Chemical structure of a new modified nucleoside located in the anticodon of Bombyx mori glycine tRNA2

There are two species of glycine tRNA, tRNA(1Gly) and tRNA(2Gly), in the posterior silk glands of Bombyx mori. The first positions of their anticodons are guanosine and an unknown nucleoside for tRNA(1Gly) and tRNA(2Gly), respectively. This new nucleoside was isolated and the chemical structure was analyzed by thin layer chromatography and by UV, 1H-NMR, field desorption mass, and ORD spectroscopic measurements. The structure characterized by physical methods was finally confirmed by synthesis to be 5-((S)-carboxy(hydroxy) methyl)uridine methyl ester.     

5-(Carboxy-hydroxymethyl)uridine, a new modified nucleoside located in the anticodon of tRNA2Gly from the posterior silk glands of Bombyx mori

A new modified nucleoside located in the anticodon of tRNA2Gly from the posterior silk glands of Bombyx mori has been isolated and its structure determined as 5-(carboxy-hydroxymethyl)uridine mainly by analyses of its UV, 1H NMR, and FD mass spectra.     

Two-domain structure of alanyl-tRNA synthetase from Bombyx mori: isolation of the N-terminal catalytic domain

Alanyl-tRNA synthetase of 115K daltons from Bombyx mori was cleaved into two fragments of 62K and 47K daltons by trypsin. The 47K fragment was active in aminoacylation of tRNA, whereas the 62K fragment was inactive. The 47K and 62K fragments were found to be located at the N- and C-terminal ends, respectively, in the intact enzyme. The intact enzyme was protected from trypsin-attack by the cognate tRNA. The Km value of the 47K fragment for tRNA was 22 microM which is about 16-fold higher than that for the intact enzyme (1.4 microM). The molecular activities of the fragment and the intact enzyme were 2.2 s-1 and 16.8 s-1, respectively. This indicates that the 62K domain enhances affinity for tRNA and it is responsible for the full activity of tRNA aminoacylation. These results do not support the "covalently linked dimer" hypothesis, but indicate that the alanyl-tRNA synthetase is a functional monomer consisting two large domains.     

The nucleotide sequence of a major glycine transfer RNA from the posterior silk gland of Bombyx mori L.

The nucleotide sequence of tRNA1Gly isolated from the posterior silk gland of Bombyx mori has been determined. This transfer RNA is present in high amounts in the posterior silk gland during the fifth larval instar. It has a GCC anticodon, capable of decoding a major glycine codon in the fibroin messenger RNA, GGU. Structural features of Bombyx tRNA1Gly and its homology to other eukaryotic glycine tRNAs are discussed.     

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.     

The biosynthesis of transfer RNA in insects. I. Increase of amino acid acceptor activity of specific tRNA's utilized for silk protein biosynthesis in the silk gland of Bombyx mori.

1) To detect the quantitative changes of amino acid acceptor activity of tRNA's from the posterior and middle silk glands of Bombyx mori at various ages, a relatively simple and rapid method was established using a mixture of radioactive amino acids in Chlorella hydrolysate. 2) The acceptor activities of silk gland tRNA for 15 amino acids tested seemed to be almost on the same level at the end of the 4th moult stage. During the 5th instar, however, characteristic increases were observed in glycine, alanine, and serine acceptor activities in both silk glands. 3) In the posterior silk gland, which produces fibroin, the acceptor activities for glycine and alanine increased more than that for serine. In the middle silk gland, which produces sericine, the acceptor activity for serine increased more than those for glycine and alanine. 4) In the light of observations on the increase of corresponding aminoacyl-tRNA synthetase activities in the silk glands, a functional adaptation of tRNA synthesis in the tissue is discussed.     

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 mutation in the small (alpha) subunit of glycyl-tRNA synthetase affects amino acid activation and subunit association parameters

A strategy was designed to isolate mutants of glycyl-tRNA synthetase that are altered at the amino acid binding site, including a class with altered amino acid specificity. For this purpose, the plasmid pBR322 was mutated so that the codon (AGC) of the active site Ser-68 in the beta-lactamase gene was changed to the glycine codon GGC to inactivate the encoded enzyme. Suppressors that increase the amount of beta-lactamase activity of the Gly-68 allele of beta-lactamase were isolated and some mapped to the gene encoding glycyl-tRNA synthetase (glyS). While in vitro misaminoacylation of tRNA(Gly) with serine was not detected for any of the mutants, glycyl-tRNA synthetase activity was altered. One severely affected glyS mutant (N302) was studied in more detail. For this mutant, a single Pro-61----Leu substitution in the alpha chain confers an elevation of the Km values for glycine (25-fold) and for ATP (45-fold) in the aminoacylation reaction, but only a minor perturbation of the Km for tRNA. There also was a severely reduced adenylate synthesis activity (greater than 100-fold). In addition, a nonlinear dependence between aminoacylation activity and enzyme concentration was observed which implies that the alpha chain Pro-61----Leu mutation has disrupted the functionally essential subunit interactions of the holoenzyme. The results of the preceding paper have shown that the alpha chain and parts of the beta chain are required for aminoacylation and adenylate synthesis activity. The results of this study suggest that the alpha chain specifically contributes to amino acid and to ATP binding in a way that is affected by proper subunit interactions.     

