Possible inhibitory effect of teichoic acid on Bacillus subtilis transfer ribonucleic acid

1. tRNA of Bacillus subtilis was found to be variably contaminated with membrane teichoic acid. 2. Samples with high contents of teichoic acid showed no accepting activity for tRNA(Phe) and tRNA(Tyr). 3. Removal of teichoic acid restored accepting activity and fractions containing teichoic acid, separated on Sephadex G-150, inhibited the charging of tRNA(Tyr). 4. The presence of teichoic acid did not inhibit the charging of tRNA(His).     

The effect of hypermethylation on the functional properties of transfer ribonucleic acid. Ribosome-binding and polypeptide synthesis

1. Phenylalanyl-tRNA formed after chemical hypermethylation of Escherichia coli B tRNA was able to bind to ribosomes with the same efficiency as normal phenylalanyl-tRNA. 2. Under incubation conditions used in the ribosome-binding assay, hypermethylation of tRNA did not measurably decrease the stability of either inter-nucleotide phosphodiester bonds or the covalent bond between amino acid and tRNA in phenylalanyl-tRNA. 3. The ability of hypermethylated tRNA to take part in polyphenylalanine synthesis was inhibited progressively as the degree of hypermethylation increased. 4. Hypermethylation of tRNA affected polyphenylalanine synthesis at the stage of amino acid recognition and at a further point in the synthesis but not at the level of codon-anticodon recognition. 5. The formation of polylysine was more seriously affected by hypermethylation of tRNA than would be accounted for by inhibition of amino acid acceptance alone. 6. Polyproline formation was completely inhibited by the presence of 7mol% excess of methyl groups in tRNA. 7. The possibility of a link between amino acid acceptance and ribosome-binding was suggested for phenylalanyl-tRNA, but not for lysyl- or prolyl-tRNA.     

Studies on the fractionation of total transfer ribonucleic acid and purification of an alanine transfer ribonucleic acid from chick embryo

Chick embryo tRNA, prepared by a simple large-scale method, was fractionated on three different ion-exchange columns. In all cases simple chromatographic patterns for various tRNA species were observed, indicating the presence of only a few major species of tRNA for each amino acid. By repeated chromatography one species of alanine tRNA was purified to approx. 80% purity. T₁ ribonuclease digest of this purified tRNA gave a simple chromatographic pattern. Because of the simplicity of the method of preparation of tRNA from this readily available source and the presence of only a few species of tRNA for each amino acid, chick embryo is suited for the study of tRNA and its various functions in higher systems.     

The identification of the tRNA substrates for the supK tRNA methylase.

Purified preparations of the tRNA methylase deficient in supK strains of Salmonella typhimurium transfer methyl groups from S-adenosylmethionine (SAM) to at least two tRNA species, an alanine tRNA and a serine tRNA. The identity of the tRNA substrates for this enzyme was determined by a change in the elution position of the methyl-labeled tRNA from BND-cellulose columns before and after aminoacylation with a specific amino acid followed by derivatization of the free primary amino group with phenoxy- or naphthoxyacetate. The radioactive methyl group enzymatically added to these tRNAs is both acid and base labile and can be hydrolyzed to a volatile product at pHs above 7.5 and also at pH 1. The methylated 3'-nucleotide isolated from digested tRNA is a pyrimidine derivative and chromatographs like a modified uridylic acid. Its identity has not been established, but it is likely that it corresponds to the methyl ester of V, uridin-5-oxyacetic acid.     

Role of aminoacyl-transfer ribonucleic acid in the regulation of ribonucleic acid synthesis in Escherichia coli

Morris, D. W. (University of California, San Diego), and J. A. DeMoss. Role of aminoacyl-transfer ribonucleic acid in the regulation of ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 90:1624-1631. 1965.-A leucine auxotroph of Escherichia coli was examined for its rate of ribonucleic acid (RNA) synthesis and the level of charged leucine-, arginine-, and valine-specific transfer RNA (tRNA) during the exponential growth period and when growth was limited by leucine starvation. During the logarithmic growth period, the leucine-specific tRNA was 70% charged, arginine-specific tRNA was 30% charged, and the valine-specific tRNA was 80% charged. When leucine became limiting, RNA synthesis was inhibited and the levels of charged arginine- and valine-specific tRNA remained constant, whereas the level of charged leucine-specific tRNA dropped to 40%. Examination of the leucyl-tRNA during the leucine starvation period showed that this 40% level is maintained by protein turnover. Addition of chloramphenicol or puromycin to a leucine-starved culture derepressed RNA synthesis. In the presence of chloramphenicol, the leucine-specific tRNA was fully charged; however, in the presence of puromycin the amount of charged leucine-specific tRNA remained at the starved level. Therefore, during leucine starvation the level of uncharged leucine-specific tRNA is not invariably correlated with the rate of RNA synthesis. We propose that it is the availability of charged tRNA and not the amount of uncharged tRNA which is the important factor in the amino acid control of RNA synthesis.     

