Article

The nucleotide sequence of phenylalanine tRNA from Bacillus subtilis

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Abstract

The nucleotide sequence of tRNA(Phe) from Bacillussubtilis W 23 has been determined using (32)P labeled tRNA. This is the second B. subtilis tRNA so far reported. The nucleotide sequence was found to be pG-G-C-U-C-G-G-U-A-G-C-U-C-A-G-U-D-G-G-D-A-G-A-G-C-A-A-C-G-G-A-C-U-Gm-A-A- ms(2)i(6)A-A-psi-C-C-G-U-G-U-m(7)G-U-C-G-G-C-G-G-T-psi- C-G-A-U-U-C-C-G-U-C-C-C-G-A-G-C-C-A-C-C-A(OH).Images

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... Cloverleaf model of Bacillus stearoihermophilus tRNA Phe. Between brackets the differences found in Bacillus subtilis tRNA Phe [8]. ...
... The tRNA Phe from B. stearothermophilus is very similar to tRNA Phe from B. subtilis (difference 3 nucleotides) [8] whereas both B. stearothermophilus and B. subtilis tRNA Phe differ substantially from E. colitRNA Phe [9] (21 nucleotides and 22 nucleotides, respectively). ...
... For example, the two Phe codons (UUU and UUC) are recognized, through wobble, by a single species of tRNA with the anticodon sequence GAA. While the G at the wobble position may be modified [e.g. to 2 0 -O-methylguanosine in Bacillus subtilis (29)], it appears that the UUC codon is always better recognized and thus the translationally optimal codon (11). ...
Article
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Among bacteria, many species have synonymous codon usage patterns that have been influenced by natural selection for those codons that are translated more accurately and/or efficiently. However, in other species selection appears to have been ineffective. Here, we introduce a population genetics-based model for quantifying the extent to which selection has been effective. The approach is applied to 80 phylogenetically diverse bacterial species for which whole genome sequences are available. The strength of selected codon usage bias, S, is found to vary substantially among species; in 30% of the genomes examined, there was no significant evidence that selection had been effective. Values of S are highly positively correlated with both the number of rRNA operons and the number of tRNA genes. These results are consistent with the hypothesis that species exposed to selection for rapid growth have more rRNA operons, more tRNA genes and more strongly selected codon usage bias. For example, Clostridium perfringens, the species with the highest value of S, can have a generation time as short as 7 min.
... The other advantage of analyzing these amino acid codons is that G at the wobble position is very specific for its pairing with either C or U. This specificity of G limits its modification only to queuosine (Harada and Nishimura 1972) or 2 0 -O-methylguanosine (Arnold and Keith 1977) at the wobble position (Osawa et al. 1992). The C-ending codons are preferred to the U-ending codons in Asn, Ile, Phe, and Tyr due to more stable base pairing between G and C at the wobble position. ...
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It is generally believed that the effect of translational selection on codon usage bias is related to the number of transfer RNA genes in bacteria, which is more with respect to the high expression genes than the whole genome. Keeping this in the background, we analyzed codon usage bias with respect to asparagine, isoleucine, phenylalanine, and tyrosine amino acids. Analysis was done in seventeen bacteria with the available gene expression data and information about the tRNA gene number. In most of the bacteria, it was observed that codon usage bias and tRNA gene number were not in agreement, which was unexpected. We extended the study further to 199 bacteria, limiting to the codon usage bias in the two highly expressed genes rpoB and rpoC which encode the RNA polymerase subunits β and β', respectively. In concordance with the result in the high expression genes, codon usage bias in rpoB and rpoC genes was also found to not be in agreement with tRNA gene number in many of these bacteria. Our study indicates that tRNA gene numbers may not be the sole determining factor for translational selection of codon usage bias in bacterial genomes.
... 32P-labeled samples were counted in 10 ml of water by Cerenkov radiation (3). Doubly labeled samples (32P and 3H) were counted in a toluene-Triton X-100 mixture (2:1) containing 0.1 g of dimethyl-POPOP ([2] -(4-methyl-5-phenyl- oxazolyl)benzene) and 4 g of PPO (2,5-diphenyloxa- zole) per liter. Spillover of 32P was subtracted from the 3H channel. ...