Properties of the cell-free protein synthesizing system from the fibroin region of the Bombyx mori silkgland]

Microsomes isolated from the fibroin region of the Bombyx mori silkgland synthesize in the cell-free system a glycin rich polypeptide or polypeptides presumably representing fibroin precursors. Besides microsomes the system requires ATP and ATP-generating system, GTP, soluble protein fraction and tRNA, glycine incorporation is inhibited by puromycin and cycloheximide. It is shown that the synthesis of a polypeptide with high Gly/Lys ratio requires soluble protein fraction isolated from the silk gland at the end of the instar V. When the soluble protein fraction from the larvoe at the early instar V is used the Gly/Lys ratio in the product is markedly lower. These results permit to suggest that fibroin synthesis may be regulated at the level of tRNA aminoacylation.     

Overlapping destinations for two dual targeted glycyl-tRNA synthetases in Arabidopsis thaliana and Phaseolus vulgaris

In plant mitochondria, some of the tRNAs are encoded by the mitochondrial genome and resemble their prokaryotic counterparts, whereas the remaining tRNAs are encoded by the nuclear genome and imported from the cytosol. Generally, mitochondrial isoacceptor tRNAs all have the same genetic origin. One known exception to this rule is the group of tRNA(Gly) isoacceptors in dicotyledonous plants. A mitochondrion-encoded tRNA(Gly) and at least one nucleus-encoded tRNA(Gly) coexist in the mitochondria of these plants, and both are required to allow translation of all four GGN glycine codons. We have taken advantage of this atypical situation to address the problem of tRNA/aminoacyl-tRNA synthetase coevolution in plants. In this work, we show that two different nucleus-encoded glycyl-tRNA synthetases (GlyRSs) are imported into Arabidopsis thaliana and Phaseolus vulgaris mitochondria. The first one, GlyRS-1, is similar to human or yeast glycyl-tRNA synthetase, whereas the second, GlyRS-2, is similar to Escherichia coli glycyl-tRNA synthetase. Both enzymes are dual targeted, GlyRS-1 to mitochondria and to the cytosol and GlyRS-2 to mitochondria and chloroplasts. Unexpectedly, GlyRS-1 seems to be active in the cytosol but inactive in mitochondrial fractions, whereas GlyRS-2 is likely to glycylate both the organelle-encoded tRNA(Gly) and the imported tRNA(Gly) present in mitochondria.     

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.     

DNA polymerase-delta from the silk glands of Bombyx mori.

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3'----5' exonuclease activity which participates in proofreading by mismatch repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.     

Loss of positional specificity in the aminoacylation of Escherichia coli tRNAGly

The positional specificity in the aminoacylation of Escherichia coli tRNAGly by its cognate aminoacyl-tRNA synthetase has been studied using tRNAGlys terminating in 2'- or 3'-deoxyadenosine under conditions believed to alter tRNA conformation. Although E. coli tRNAGly terminating in 3'-deoxyadenosine has been reported not to be a good substrate for activation by the homologous glycyl-tRNA synthetase, by systematic variation of the conditions employed for aminoacylation it was possible to activate this tRNA to essentially the same extent as unmodified tRNAGly. Activation of tRNAGly terminating in 3'-deoxyadenosine was carried out optimally at 45 degrees C in an incubation mixture containing 0.3-0.4 M NaCl; 10% methanol, ethanol, and dimethyl sulfoxide were found to facilitate activation of the modified tRNA. Interestingly, the conditions employed to enhance activation of this modified tRNAGly had no effect on the activation of unmodified tRNAGly or tRNAGly terminating in 2'-deoxyadenosine. These experiments afford insight into the activation of tRNAGly by glycyl-tRNA synthetase and provide facile access to positionally defined, isomeric glycl-tRNAGlys.     

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.     

The beta subunit of E. coli glycyl-tRNA synthetase plays a major role in tRNA recognition.

The contributions made by the alpha and beta subunits of E. coli glycyl-tRNA synthetase to the recognition of tRNA have been investigated via binding and immunological methods. Using the nitrocellulose filter assay, we have shown that isolated beta subunit, but not the alpha subunit, binds [14C]glycyl-tRNA with an affinity comparable to that of the native enzyme. Further, the data indicate that the beta subunit possesses one binding site for glycyl-tRNA while the native or reconstituted enzyme (alpha 2 beta 2) has two sites. Rabbit antibodies directed at the beta subunit or the holoenzyme inhibit efficiently the ability of the enzyme to aminoacylate tRNA while alpha-subunit antibodies have a smaller effect. Since none of the antisera have an appreciable effect on the ATP-PPi exchange activity of the enzyme under these conditions, the beta-subunit (and holoenzyme) antisera evidently interfere with productive tRNA binding. Taken together, the data indicate that the larger, beta subunit of glycyl-tRNA synthetase plays a major role in tRNA recognition.     

Occurrence of Lectin in the Silkgland of the Silkworm, Bombyx mori

Monoclonal antibodies were prepared against the 350 kDa lectin purified from larval hemolymph of the silkworm, Bombyx mori . The antibodies inhibited the hemagglutinating activity (HA activity) and bound specifically to the hemolymph 350 kDa lectin on Western blotting analysis. Immunohistological observations revealed the occurrence of lectin in the cuticular intima of the anterior silk gland, but not the middle or posterior silk glands of fifth instar larvae of Bombyx mori . Extracts from the anterior silk glands showed HA activity and exhibited the same biochemical characteristics as those of the 350 kDa lectin in the hemolymph. These results suggested that lectin-like molecules in epithelial tissues may be important in histolysis during molting and metamorphosis.