Breaking the stereo barrier of amino acid attachment to tRNA by a single nucleotide

Aminoacyl-tRNA synthetases are responsible for attaching amino acid residues to the tRNA 3'-end. The two classes of synthetases approach tRNA as mirror images, with opposite but symmetrical stereochemistries that allow the class I enzymes to attach amino acid residues to the 2'-hydroxyl group of the terminal ribose, whereas, the class II enzymes attach amino acid residues to the 3'-hydroxyl group. However, we show here that the attachment of cysteine to tRNA(Cys) by the class I cysteinyl-tRNA synthetase (CysRS) is flexible; the enzyme is capable of using either the 2' or 3'-hydroxyl group as the attachment site. The molecular basis for this flexibility was investigated. Introduction of the nucleotide U73 of tRNA(Cys) into tRNA(Val) was found to confer the flexibility. While valylation of the wild-type tRNA(Val) by the class I ValRS was strictly dependent on the terminal 2'-hydroxyl group, that of the U73 mutant of tRNA(Val) occurred at either the 2' or 3'-hydroxyl group. Thus, the single nucleotide U73 of tRNA has the ability to break the stereo barrier of amino acid attachment to tRNA, by mobilizing the 2' and 3'-hydroxyl groups of A76 in flexible geometry with respect to the tRNA acceptor stem.     

Gram-scale purification of arginine, formylmethionione, glutamic acid and the nylalanine transfer ribonucleic acids from E. coli K-12MO7

Gram-sized quantities of purified arginine, formylmethionine, glutamic acid, and phenylalanine-2 tRNAs have been prepared from pools of E. coli K–12 MO7 mixed tRNAs by reversed-phase chromatography after preliminary fractionation on DEAE-cellulose. Purified formylmethionine tRNA and partially purified arginine tRNA and glutamie acid tRNA were obtained from large-scale RPC–3 runs (4 × 36 in. column). The arginine tRNA was further purified by rechromatography on RPC–4 columns, and the gluatmic acid tRNA by rechromatography on an RPC–3 column. Two phenylalanine tRNAs were resolved on large-scale (2 × 96 in. column) RPC–3 runs; only the second phenylalanine tRNA reached a satisfactory degree of activity. About 0.88 g of arginine tRNA, 70% activity; 3.32 g of formylmethionine tRNA, 97% activity; 0.80 g of glutamic acid tRNA, 83% activity and O.92 g of phenylalanine-2 tRNA, 78% activity, were produced. The processing steps employed are reliable and reproducible and the procedure is amenable to routine production of these tRNAs.     

LEVEL AND AMINO ACID ACCEPTOR ACTIVITY OF MOUSE BRAIN tRNA DURING NEURAL DEVELOPMENT

Abstract— The level of tRNA in mouse brain tissue was measured at various stages of postnatal development. The amount of tRNA per unit of brain wet weight was little, if at all, altered during the first 22 days after birth and decreased by 26 and 32 per cent by 56 days and maturity, respectively. On a DNA or cellular basis, there was no maturation-dependent decrease in tRNA content. The total amino acid acceptor activity of tRNA for seven different amino acids was measured during neural development. There were considerable differences in the tRNA acceptor activities of individual amino acids within an age group; however on a DNA basis, there was little difference between tRNA preparations obtained from newborn and adult mouse brain tissue. The in vivo levels of aminoacylated-tRNA for the seven amino acids of interest, were measured in brain tissue of 1–, 9–, 34, 70–day-old and adult (over 9 months old) mice. Alterations in tRNA level, total tRNA acceptor activity, for each amino acid, and the levels of in uivo aminoacylation of tRNA were shown to be independent of developmental alterations in brain amino acid pool sizes. The results are discussed with regard to the availability of cellular amino acids for translational events during early mammalian brain development.     