Article
The synthesis of transfer ribonucleic acid (tRNA) was examined during spore formation and spore outgrowth in Bacillus subtilis by two-dimensional polyacrylamide gel electrophoresis of in vivo 32P-labeled RNA. The two-dimensional gel system separated the B. subtilis tRNA's into 32 well-resolved spots, with the relative abundances ranging from 0.9 to 17% of the total. There were several spots (five to six) resolved which were not quantitated due to their low abundance. All of the tRNA species resolved by this gel system were synthesized at every stage examined, including vegetative growth, different stages of sporulation, and different stages of outgrowth. Quantitation of the separated tRNA's showed that in general the tRNA species were present in approximately the same relative abundances at the different developmental periods. tRNA turnover and compartmentation occurring during sporulation were examined by labeling during vegetative growth followed by the addition of excess phosphate to block further 32P incorporation. The two-dimensional gels of these samples showed the same tRNA's seen during vegetative growth, and they were in approximately the same relative abundances, indicating minimal differences in the rates of turnover of individual tRNA's. Vegetatively labeled samples, chased with excess phosphate into mature spores, also showed all of the tRNA species seen during vegetative growth, but an additional five to six minor spots were also observed. These are hypothesized to arise from the loss of 3'-terminal residues from preexisting tRNA's.
... The sequence of the E. coli equivalent tRNA suggests that the probable modification is m 6 A. A methylated adenosine was also mapped to position 22 of the tRNA Leu UAA ( Figure S2B). This adenosine was identified as m 1 A, based on prior literature [65], and this was confirmed by RTsignature analysis ( Figure S1 and Table S3). We also mapped the m 2 A found at position 37 of tRNA Glu UUC, having mnm 5 s 2 U at U34 ( Figure S2C). ...
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Extensive knowledge of both the nature and position of tRNA modifications in all cellular tRNAs has been limited to two bacteria, Escherichia coli and Mycoplasma capricolum. Bacillus subtilis sp subtilis strain 168 is the model Gram-positive bacteria and the list of the genes involved in tRNA modifications in this organism is far from complete. Mass spectrometry analysis of bulk tRNA extracted from B. subtilis, combined with next generation sequencing technologies and comparative genomic analyses, led to the identification of 41 tRNA modification genes with associated confidence scores. Many differences were found in this model Gram-positive bacteria when compared to E. coli. In general, B. subtilis tRNAs are less modified than those in E. coli, even if some modifications, such as m1A22 or ms2t6A, are only found in the model Gram-positive bacteria. Many examples of non-orthologous displacements and of variations in the most complex pathways are described. Paralog issues make uncertain direct annotation transfer from E. coli to B. subtilis based on homology only without further experimental validation. This difficulty was shown with the identification of the B. subtilis enzyme that introduces ψ at positions 31/32 of the tRNAs. This work presents the most up to date list of tRNA modification genes in B. subtilis, identifies the gaps in knowledge, and lays the foundation for further work to decipher the physiological role of tRNA modifications in this important model organism and other bacteria.
Chapter
This chapter focuses on the synthesis and function of modified nucleosides in transfer RNA (tRNA). The modification pattern in the anticodon region of organelle tRNAs is described. At position 34 of mitochondrial tRNAs an unmodified U is very common which renders the tRNA the potential to decode the whole family of four codons (NNU, NNC, NNA, NNG). If necessary, the wobble position is modified to cmnm5U to avoid misreading in those families of four codons that specify two different amino acids. Transfer RNA-modifying enzymes that catalyze the synthesis of m5U54, m1G37, mnm5s2U34, mcmo5U, Q34, and Ψ38, 39, 40 have been purified to near homogeneity from Escherichia coli (E. coli)/Salmonella typhimurium as well as the tRNA(Gm18)methyl-transferase from Thermus thermophilus. Eucaryotic tRNAs contain more modified nucleosides than tRNAs from eubacteria, and consequently more genes for tRNA modifying enzymes must be present in, for instance, yeast than in E. coli. Transfer RNA from yeast which reads codons starting with U contains i6A37. Antisuppressor mutations of yeast, sin1and mod5-1, both contain an unmodified A37 instead of i6A37.
