Equilibrium unfolding of Bombyx mori glycyl-tRNA synthetase.

Unfolding of Bombyx mori glycyl-tRNA synthetase was examined by multiple spectroscopic techniques. Tryptophan fluorescence of wild type enzyme and an N-terminally truncated form (N55) increased at low concentrations of urea or guanidine-HCl followed by a reduction in intensity at intermediate denaturant concentrations; a transition at higher denaturant was detected as decreased fluorescence intensity and a red-shifted emission. Solute quenching of fluorescence indicated that tryptophans become progressively solvent-exposed during unfolding. Wild type enzyme had stronger negative CD bands between 220 and 230 nm than the mutant, indicative of greater alpha-helical content. Urea or guanidine-HCl caused a reduction in ellipticity at 222 nm at low denaturant concentration with the wild type enzyme, a transition that is absent in the mutant; both enzymes exhibited a cooperative transition at higher denaturant concentrations. Both enzymes dissociate to monomers in 1.5 m urea. Unfolding of wild type enzyme is described by a multistate unfolding and a parallel two state unfolding; the two-state component is absent in the mutant. Changes in spectral properties associated with unfolding were largely reversible after dilution to low denaturant. Unfolding of glycyl-tRNA synthetase is complex with a native state, a native-like monomer, partially unfolded states, and the unfolded state.

Immunoelectron microscopic localization of glutamyl-/ prolyl-tRNA synthetase within the eukaryotic multisynthetase complex.

A high molecular mass complex of aminoacyl-tRNA synthetases is readily isolated from a variety of eukaryotes. Although its composition is well characterized, knowledge of its structure and organization is still quite limited. This study uses antibodies directed against prolyl-tRNA synthetase for immunoelectron microscopic localization of the bifunctional glutamyl-/prolyl-tRNA synthetase. This is the first visualization of a specific site within the multisynthetase complex. Images of immunocomplexes are presented in the characteristic views of negatively stained multisynthetase complex from rabbit reticulocytes. As described in terms of a three domain working model of the structure, in "front" views of the particle and "intermediate" views, the primary antibody binding site is near the intersection between the "base" and one "arm." In "side" views, where the particle is rotated about its long axis, the binding site is near the midpoint. "Top" and "bottom" views, which appear as square projections, are also consistent with the central location of the binding site. These data place the glutamyl-/prolyl-tRNA synthetase polypeptide in a defined area of the particle, which encompasses portions of two domains, yet is consistent with the previous structural model.

Spectral and physical characterization of the inverted terminal repeat DNA structure from adenoassociated virus 2.

An oligodeoxynucleotide (ODN) that includes elements found in secondary structures at the 5'- and 3'- ends of adenoassociated virus 2 virion DNA was synthesized by ligation of three overlapping ODNs. The most stable secondary structure was calculated to be branched, with a 61 bp duplex stem, terminating in a three-way junction with 9 bp arms. The electrophoretic mobility of the ODN is slower than expected for normal duplex DNA of the same size, suggesting a bent or branched conformation. CD spectra indicate that the ITR structure is largely B form DNA, although there is a slight blue shift compared to the spectra of the isolated stem and loop elements. Thermal melting experiments indicate that the hairpin is significantly more stable than the isolated stem and loop elements. Singular value decomposition of UV spectra obtained as a function of temperature indicates that four species contribute to changes in the spectra upon denaturation, indicating that the melting is not a simple two-state process. Characterization of the branched ODN by differential scanning calorimetry permits estimation of the enthalpy of melting by a model-free analysis, yielding DeltaHcal= 614 kcal mol-1. This value agrees with the enthalpy computed for the most stable secondary structure.

Cardiac myosin-binding protein C (MyBP-C): identification of protein kinase A and protein kinase C phosphorylation sites.

