Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The biochemical basis of suppression of a temperature-sensitive alanyl-tRNA synthetase (alaS) mutation by mutational alterations of the ribosome has been investigated. Measurement of the polyU-dependent polyphenylalanine synthesis showed that ribosomes from the suppressor strains are less active than ribosomes from the unsuppressed aminoacyl-tRNA synthetase mutant. In this system no increased translational ambiguity could be detected for the suppressor ribosomes. This fact and also the findings that the ram-1 mutation is not able to suppress the aminoacyl-tRNA synthetase mutation and that presence of the suppressor allele is not accompanied by a measureably improved alanyl-tRNA synthetase activity argue against the possibility that suppression might be due to increased translational misreading rates of the alanyl-tRNA synthetase mRNA. It has been further found that partial suppression of temperature sensitive growth of the alaS mutation can be achieved by independent ribosomal mutations leading to reduced growth rates because of a mutation to antibiotic resistance. Addition of low concentrations of a variety of antibiotics acting at the ribosomal level can also partially revert the temperature-sensitive phenotype of the alaS mutant. Although the possibility cannot be excluded that suppression is due to the stabilisation or activation of the mutant enzyme by some indirect effect of the suppressor ribosomal mutations, the following working hypothesis is favoured at the moment: It is assumed that limitation of the aminoacyl-tRNA synthetase activity in a certain range of the restrictive temperature causes growth inhibition by the premature termination of polypeptide synthesis at the ribosome or by the unbalanced synthesis of the individual cellular proteins under this condition. The mechanism of suppression by ribosomal mutations is proposed to consist of the release of this growth inhibition by the reduction of the rate of polypeptide synthesis, which would keep amino acid incorporation from exceeding the slow charging of tRNA and thus exhausting the pool of charged tRNA. In the suppressor strains, therefore, growth at the semi-restrictive temperature is no longer limited by the aminoacylation of tRNA but by the translational process at the mutated ribosome. This influence of the ribosomal mutation on the speed of translation could be directly or indirectly coupled with an effect on translational fidelity resulting in the prevention of the binding of uncharged or non-cognate charged tRNA or in the tighter binding of peptidyl-tRNA when cognate aminoacyl-tRNA is limiting.
 ...
PMID:Suppression of temperature-sensitive aminoacyl-tRNA synthetase mutations by ribosomal mutations: a possible mechanism. 79 71

Escherichia coli alanyl-tRNA synthetase contains the sequence Cys-X2-Cys-X6-His-X2-His. This motif is distinct from the zinc fingers of DNA-binding proteins but has some similarity to the Cys-X2-Cys-X4-His-X4-Cys zinc-binding motif of retroviral gag proteins, where it has a role in RNA packaging. In Ala-tRNA synthetase, this sequence is located in an amino-terminal domain which has the site for docking the acceptor end of the tRNA near the bound aminoacyl adenylate and is immediately adjacent in the sequence to the location of a mutation that affects the specificity of tRNA recognition. We show here that Ala-tRNA synthetase contains approximately 1 mol of zinc/mol of polypeptide and that addition of the zinc chelator 1,10-phenanthroline inhibits its aminoacylation activity. Conservative mutations of specific cysteine or histidine residues in the "Cys-His box" destabilize and inactivate the enzyme, whereas mutations of intervening amino acids do not inactivate. The possibility that this motif can bind zinc (or cobalt) was demonstrated with a synthetic 22 amino acid peptide that is based on the sequence of the alanine enzyme. The peptide-cobalt complex has the spectral characteristics of tetrahedral coordination geometry. The results establish that the Cys-His box motif of Ala-tRNA synthetase has the potential to form a specific complex with zinc (at least in the context of a synthetic peptide analogue) and suggest that this motif is important for enzyme stability/activity.
 ...
PMID:Evidence for a "cysteine-histidine box" metal-binding site in an Escherichia coli aminoacyl-tRNA synthetase. 171 32