Article
Clones earring Bacillue aubtilis tRNA genes were isolated from a 003B3;816 library. A recombinant phage 003B3;816-BS83 which was hybridized effectively with unfractionated tRNA probes contained a 3-kb fragment. By a Southern's blot analysis, it was found that tRNA genes were located in Eco RI-Hinc II region of this fragment. Sequence determination revealed the presence of a cluster of four tRNA genes in this region. The gene organization was as follows: tDNALyS-9bp-tDNAGlU-81bp-tDNAAsP-30bp-tDNAPhe. The RNA sequences expected from tDNALys and tDNAphe were identical with the reported RNA sequences. Two tRNA genes, tDNALys and tDNAAsp encoded the CCA sequence of 3'-terminal region, but the other two, tDNAGlu and tDNAphe did not. A promoter-like sequence which corresponds to the 003C3;55-recognition site was found in a region about l00bp upstream from tDNALys
Article
RNA methylation gives rise to a variety of methylated nucleotides and methylated derivatives of all the base components of RNA are known. RNA methyltransferases catalyze the post-transcriptional modification of RNA, and methylated nucleosides are present in rRNA and mRNA as well as in tRNA. This chapter discusses the properties of the enzymes responsible for the methylation of tRNA, particularly on enzymes that have been highly purified. S-adenosylmethionine serves as the methyl donor in the vast majority of RNA methylations. The substrate RNA is usually derived from a different source than the methyltransferase, because homologous RNA would already be completely methylated. Methyl-deficient tRNA, isolated from mutants of E. coli, has also been useful in the detection and isolation of tRNA methyltransferase enzymes. Some specific methyltransferase enzymes include 5-methylcytidine, 1-methyladenosine, and 1-methylguanosine. Further advances in isolation of RNA methylase mutants can significantly help in understanding the biological regulation and function of tRNA modification enzymes.
Article
Six thionucleosides found in Bacillus subtilis transfer ribonucleic acids were investigated: N6-(delta 2-isopentenyl)-2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, 2-methylthioadenosine, N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine, and one unknown (X1). The presence of N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine was demonstrated based on the affinity of the transfer ribonucleic acid containing it for an immunoadsorbent made with the antibody directed toward N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine. The existance of N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine in two species of lysine transfer ribonucleic acids was also confirmed by high-resolution mass spectrometry. Four of these thionucleosides--N6-(delta 2-isopenenyl)-2-methylthioadenosine, 2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, and the unknown designated X1--occurred only in specific areas in the elution profile of an RPC-5 column and probably affect the chromatographic properties of the transfer ribonucleic acids containing them. In contrast with Escherichia coli, where 4-thiouridine is the most frequent type of sulfur-containing modification, approximately one-third of the sulfur groups in B. subtilis transfer ribonucleic acid are present as thiomethyl groups on the 2 position of an adenosine or modified adenosine residue.
Article
Mitochondrial tRNAPhe from Saccharomyces cerevisiae isolated by two-dimensional gel electrophoresis was sequenced by fingerprinting uniformly labeled 32 p -tRNA as well as by 5′-end postlabeling techniques. Its sequence was found to be : pG-C-U-U-U-U-A-U-A-G-C-U-U-A-G-D-G-G-D-A-A-A-G-C-m22G-A-U-A-A-A-ψ-U-G-A-A-m1G-A-ψ-U-U-A-U-U-U-A-C-A-U-G-U-A-G-U-ψ-C-G-A-U-U-C-U-C-A-U-U-A-A-G-G-G-C-A-C-C-A. The secondary structure we propose, in order to maximize base pairing in the ψC stem and to allow tertiary interaction between G15 and C46 excludes U50 from base pairing giving a bulge in the ψC stem. No conclusion can be drawn concerning the endosymbiotic theory of mitochondria evolution by comparing the primary structure of mt. tRNAPhe with other sequenced tRNAsPhe. This mt.tRNAPhe lacks some of the structural elements reported to be involved in the yeast cytoplasmic phenylalanyl-tRNA ligase recognition site and cannot be aminoacylated by purified yeast cytoplasmic phenylalanyl-tRNA ligase.