Myosin binding protein C (MyBP-C) is a major myofibril-associated protein in cardiac muscle which is subject to reversible phosphorylation. Cardiac MyBP-C is a substrate in vivo and in vitro for cAMP-dependent protein kinase (PKA) and calcium/phospholipid-dependent protein kinase (PKC). Chicken cardiac MyBP-C was phosphorylated by PKA to 3.0 mol phosphate/mol and by PKC to 2.0 mol phosphate/mol. Tryptic phosphopeptides from MyBP-C were purified by successive iron iminodiacetate column chromatography and reversed-phase high-performance liquid chromatography. Three phosphopeptides purified from PKA-phosphorylated MyBP-C contained phosphoserine [T1, (RTS[P]LAGGGR) and T2, (KRDS[P]FLR)] or phosphothreonine (CT3, MT[P]SAFL). PKC phosphorylated two of the same sites (T1 and T2) as PKA and an additional site [T2a (TGTTYKPPS[P]YK)]. PKA phosphorylation sites corresponding to peptides T1, T2, and T3 were identified in the N-terminus of the cDNA deduced amino acid sequence (S265, S300, and T274, respectively). The PKC-specific site in peptide T2a was at position S1169. cDNA clones encoding rat cardiac MyBP-C were isolated, and the segment corresponding to PKA and major PKC phosphorylation sites was sequenced. Chicken cardiac MyBP-C has a threonine at position 274 (CT3), whereas rat cardiac MyBP-C has a serine at the corresponding position. Only chicken cardiac MyBP-C had a phosphorylatable residue at the position corresponding to S1169. All of the cardiac MyBP-C phosphorylation sites are absent in known sequences of skeletal muscle MyBP-C isoforms.

Role of carbohydrate structures in the binding of beta1-latency-associated peptide to ligands.

Transforming growth factor beta1 (TGF-beta1) is a potent growth differentiation and morphogenesis factor. The amino-terminal 248 amino acid pro region of TGF-beta1, the beta1-latency-associated peptide (beta1-LAP), is noncovalently associated with TGF-beta1 in an inactive complex. Previous studies suggested that deglycosylated beta1-LAP can not form this latent complex with TGF-beta1. To study the role of the carbohydrate structures of beta1-LAP in its biological functions, we expressed simian beta1-LAP in Escherichia coli with a 10 histidine residue tag on the N-terminus. This polypeptide was solubilized from inclusion bodies with 6 M guanidine hydrochloride and purified by metal chelate affinity chromatography. Purified beta1-LAP was refolded to its dimeric form using a chaotrope-mediated folding procedure. The dimeric beta1-LAP forms 90 kDa complexes with TGF-beta1, TGF-beta2, and TGF-beta3, and reverses the inhibitory activity of TGF-beta1 on Mv1Lu cells. Solid phase binding assays demonstrate that refolded beta1-LAP binds to heparin and thrombospondin 1. FET cell adhesion promoted by refolded beta1-LAP was blocked by an RGD peptide. Purified beta1-LAP produced in Chinese hamster ovary cells, deglycosylated with N-glycosidase F, forms a 80-90 kDa complex with mature TGF-beta1. The carbohydrate structures of beta1-LAP are not required for binding to ligands or for its biological activity.

Structural characterization of the latent complex between transforming growth factor beta 1 and beta 1-latency-associated peptide.

The formation of a non-covalent complex between mature transforming growth factor beta 1 (TGF-beta 1) and its pro region, the beta 1-latency-associated peptide (beta 1-LAP), is important in regulating the activity of this multipotent growth factor. We have overexpressed simian beta 1-LAP in Chinese hamster ovary (CHO) cells to produce a cell line which secretes beta 1-LAP into the culture medium at > 1 mg/l, thus enabling structural studies of complex formation between beta 1-LAP and TGF-beta 1. The simian beta 1-LAP expressed in CHO cells reversed the growth inhibitory effect of exogenous TGF-beta 1 on Mv1Lu (mink lung epithelial) cells and was able to form a cross-linked complex with 125I-TGF-beta 1. Simian beta 1-LAP was purified to homogeneity by a combination of ammonium sulphate precipitation, gel filtration, dye ligand chromatography and anion-exchange chromatography, with a yield of 15%. The purified protein had an apparent molecular mass of 114 kDa as determined by SDS/PAGE, which is greater than that determined for the transient expression of simian beta 1-LAP in COS-1 and for the simian precursor of TGF-beta 1 (pro-TGF-beta 1) in CHO cells, this major difference being due to more extensive glycosylation of beta 1-LAP expressed by this CHO clone. Far-UV CD spectroscopy of simian beta 1-LAP indicates a mostly beta-sheet structure, with extensive structural rearrangements occurring upon formation of the latent complex between TGF-beta 1 and beta 1-LAP.

