A domain in the N-terminal extension of class IIb eukaryotic aminoacyl-tRNA synthetases is important for tRNA binding.
(41/1309)
Cytoplasmic aspartyl-tRNA synthetase (AspRS) from Saccharomyces cerevisiae is a homodimer of 64 kDa subunits. Previous studies have emphasized the high sensitivity of the N-terminal region to proteolytic cleavage, leading to truncated species that have lost the first 20-70 residues but that retain enzymatic activity and dimeric structure. In this work, we demonstrate that the N-terminal extension in yeast AspRS participates in tRNA binding and we generalize this finding to eukaryotic class IIb aminoacyl-tRNA synthetases. By gel retardation studies and footprinting experiments on yeast tRNA(Asp), we show that the extension, connected to the anticodon-binding module of the synthetase, contacts tRNA on the minor groove side of its anticodon stem. Sequence comparison of eukaryotic class IIb synthetases identifies a lysine-rich 11 residue sequence ((29)LSKKALKKLQK(39) in yeast AspRS with the consensus xSKxxLKKxxK in class IIb synthetases) that is important for this binding. Direct proof of the role of this sequence comes from a mutagenesis analysis and from binding studies using the isolated peptide. (+info)
A novel anti-tumor cytokine contains an RNA binding motif present in aminoacyl-tRNA synthetases.
(42/1309)
Endothelial monocyte-activating polypeptide II (EMAP II) is a novel pro-apoptotic cytokine that shares sequence homology with the C-terminal regions of several tRNA synthetases. Pro-EMAP II, the precursor of EMAP II, is associated with the multi-tRNA synthetase complex and facilitates aminoacylation activity. The structure of human EMAP II, solved at 1.8 A resolution, revealed the oligomer-binding fold for binding different tRNAs and a domain that is structurally homologous to other chemokines. The similar structures to the RNA binding motif of EMAP II was previously observed in the anticodon binding domain of yeast Asp-tRNA synthetase (AspRSSC) and the B2 domain of Thermus thermophilus Phe-tRNA synthetase. The RNA binding pattern of EMAP II is likely to be nonspecific, in contrast to the AspRSSC. The peptide sequence that is responsible for cytokine activity is located, for the most part, in the beta1 strand. It is divided into two regions by a neighboring loop. (+info)
The structural basis for the resistance of Escherichia coli formylmethionyl transfer ribonucleic acid to cleavage by Escherichia coli peptidyl transfer ribonucleic acid hydrolase.
(43/1309)
Escherichia coli formylmethionly-tRNA-tMet is unique among N-acylaminoacyl-tRNAs in its resistance to cleavage by peptidyl-tRNA hydrolase. Chemical modification of tRNA-fMet with sodium bisulfite converts fMet-tRNA-fMet into a good substrate for the hydrolase. The products of the enzymatic cleavage are free tRNA-fMet and formylmethionine. Bisulfite treatment produces cytidine to uridine base changes at several sites in the tRNA structure. One of these modifications results in formation of a new hydrogen-bonded base pair at the end of the acceptor stem of tRNA-fMet. We have shown that this modification is responsible for the observed change in biological activity. Enzymatic cleavage appears to be facilitated by the presence of a 5-terminal phosphate at the end of a fully base-paired acceptor stem, because removal of the 5-phosphate group from N-acetylphenylalanyl-tRNA-Phe or bisulfite-modified fMet-tRNA-FMet reduced the rate of hydrolysis of these substrates. The unpaired base at the 5 terminus of unmodified fMet-tRNA-fMet appears to reduce susceptibility of the tRNA to hydrolytic attack both by positioning the 5-phosphate in an unfavorable orientation and by directly interfering with enzymatic binding. The unusual structure of the acceptor stem of this E. coli tRNA thus plays a critical role in maintaining the viability of the organism by preventing enzymatic cleavage of the fMet group from the bacterial initiator tRNA. (+info)
Purification and properties of a soluble factor required for the deoxyribonucleic acid-directed in vitro synthesis of beta-galactosidase.