Many RNAs are complex, globular molecules formed from elements of secondary and tertiary structure analogous to those found in proteins. Little is known about recognition of RNAs by proteins. In the case of transfer RNAs (tRNAs), considerable evidence suggests that elements dispersed in both the one- and three-dimensional structure are important for recognition by aminoacyl tRNA synthetases. Fragments of alanine tRNA synthetase were created by in vitro manipulations of the cloned alaS gene and examined for their interaction with alanine-specific tRNA. Sequences essential for recognition were located near the middle of the polypeptide, juxtaposed to the carboxyl-terminal side of the domain for aminoacyl adenylate synthesis. The most essential part of the tRNA interaction strength and specificity was dependent on a sequence of fewer than 100 amino acids. Within this sequence, and in the context of the proper conformation, a segment of no more than 17 amino acids was responsible for 25% or more of the total synthetase-tRNA free energy of association. The results raise the possibility that an important part of specific RNA recognition by an aminoacyl tRNA synthetase involves a polypeptide segment that is short relative to the total size of the protein.
 ...
PMID:Polypeptide sequences essential for RNA recognition by an enzyme. 243 5

The nrdB gene of Escherichia coli, coding for the B2 protein of ribonucleotide reductase, has been cloned in a runaway-replication vector. The runaway derivative pBEU17 carries the promoter-proximal portion of the E. coli alanyl-tRNA synthetase gene and proved useful for expressing cloned genes lacking their native transcription initiation signals. The alaS promoter is located approximately 500 base pairs upstream of a single BamHI restriction endonuclease cleavage site utilized in the construction of an expression recombinant plasmid, pBS1, for the nrdB product. After 5-h thermal induction of cells carrying the runaway recombinant pBS1, protein B2 constituted 40% of the soluble protein fraction of the cells. The high concentration of protein B2 in crude extracts of induced cells has enabled a simplified purification scheme to be developed for production of homogeneous and concentrated B2 preparations. Protein B2 produced from pBS1 is identical to the chromosomally encoded nrdB product of E. coli as regards molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme activity, tyrosine radical content, and structure of the binuclear iron center. Amino acid sequence analysis showed that the two polypeptide chains of protein B2 are identical. They start with an alanine residue, and the first 30 residues confirmed the amino acid sequence predicted from the nucleotide sequence of the nrdB gene, apart from an NH2-terminal processing removal of the initiator methionine.
 ...
PMID:Overproduction and purification of the B2 subunit of ribonucleotide reductase from Escherichia coli. 300 19

Two previously described chromosomal mutant alleles, alaS4 and alaS5, of Escherichia coli Ala-tRNA synthetase have been analyzed. Each causes a sharp diminution in aminoacylation activity and disrupts the alpha 4 tetramer structure of identical chains of 875 amino acids; neither mutation significantly disturbs the activity for synthesis of alanyladenylate. The location of each mutation within the structural gene has been mapped by marker rescue with specific gene fragments. Each mutant allele was cloned from the genome by reciprocal recombination with a multicopy plasmid that contains segments of alaS which flank the respective mutations. Further analysis established: 1) a single G----A transition results in a Gly----Asp change for each mutant allele at codon 674 (alaS4) and at codon 677 (alaS5). 2) The mutations are in the oligomerization domain, about 200 amino acids beyond the C-terminal side of the catalytic domain that previously was mapped by deletion analysis; the mutations are, thus, in a part of the polypeptide which is dispensable for catalytic activity. 3) For both mutant enzymes, there is little effect of the mutation on the Km for tRNAAla; kcat for aminoacylation is decreased by an order of magnitude. These point mutations reveal a subtle integration of the catalytic core with parts of the polypeptide that are not essential for catalytic activity.
 ...
PMID:Two mutations in the dispensable part of alanine tRNA synthetase which affect the catalytic activity. 388 89

Gene deletions show that much of Escherichia coli alanine tRNA synthetase is dispensable for each of three activities and that these activities appear to require specific domains arranged linearly along the polypeptide. Thus, variable fusions of extra polypeptide domains to a catalytic core may account for the diverse of aminoacyl tRNA synthetases.
 ...
PMID:Modular arrangement of functional domains along the sequence of an aminoacyl tRNA synthetase. 635 98

Alanyl- and leucyl-tRNA synthetases from baker's yeast were purified to homogeneity in the presence of the protease inhibitor phenylmethylsulfonyl fluoride. Both consist of single polypeptide chains of 118 000 and 125 000 daltons, respectively, as determined by polyacrylamide gel electrophoresis under denaturing conditions. The monomeric structure of leucyl-tRNA synthetase differs from the dimeric one obtained previously in the absence of protease inhibitors. This illustrates the sensitivity of the synthetases to proteolytic actions and indicates that native structures can only be obtained under optimal protecting conditions. Alanyl- and leucyl-tRNA synthetases differ with respect to pH optimum (6.5 and 8.5, respectively), Michaelis constant for amino acid (1 mM and 0.03, respectively) and in the rate-limiting step for the tRNA aminoacylation reaction. Whereas the catalytic step itself was rate-limiting for alanyl-tRNA synthetase, a step occurring after this was rate-limiting for leucyl-tRNA synthetase.
 ...
PMID:Purification and some properties of alanyl- and leucyl-tRNA synthetases from baker's yeast. 701 9