Analysis of truncated forms of Bombyx mori glycyl-tRNA synthetase: function of an N-terminal structure in RNA binding.

Bombyx mori glycyl-tRNA synthetase (GRS) was expressed as the full length protein and as N-terminally and C-terminally truncated forms. The intact enzyme and forms with deletions of 12, 27, 46, and 55 N-terminal residues were expressed, purified, and characterized. All were active, having 15-25% of both pyrophosphate exchange activity and aminoacyl-tRNA synthetase activity compared to wild type enzyme. Active site titration indicated that this difference in activity was not the result of production of inactive enzyme. Sedimentation and gel filtration experiments indicate that the N-terminally deleted forms and the wild type enzyme were dimers. Deletion of 55 N-terminal residues did not result in significant effects on the Michaelis constants for ATP, glycine, or tRNA, while deletion of 108 N-terminal residues and two internal 64- and 200-residue deletions generated inactive forms. Five forms with C-terminal deletions of 24, 37, 59, 162, and 327 amino acid residues were soluble and intact but lacked detectable pyrophosphate exchange activity or aminoacyl-tRNA synthetase activity. The C-terminal sequence may be required for catalysis or to maintain a stable structure. Zone electrophoresis demonstrated the wild type enzyme bound both tRNA(Gly) and noncognate tRNA(Ala). Deletion of 55 N-terminal residues resulted in altered binding of tRNA(Gly) and eliminated binding of tRNA(Ala). The first 55 N-terminal residues are not essential for catalysis, dimerization, or substrate binding in aminoacylation but are required for RNA binding not associated with aminoacylation.

Post-transcriptional regulation of glutamyl-prolyl-tRNA synthetase in rat salivary gland.

Treatment of rats with isoproterenol resulted in elevated levels of prolyl- and glutamyl-tRNA synthetase activities in the parotid and submandibular glands. This increase in enzyme activity was accompanied by an increase in the bi-functional glutamyl-prolyl-tRNA synthetase and of a low molecular weight form of prolyl-tRNA synthetase. Isoproterenol also induced the synthesis of proline-rich glycoproteins in the parotid and submandibular glands. Withdrawal from the drug was accompanied by a decline in prolyl- and glutamyl-tRNA synthetase activities and by a decline in the levels of proline-rich glycoproteins in the salivary gland. During the time course of isoproterenol treatment, little change in the levels of mRNA encoding the bi-functional glutamyl-prolyl-tRNA synthetase was detected by Northern blot analysis. These results indicate that the synthesis of glutamyl-prolyl-tRNA synthetase is regulated at a post-transcriptional step and that the synthesis of this bifunctional protein may be linked to the utilization of proline and glutamic acid in protein synthesis.

Primary structure of the gene for glycyl-tRNA synthetase from Bombyx mori.

The primary structure of the gene encoding Bombyx mori glycyl-tRNA synthetase was determined by sequence analysis of one cDNA and two genomic clones. The sequence of the protein deduced from the nucleotide sequence was verified by sequence analysis of eight peptides. The M(r) 77,667 protein is encoded in a single open reading frame of 2061 nucleotides. There are no introns in the gene. The deduced protein sequence has no obvious similarity to Escherichia coli glycyl-tRNA synthetase but contains a sequence in its amino terminus that is similar to a sequence found in the Drosophila melanogaster and human glutamyl-tRNA synthetases, the hamster and human histidyl-tRNA synthetases, bovine tryptophanyl-tRNA synthetase, and the mammalian peptide chain release factor. The B. mori glycyl-tRNA synthetase also has sequence similarity with the Saccharomyces cerevisiae (cytoplasmic and mitochondrial), E. coli, and human threonyl-tRNA synthetases. This sequence similarity occurs in a sequence motif that is characteristic of other class II aminoacyl-tRNA synthetases. Two transcription start sites approximately 100 nucleotides apart were identified by ribonuclease mapping. One of the transcription start sites is used preferentially in the posterior silk gland. The peak in mRNA accumulation occurs 80-100 h prior to the peak in glycyl-tRNA synthetase activity and enzyme protein.