(44/1309)
The DNA-directed in vitro synthesis of beta-galactosidase has been investigated in a system dependent on Escherichia coli ribosomes, a salt wash of the ribosomes, and a supernatant fraction. Fractionation of the supernatant has made it possible to obtain dependencies on RNA polymerase and another protein factor for beta-galactosidase synthesis. The other factor (called L factor) cannot be replaced by a variety of proteins known to be required for transcription and translation. It has been purified to homogeneity and has a molecular weight of approximately 65,000. Although it is required for the in vitro synthesis of beta-galactosidase, it has no effect on total DNA-dependent amino acid incorporation under the conditions of the incubation. However, total RNA synthesis is depressed by the addition of L factor in a manner similar to what is observed with rho factor could not replace L factor in beta-galactosidase synthesis. (+info)
Regulation of branched-chain aminoacyl-transfer ribonucleic acid synthetases in an ilvDAC deletion strain of Escherichia coli K-12.
(45/1309)
Valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid synthetase formation was compared in isogenic strains of Escherichia coli K-12 that differed only in that one strain carried a deletion of three genes of the ilv gene cluster, ilvD, -A, and -C. It was found that: (i) the activities of these synthetases in the deletion strain were less than those in the normal strain during growth in minimal medium supplemented with excess isoleucine, valine, and leucine, and (ii) their stability was reduced in the deletion strain during specific branched-chain amino acid limitations. The results of density-labeling experiments suggest that the in vivo stability of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid synthetases requires some product missing in the ilvDAC deletion strain. (+info)
An elongation factor-associating domain is inserted into human cysteinyl-tRNA synthetase by alternative splicing.
(46/1309)
The amino acid sequence of human cytoplasmic cysteinyl-tRNA synthetase (CRS) was examined by analyzing sequences of genomic and expressed sequence tag fragments. From theses analyses, a few interesting possibilities were suggested for the structure of human CRS. First, different isoforms of CRS may result from alternative splicing. Second, the largest one would comprise 831 amino acids. Third, a new exon was identified encoding an 83 amino acid domain that is homologous to parts of elongation factor-1 subunits as well as other proteins involved in protein synthesis. Northern blot analysis showed three different mRNAs for CRS (of approximately 3.0, 2.7 and 2.0 kb) from human testis while only the 2.7 kb mRNA was commonly detected in other tissues. Expression of the exon 2-containing transcript in testis was confirmed by RT-PCR and northern blotting. CRS containing the exon 2-encoded peptide retained catalytic activity comparable to that lacking this peptide. This peptide was responsible for the specific interaction of CRS with elongation factor-1gamma. (+info)
Heat shock protein 90 mediates protein-protein interactions between human aminoacyl-tRNA synthetases.
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Heat shock protein 90 (hsp90) is a molecular chaperone responsible for protein folding and maturation in vivo. Interaction of hsp90 with human glutamyl-prolyl-tRNA synthetase (EPRS) was found by genetic screening, co-immunoprecipitation, and in vitro binding experiments. This interaction was sensitive to the hsp90 inhibitor, geldanamycin, and also ATP, suggesting that the chaperone activity of hsp90 is required for interaction with EPRS. Interaction of EPRS with hsp90 was targeted to the region of three tandem repeats linking the two catalytic domains of EPRS that is also responsible for the interaction with isoleucyl-tRNA synthetase (IRS). Interaction of EPRS and IRS also depended on the activity of hsp90, implying that their association was mediated by hsp90. EPRS and IRS form a macromolecular protein complex with at least six other tRNA synthetases and three cofactors. hsp90 preferentially binds to most of the complex-forming enzymes rather than those that are not found in the complex. In addition, inactivation of hsp90 interfered with the in vivo incorporation of the nascent aminoacyl-tRNA synthetases into the multi-ARS complex. Thus, hsp90 appears to mediate protein-protein interactions of mammalian tRNA synthetases. (+info)
Enzymatic arginylation of beta-melanocyte-stimulating hormone and of angiotensin II.
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Porcine beta-melanocyte-stimulating hormone and angiotensin II were examined as acceptors in the reaction catalyzed by arginyl-tRNA-protein transferase. Both inhibited enzymatic transfer of [14C]arginine from tRNA to bovine albumin. Inhibition was competitive with albumin and the K-i values were, respectively, 15 and 0.8 muM. The expected arginylated compounds were isolated and characterized. Beta-melanocyte-stimulating hormone and its arginylated product had identical activities in the frog epithelium bioassay. In contrast, the biological activity of angiotensin II was diminished by enzymatic arginylation. The pressor effect of the arginylated derivative on anesthetized rats and its activity on the isolated rat uterus were, respectively, approximately 60% and 20% of those found for the unmodified peptide. (+info)