Different Arginine Transfer Ribonucleic Acid Species Prevalent in Shaken and Unshaken Cultures of Neurospora

When the arginyl-transfer ribonucleic acid (tRNA) species isolated from unshaken and from shaken cultures of Neurospora were compared by co-chromatography, a marked change in the relative abundance of the two main tRNA(arg) species was found. The two arginine tRNA species had different codon responses in ribosome binding assays. The tRNA(arg) eluting first (prevalent in shaken cultures) bound strongly to polyadenylic-guanylic acid [poly(A,G)] and to a lesser extent to polycytidylic-guanylic-adenylic acid [poly(C,G,A)]. The second tRNA(arg) species (prevalent in unshaken cultures) bound to poly(C,G,A) but not to poly(A, G). The possible significance of these observations is briefly discussed. Several modifications that improve the yield of tRNA from Neurospora were introduced in a standard isolation procedure.     

Specificity of Interaction Between the First Enzyme for Histidine Biosynthesis and Aminoacylated Histidine Transfer Ribonucleic Acid

The specificity of the interaction between phosphoribosyltransferase and partially purified preparations of various species of transfer ribonucleic acid (tRNA) was investigated with the use of a filter binding assay. The enzyme showed a higher affinity for histidyl-tRNA than for arginyl- or glutamyl-tRNA. Competition experiments revealed that the enzyme does not distinguish between the aminoacylated and deacylated forms of arginine tRNA or glutamic acid tRNA, since all the binding of the aminoacylated tRNA could be inhibited by deacylated tRNA. The enzyme does, however, distinguish between the aminoacylated and deacylated forms of histidine tRNA. Approximately 70% of the binding of aminoacylated histidine tRNA is specific, since only 30% of the binding could be inhibited by deacylated tRNA. The possibility that the regulatory role of phosphoribosyltransferase is carried out as a complex with histidyl-tRNA is consistent with these data.     

Isoaccepting lysine transfer ribonucleic acid species of Pseudomonas aeruginosa.

Pseudomonas aeruginosa tRNA was treated with iodine, CNBr and N-ethylmaleimide, three thionucleotide-specific reagents. Reaction with iodine resulted in extensive loss of acceptor activity by lysine tRNA, glutamic acid tRNA, glutamine tRNA, serine tRNA and tyrosine tRNA. CNBr treatment resulted in high loss of acceptor ability by lysine tRNA, glutamic acid tRNA and glutamine tRNA. Only the acceptor ability of tyrosine tRNA was inhibited up to 66% by N-ethylmaleimide treatment, a reagent specific for 4-thiouridine. By the combined use of benzoylated DEAE-cellulose and DEAE-Sephadex columns, lysine tRNA of Ps. aeruginosa was resolved into two isoaccepting species, a major, tRNA Lys1 and a minor, tRNALys1. Co-chromatography of 14C-labelled tRNALys1 and 3H-labelled tRNALys2 on benzoylated DEAE-cellulose at pH 4.5 gave two distinct, non-superimposable profiles for the two activity peaks, suggesting that they were separate species. The acceptor activity of these two species was inhibited by about 95% by iodine and CNBr. Both the species showed equal response to codons AAA and AAG and also for poly(A) and poly(A1,G1) suggesting that the anticodon of these species was UUU. Chemical modification of these two species by iodine did not inhibit the coding response. The two species of lysine of Ps. aeruginosa are truly redundant in that they are indistinguishable either by chemical modification or by their coding response.     

Presence and Function of Sulfur-containing Transfer Ribonucleic Acid of Bacillus subtilis

Bacillus subtilis transfer ribonucleic acid (tRNA) was analyzed for the occurrence of thionucleotides by in vivo labeling with (35)S and fractionation by methylated albumin kieselguhr column chromatography. Alkaline hydrolysates of tRNA were also examined by column chromatography and paper electrophoresis, and the amino acid-accepting ability of thionucleotide-containing tRNA was tested after iodine oxidation. The results showed that B. subtilis tRNA contains 4-thiouridylate, a second nucleotide with properties similar to 2-thiopyrimidine, and a third unidentified thionucleotide. The amino acid-accepting ability for serine, tyrosine, lysine, and glutamic acid was markedly inhibited after oxidation of the tRNA with iodine, suggesting the presence of thionucleotides in these tRNA species. This inhibition could be reversed by thiosulfate reduction. The iodine treatment totally inactivated all lysine tRNA species, partially inactivated the serine tRNA species, and did not affect the accepting ability for valine. A comparison of tRNA from cells in the log and stationary phases and from spores revealed similar iodine inactivation patterns in all cases. The thionucleotide content in B. subtilis tRNA differed from that in Escherichia coli, both in extent and in distribution. A possible function of the thionucleotides in tRNA is discussed.     

