP followed by amino acid transfer to the tRNA. Previous work showed that the transfer of alanine from Escherichia coli alanyl-tRNA synthetase to a cognate RNA minihelix involves a transition state sensitive to changes in the tRNA acceptor stem. Specifically, the "discriminator" base at position 73 of minihelix(Ala) is a critical determinant of the transfer step of aminoacylation. This single-stranded nucleotide has previously been shown by solution NMR to be stacked predominantly onto G(1) of the first base pair of the alanine acceptor stem helix. In this work, RNA duplex(Ala) variants were prepared to investigate the role of specific discriminator base atomic groups in aminoacylation catalytic efficiency. Results indicate that the purine structure appears to be important for stabilization of the transition state and that major groove elements are more critical than those located in the minor groove. This result is in accordance with the predicted orientation of a class II synthetase at the end of the acceptor helix. In particular, substitution of the exocyclic amino group of A(73) with a keto-oxygen resulted in negative discrimination at this site. Taken together, these new results are consistent with the involvement of major groove atomic groups of the discriminator base in the formation of the transition state for the amino acid transfer step.  (+info)

Expression of Arabidopsis thaliana mitochondrial alanyl-tRNA synthetase is not sufficient to trigger mitochondrial import of tRNAAla in yeast. (6/81)

It has often been suggested that precursors to mitochondrial aminoacyl-tRNA synthetases are likely carriers for mitochondrial import of tRNAs in those organisms where this process occurs. In plants, it has been shown that mutation of U(70) to C(70) in Arabidopsis thaliana tRNA(Ala)(UGC) blocks aminoacylation and also prevents import of the tRNA into mitochondria. This suggests that interaction of tRNA(Ala) with alanyl-tRNA synthetase (AlaRS) is necessary for import to occur. To test whether this interaction is sufficient to drive import, we co-expressed A. thaliana tRNA(Ala)(UGC) and the precursor to the A. thaliana mitochondrial AlaRS in Saccharomyces cerevisiae. The A. thaliana enzyme and its cognate tRNA were correctly expressed in yeast in vivo. However, although the plant AlaRS was efficiently imported into mitochondria in the transformed strains, we found no evidence for import of the A. thaliana tRNA(Ala) nor of the endogenous cytosolic tRNA(Ala) isoacceptors. We conclude that at least one other factor besides the mitochondrial AlaRS precursor must be involved in mitochondrial import of tRNA(Ala) in plants.  (+info)

Importance of discriminator base stacking interactions: molecular dynamics analysis of A73 microhelix(Ala) variants. (7/81)

Transfer of alanine from Escherichia coli alanyl-tRNA synthetase (AlaRS) to RNA minihelices that mimic the amino acid acceptor stem of tRNA(Ala) has been shown, by analysis of variant minihelix aminoacylation activities, to involve a transition state sensitive to changes in the 'discriminator' base at position 73. Solution NMR has indicated that this single-stranded nucleotide is predominantly stacked onto G1 of the first base pair of the alanine acceptor stem helix. We report the activity of a new variant with the adenine at position 73 substituted by its non-polar isostere 4-methylindole (M). Despite lacking N7, this analog is well tolerated by AlaRS. Molecular dynamics (MD) simulations show that the M substitution improves position 73 base stacking over G1, as measured by a stacking lifetime analysis. Additional MD simulations of wild-type microhelix(Ala) and six variants reveal a positive correlation between N73 base stacking propensity over G1 and aminoacylation activity. For the two DeltaN7 variants simulated we found that the propensity to stack over G1 was similar to the analogous variants that contain N7 and we conclude that the decrease in aminoacylation efficiency observed upon deletion of N7 is likely due to loss of a direct stabilizing interaction with the synthetase.  (+info)

Origin of mitochondria in relation to evolutionary history of eukaryotic alanyl-tRNA synthetase. (8/81)

The origin of the eukaryotic cell remains an unsolved question. Numerous experimental and phylogenetic observations support the symbiotic origin of the modern eukaryotic cell, with its nucleus and (typically) mitochondria. Incorporation of mitochondria has been proposed to precede development of the nucleus, but it is still unclear whether mitochondria were initially part of basal eukaryotes. Data on alanyl-tRNA synthetase from an early eukaryote and other sources are presented and analyzed here. These data are consistent with the notion that mitochondrial genesis did not significantly precede nucleus formation. Moreover, the data raise the possibility that diplomonads are primary amitochondriates that radiated from the eukaryotic lineage before mitochondria became fully integrated as a cellular organelle.  (+info)

• 1
• 2
• 3
• 4
• 5
• 6
• 7
• 8
• 9
• 10