Isolation of prolyl-tRNA synthetase as a free form and as a form associated with glutamyl-tRNA synthetase.

Rat liver prolyl-tRNA synthetase was purified as a dimer of M(r) 60,000 subunits not associated with other aminoacyl-tRNA synthetases and as a form associated with glutamyl-tRNA synthetase. Proteolysis of the dimeric enzyme generated a less active form with M(r) 52,000 subunits and an inactive form with M(r) 40,000 subunits. A second species was isolated with polypeptides of M(r) 60,000 and 150,000. This form dissociated during gel filtration chromatography being partially resolved into the M(r) 150,000 and 60,000 components; glutamyl-tRNA synthetase was associated with the larger polypeptide and prolyl-tRNA synthetase with the smaller component. Antibodies against the M(r) 60,000 polypeptide reacted with the M(r) 60,000 and 150,000 polypeptides. Gel filtration of extracts revealed multiple forms of prolyl- and glutamyl-tRNA synthetase. Antibody against the M(r) 60,000 component detected the M(r) 60,000 and 150,000 polypeptides throughout the chromatogram; these forms could be partially separated by polyethylene glycol fractionation. The M(r) 150,000 and 60,000 polypeptides were detected by Western blot analysis of crude extracts prepared under several conditions. Antibody to prolyl-tRNA synthetase reacted with a M(r) 150,000 polypeptide of the aminoacyl-tRNA synthetase core complex identified previously as glutamyl-tRNA synthetase.

Alanyl-tRNA synthetase from Escherichia coli, Bombyx mori and Ratus ratus. Existence of common structural features.

Alanyl-tRNA synthetase from Escherichia coli, Bombyx mori and rat were examined with respect to the following functional and structural properties: the effect of substrates on sensitivity to proteolysis, secondary structure as determined by circular dichroism, amino acid composition and, in the case of the rat and insect enzymes, partial amino acid sequence determination on a 60-kDa C-terminal tryptic fragment. Digestion of the enzyme from all three sources with trypsin resulted in significant decline in aminoacyl-tRNA synthetase activity with little effect on pyrophosphate-exchange activity. In each case the presence of alanine and ATP together, but not separately, reduced the rate of digestion by trypsin; the largest effect was observed with the enzyme from rat liver. Trypsin digestion generated fragments of 47 kDa and 40 kDa with all three enzymes, but detection of significant quantities of the 47-kDa fragment from the rat enzyme required the presence of ATP and alanine. Trypsin digestion produced a fragment of 60 kDa with all three enzymes, but detection of significant quantities of this fragment with the bacterial enzyme required the presence of ATP and alanine. Limited sequence analysis of the 60-kDa fragment from the insect and rat enzymes indicated that trypsin cleaved both proteins at the same site to generate this species. Similar effects of substrates were observed when the enzymes were digested with chymotrypsin suggesting that the effects of substrates on protease sensitivity were not unique to trypsin. Circular dichroism spectra obtained for the three enzymes were qualitatively and quantitatively similar. There is some similarity in amino acid composition between the rat and insect enzymes.

Primary structure of alanyl-tRNA synthetase and the regulation of its mRNA levels in Bombyx mori.

cDNA clones encoding Bombyx mori alanyl-tRNA synthetase were isolated from a library in lambda gt11 using antibody, synthetic oligonucleotides, and a characterized cDNA as probes. Analysis of the sequence revealed significant homology between the B. mori and Escherichia coli alanyl-tRNA synthetases, particularly in their amino-terminal domains. Northern blot analysis indicated that the mRNA for alanyl-tRNA synthetase is 3.8 kilobase pairs in mRNA isolated from posterior silk gland, middle silk gland, and ovarian tissue. Steady-state levels of alanyl-tRNA synthetase mRNA in the posterior silk gland increased in the first 48 h of the fifth larval instar, decreasing gradually thereafter. In the middle silk gland, alanyl-tRNA synthetase mRNA peaked at 72 h of the fifth larval instar, declining to undetectable levels by 120 h. Genomic Southern blot analysis using a nick-translated cDNA probe revealed hybridization to single fragments when B. mori genomic DNA was digested with various restriction endonucleases.