Modeling with in vitro kinetic parameters for the elaboration of transfer RNA identity in vivo

A tRNA with "double identity" was created, and this tRNA was demonstrated in vitro to aminoacylate quantitatively with either of two amino acids. In contrast, acceptance of only one of these amino acids was observed in vivo, and a simple manipulation determined which one was accepted. Kinetic parameters were obtained for aminoacylation with each amino acid of the tRNA with double identity and of related tRNAs. Modeling with these parameters largely explains which amino acid specificity is observed in vivo. The results delineate some of the kinetic boundaries for the design and accommodation of tRNA sequence variations in the elaboration of identity in vivo.     

Specific interaction between tRNA and its cognate amino acid as detected by circular dichroism and fluorescence spectroscopy

Specific interaction was detected between tRNA and its cognate L-amino acid by circular dichroism (CD) and fluorescence spectroscopy; fluorescence spectral change with saturation was observed when tRNAAsp was titrated only with L-aspartic acid, but not with D-aspartic acid, L- and D-glutamic acids. It was also the case for tRNA2Glu and L-glutamic acid as detected by CD. These results are consistent with the hypothesis that the anticodon, or the complex of four nucleotides (C4N) of the anticodon three bases and the discriminator base in a tRNA (Shimizu, M., J. Mol. Evol. (1982) 18, 297-303) participates in recognition of amino acids.     

Specific incorporation of glycine into bacterial lipopolysaccharide. Novel function of specific transfer ribonucleic acids.

It has been found that the bacterial endotoxins (lipopolysaccharides, LPSs) contain some amino acids and glycine is the most abundant amino acid in the polysaccharide core preparations of LPSs of gram-negative bacteria. Until now nothing was known about the mechanism of amino acid incorporation into the lipopolysaccharide core. We found that one out of three glycyl-tRNAs(Gly) from Escherichia coli is the donor of amino acid and is the substrate for a putative aminoacyl-tRNA:LPS transferase. We have isolated, purified this tRNA and determined its nucleotide sequence to be major E.coli tRNA(3Gly). This tRNA(Gly) (anticodon GCC) conserved the tRNA structural features. The assay for determination of the specific incorporation of glycine into the lipopolysaccharide was also invented and described.     

大肠杆菌tRNA~(Leu)的提取、纯化及性质研究

采用高表达大肠杆菌ｔＲＮＡＬｅｕ菌株提取、纯化了亮氨酸等受体转移核糖核酸ｔＲＮＡＬｅｕ１和ｔＲＮＡＬｅｕ２．利用稳态动力学手段研究了ｔＲＮＡＬｅｕ１及脱镁ｔＲＮＡＬｅｕ１在不同稀土离子作用下与纯化亮氨酰－ｔＲＮＡ合成酶的氨酰化作用．ｔＲＮＡＬｅｕ１与亮氨酰－ｔＲＮＡ合成酶的结合及催化效率均受参与稀土离子的影响,表观Ｋｍ值有较明显的变化．结果表明,亮氨酰－ｔＲＮＡ合成酶催化的ｔＲＮＡＬｅｕ１氨酰化反应所需Ｍｇ２＋能够被稀土离子取代,但亲合性能不同．     

Diurnal incorporation of 3H-leucine into liver protein

The tRNA fraction, extracted from very high speed supernatant fluid, from livers of rats injected with 3H-orotic acid, attained maximum specific activity after a little over 24 hr and, thereafter, decayed with an apparent half life of 5 days. This behaviour of tRNA was indistinguishable from that of liver rRNA and the acid soluble pool. Chromatography of tRNA, doubly labelled during a period of short synthesis and of prolonged decay, on a BD cellulose column, indicated that individual tRNA species turn over at a constant rate with respect to one another.     

Aminoacylation of undermethylated mammalian transfer RNA.