The class II Escherichia coli and human alanyl-tRNA synthetases cross-acylate their respective tRNAs and require, for aminoacylation, an acceptor helix G3:U70 base pair that is conserved in evolution. We report here the primary structure and expression in the yeast Pichia of an active human alanyl-tRNA synthetase. The N-terminal 498 amino acids of the 968-residue polypeptide have substantial (41%) identity with the E. coli protein. A closely related region encompasses the class-defining domain of the E. coli enzyme and includes the part needed for recognition of the acceptor helix. As a result, previously reported mutagenesis, modeling, domain organization, and biochemical characterization on the E. coli protein appear valid as a template for the human protein. In particular, we show that both the E. coli enzyme and the human enzyme purified from Pichia aminoacylate 9-base pair RNA duplexes whose sequences are based on the acceptor stems of either E. coli or human alanine tRNAs. In contrast, the sequences of the two enzymes completely diverge in an internal portion of the C-terminal half that is essential for tetramer formation by the E. coli enzyme, but that is dispensable for microhelix aminoacylation. This divergence correlates with the expressed human enzyme behaving as a monomer. Thus, the region of close sequence similarity may be a consequence of strong selective pressure to conserve the acceptor helix G3:U70 base pair as an RNA signal for alanine.
 ...
PMID:Human alanyl-tRNA synthetase: conservation in evolution of catalytic core and microhelix recognition. 765 87

The class-defining active site domain of the 10 class II tRNA synthetases is well conserved and, based on the crystal structure of aspartyl-tRNA synthetase, approaches the end of the tRNA acceptor stem from the major groove side of the helix. Paradoxically, for the class II alanyl-tRNA synthetase (AlaRS), aminoacylation is dependent on minor groove recognition of an acceptor helix G3.U70 base pair. Additional contributions to aminoacylation efficiency are made by the A73 "discriminator" base and G2.C71 base pair located at the end of the acceptor stem. Using microhelix substrates containing only the first four base pairs of the alanine tRNA acceptor helix, we demonstrated that the catalytic center of AlaRS with the three class-defining sequence motifs contains determinants for recognition of A73 and G2.C71. However, this structural unit does not discriminate between different base pairs at the critical 3.70 position. Discrimination at G3.U70 was mapped to a 76 amino acid polypeptide outside the catalytic center. We propose that the G3.U70 recognition motif is a structural appendage that folds back to the catalytic center to make contact with the bound acceptor stem. A "fold-back" appendage provides a specific mechanism for minor groove recognition of the acceptor helix by a class II tRNA synthetase.
 ...
PMID:Minor groove recognition of the critical acceptor helix base pair by an appended module of a class II tRNA synthetase. 774 3

In plants, all aminoacyl-tRNA synthetases are nuclearly encoded, despite the fact that their activities are required in the three protein-synthesizing cell compartments (cytosol, mitochondria, and chloroplasts). To investigate targeting of these enzymes, we cloned cDNAs encoding alanyl-tRNA synthetase (AlaRS) and the corresponding nuclear gene, ALATS, from Arabidopsis by using degenerate polymerase chain reaction primers based on highly conserved regions shared between known AlaRSs from other organisms. Analysis of the transcription of the gene showed the presence of two potential translation initiation codons in some ALATS mRNAs. Translation from the upstream AUG would generate an N-terminal extension with features characteristic of mitochondrial targeting peptides. A polyclonal antibody raised against part of the Arabidopsis AlaRS revealed that the Arabidopsis cytosolic and mitochondrial AlaRSs are immunologically similar, suggesting that both isoforms are encoded by the ALATS gene. In vitro experiments confirmed that two polypeptides can be translated from AlATS transcripts, with most ribosomes initiating on the downstream AUG to give the shorter polypeptide corresponding in size to the cytosolic enzyme. The ability of the presequence encoded between the two initiation codons to direct polypeptides to mitochondria was demonstrated by expression of fusion proteins in tobacco protoplasts and in yeast. We conclude that the ALATS gene encodes both the cytosolic and the mitochondrial forms of AlaRS, depending on which of the two AUG codons is used to initiate translation.
 ...
PMID:The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases. 867 89


1 2 Next >>