Control of the levels of alanyl-, glycyl-, and seryl-tRNA synthetases in the silkgland of Bombyx mori.

We have examined the levels of glycyl-, alanyl-, and seryl-tRNA synthetases and the levels of their corresponding translatable mRNAs in the posterior and middle silkglands of the silkworm, Bombyx mori. Analysis of Western blots reveals that the change in the abundance of these enzymes during the fifth instar in crude extracts derived from posterior and middle silkgland correlates with changes in enzymatic activity; most of the change in activity for seryl- and alanyl-tRNA synthetases can be accounted for by the corresponding change in enzyme concentration, while the apparent specific activity of glycyl-tRNA synthetase appears to be elevated in the posterior silkgland. Accompanying the changes in enzyme activity and enzyme concentration are changes in the levels of the corresponding mRNAs as determined by immunoprecipitation of in vitro translation products. The levels of all three enzymes are 10 to 20 times higher in the posterior and middle silkglands than in ovarian tissue. A form of alanyl-tRNA synthetase with a slightly higher apparent molecular weight is detected in the posterior silkgland early in the fifth instar and in ovarian tissue.

Seryl-tRNA synthetase from Bombyx mori. Purification and properties.

Seryl-tRNA synthetase has been purified from the middle silk glands of Bombyx mori by successive chromatography on DEAE-Sephacel, hydroxylapatite, and Bio-Rex 70. The high abundance of seryl-tRNA synthetase in the middle silk glands may result from an adaptation of this organ for the production of the serine-rich protein, sericin. The enzyme is a dimer of Mr = 124,000 consisting of similar or identical subunits and has an oligomeric structure similar to its procaryotic and eucaryotic counterparts. Seryl-tRNA synthetase can be cleaved with trypsin to generate a fragment of Mr = 45,000 on sodium dodecyl sulfate gels; the presence of tRNASer protects the enzyme from tryptic cleavage. Conversion to the Mr = 45,000 species is accompanied by a 90% loss in aminoacyl-tRNA synthetase activity, but only a 20% loss in ATP PPi exchange activity.

A bacterial and silkworm aminoacyl-tRNA synthetase have a common epitope which maps to the catalytic domain of each.

We report here the identification of a common immunological determinant in Escherichia coli and Bombyx mori (silkworm) alanine tRNA synthetases. The E. coli protein is a tetramer of identical Mr = 95,000 chains, and the silkworm enzyme is a monomer of Mr = 115,000. Antibodies against the silkworm enzyme react with E. coli Ala-tRNA synthetase. Analysis of 10 fragments of the E. coli enzyme has mapped the cross-reacting epitope to between amino acids 350 and 385. This is within the part of the enzyme which is essential for alanyladenylate synthesis. The anti-B. mori Ala-tRNA synthetase antibodies which cross-react with the E. coli enzyme were affinity-purified. They react specifically with the catalytic domain of the silkworm enzyme and not with the remaining dispensable segment of 500 amino acids. The results support the concept that the core catalytic structural elements, and not the dispensable portions, are the most related among the synthetases.

Glycyl- and alanyl-tRNA synthetases from Bombyx mori. Purification and properties.

Glycyl- and alanyl-tRNA synthetases have been purified from an extract of Bombyx mori posterior silk glands by a procedure that allows for the simultaneous isolation of both enzymes. Glycyl-tRNA synthetase is a dimer of Mr = 160,000 consisting of similar or identical subunits, whereas alanyl-tRNA synthetase is a monomer of Mr = 110,000 to 115,000. The abundance of these two enzymes in the posterior silk gland is consistent with the observed adaptation of this organ to the production of the silk protein, fibroin. The two enzymes are similar in oligomeric structure to the corresponding enzymes in Saccharomyces cerevisiae, but dissimilar from their counterparts in Escherichia coli.

Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.

We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using the major late promoter of adenovirus 2. The extract also directs accurate transcription initiation from other adenovirus promoters and cellular promoters. The extract also directs accurate transcription initiation from class III promoters (tRNA and Ad 2 VA).