To study the role of 5-methylcytidine in the aminoacylation of mammalian tRNA, bulk tRNA specifically deficient in 5-methylcytidine was isolated from the livers of mice treated with 5-azacytidine (18 mg/kg) for 4 days. For comparison, more extensively altered tRNA was isolated from the livers of mice treated with DL-ethionine (100 mg/kg) plus adenine (48 mg/kg) for 3 days. The amino acid acceptor capacity of these tRNAs was determined by measuring the incorporation of one of eight different 14C-labeled amino acids or a mixture of 14C-labeled amino acids in homologous assays using a crude synthetase preparation isolated from untreated mice. The 5-methylcytidine-deficient tRNA incorporated each amino acid to the same extent as fully methylated tRNA. The tRNA from DL-ethionine-treated livers showed an overall decreased amino-acylation capacity for all amino acids tested. The 5-methylcytidine-deficient tRNA from DL-ethionine-treated mice were further characterized as substrates in homologous rate assays designed to determine the Km and V of the aminoacylation reaction using four individual 14C-labeled amino acids and a mixture of 14C-labeled amino acids. The Km and V of the reactions for all amino acids tested using 5-methylcytidine-deficient tRNA as substrate were essentially the same as for fully methylated tRNA. However, the Km and V were increased when liver tRNA from mice treated with DL-ethionine plus adenine was used as substrate in the rate reaction with [14C]lysine as label. Our results suggest that although extensively altered tRNA is a poorer substrate than control tRNA in both extent and rate of aminoacylation, 5-methylcytidine in mammalian tRNA is not involved in the recognition of the tRNA by the synthetase as measured by aminoacylation activity.     

Effect of estradiol on the amino acid-accepting activity of uterine tRNAs and their participation in protein synthesis

Estradiol (E2) induces a complementary increase in both the amount of mRNA and the rate of translation of the mRNA in the uterus of ovariectomized mature rats. The mechanism of the translational effect was evaluated by measuring the functional capacity of uterine tRNA isolated from control, E2 (1 h)- and E2 (14 h)-treated ovariectomized rats to support amino acid acceptor activity and uterine protein synthesis. The specific amino acid acceptor activity (SAA) of deacylated tRNA for 18 individual amino acids was determined using a tRNA-dependent rat liver tRNA synthetase preparation. The SAA was the same for all amino acids for uterine tRNA from control and E2 (1 h)-treated rats but was increased for uterine tRNA from E2 (14 h)-treated rats to levels that were 1.4-4.3 times the SAA of uterine tRNA from control rats. When uterine tRNA from control and E2 (14 h)-treated rats was incubated with purified tRNA nucleotidyltransferase, the SAA for all amino acids was increased an average of 1.6-fold for control tRNA and 0.3-fold for tRNA from E2 (14 h)-treated rats. The ability of uterine tRNA to support maximal rates of protein synthesis in tRNA-dependent uterine ribosome protein synthesis assay was increased by either in vivo treatment of the rats with estradiol or by in vitro repair of the 3'-CCA terminus of this tRNA by nucleotidyltransferase. These observations suggest that E2 may increase the rate of mRNA translation in the uterus, in part, by increasing the proportion of certain tRNAs with intact and functional 3'-CCA acceptor termini.     

The repertoire of transfer RNA genes is tuned to codon usage bias in the genomes of Phytophthora sojae and Phytophthora ramorum

In all, 238 and 155 transfer (t)RNA genes were predicted from the genomes of Phytophthora sojae and P. ramorum, respectively. After omitting pseudogenes and undetermined types of tRNA genes, there remained 208 P. sojae tRNA genes and 140 P. ramorum tRNA genes. There were 45 types of tRNA genes, with distinct anticodons, in each species. Fourteen common anticodon types of tRNAs are missing altogether from the genome in the two species; however, these appear to be compensated by wobbling of other tRNA anticodons in a manner which is tied to the codon bias in Phytophthora genes. The most abundant tRNA class was arginine in both P. sojae and P. ramorum. A codon usage table was generated for these two organisms from a total of 9,803,525 codons in P. sojae and 7,496,598 codons in P. ramorum. The most abundant codon type detected from the codon usage tables was GAG (encoding glutamic acid), whereas the most numerous tRNA gene had a methionine anticodon (CAT). The correlation between the frequencies of tRNA genes and the codon frequencies in protein-coding genes was very low (0.12 in P. sojae and 0.19 in P. ramorum); however, the correlation between amino acid tRNA gene frequency and the corresponding amino acid codon frequency in P. sojae and P. ramorum was substantially higher (0.53 in P. sojae and 0.77 in P. ramorum). The codon usage frequencies of P. sojae and P ramorum were very strongly correlated (0.99), as were tRNA gene frequencies (0.77). Approximately 60% of orthologous tRNA gene pairs in P sojae and P. ramorum are located in regions that have conserved synteny in the two species.