Article
Highly purified tRNAPhe from rabbit liver, calf liver and bovine liver were completely digested with pancreatic ribonuclease and ribonuclease T1. The oligonucleotides were separated and identified. The tRNAPhe from rabbit liver and calf liver were partially cleaved with ribonuclease T1 or by action of lead acetate. We describe the analyses of the large fragments and the derivation of the primary structure of these mammalian tRNAsPhe.
Article
A series of sequence variants of amber suppressor genes of tRNA(Phe) were synthesized in vitro and cloned in Escherichia coli to examine the contributions of individual nucleotides to identity for amino acid acceptance. Three different but complementary types of tRNA variants were constructed. The first involved the substitution of base-pairs on the cloverleaf stem regions of the E. coli tRNA(Phe). The second type of variant involved total gene synthesis based on wild-type tRNA(Phe) sequences found in Bacillus subtilis and in Halobacterium volcanii. In the third type of variant, the identity of E. coli tRNALys was changed to that of tRNA(Phe). The nucleotides which are important for tRNA(Phe) identity in E. coli are located on the corner of the L-shaped tRNA molecule, where the dihydrouridine loop interacts with the T loop, and extend to the interior opening of the anticodon stem and the adjoining variable loop. The nucleotide sequence on the dihydrouridine stem region, which joins the corner and stem regions, was not successfully studied though it may contribute to tRNA(Phe) identity. The fourth nucleotide from the 3' end of tRNA(Phe) has some importance for identity.
Article
tRNAITyr and tRNAIITyr have been purified from B.subtilis and their nucleotide sequence determined. tRNAITyr differs from tRNAIITyr only by the extent of modification of the adenosine in 3′ position adjacent to the anticodon, i6A and ms2i6A respectively.
One of the characteristics of tRNAs is that they contain a variety of atypical (also called modified or rare) nucleosides. Modified nucleosides also occur in other RNAs — mRNAs, rRNAs, small nuclear RNAs (snRNAs) — and even in DNAs, but never in such high proportions. For example, 15 of the total 75 nucleosides in beef liver tRNATrp are modified (Fournier et al. 1978). Furthermore, the modified nucleosides found in tRNA exhibit a wide range of structural variations: more than 50 have been isolated and characterized. Some are modified by a single methylation of the base or on the 2′ hydroxyl of the ribose moiety, but there are also a number of so-called hypermodified nucleosides with more complex modifications.
Article
This chapter discusses three tRNA modifications: ribosylthymine (T) at position 54; N6-(2-isopentenyl) adenosine (i6A) and its derivatives at position 37; and queuosine (Q) and its derivatives at position 34. These modified nucleosides in tRNAs provide cells with a survival advantage under unfavorable environmental conditions, thus, indicating that these tRNA modifications probably played an important role in the evolution of organisms. A total lack of any one of these modifications in an organism that usually contains the normal nucleoside in specific tRNAs decreases its ability to survive under unfavorable environmental conditions. The modification T in elongator tRNAs of eubacteria and of eukaryotes increases the Vmax of ribosomal A-site interaction of a cognate (EF-Tu)·GTP·(AA-tRNA) complex. These changes are probably important to regulate the overall rate of protein synthesis. In cell-free systems of homopeptide synthesis, the absence of T in tRNA increases significantly the inisincorporation of an amino acid by an error-prone tRNA. The modification of A-37 in specific tRNAs of microorganism also exhibits an all-or-none effect in codon recognition. The replacement of i6A by A, or ms2i6A by i6A reverses the suppression of particular codons by specific suppressor tRNAs. In eukaryotes, Q modification of tRNA has been suggested to play a role in the processes of differentiation and aging and neoplastic transformation.
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Epitranscriptomics heavily rely on chemical reagents for the detection, quantification, and localization of modified nucleotides in transcriptomes. Recent years have seen a surge in mapping methods that use innovative and rediscovered organic chemistry in high throughput approaches. While this has brought about a leap of progress in this young field, it has also become clear that the different chemistries feature variegated specificity and selectivity. The associated error rates, e.g., in terms of false positives and false negatives, are in large part inherent to the chemistry employed. This means that even assuming technically perfect execution, the interpretation of mapping results issuing from the application of such chemistries are limited by intrinsic features of chemical reactivity. An important but often ignored fact is that the huge stochiometric excess of unmodified over‐modified nucleotides is not inert to any of the reagents employed. Consequently, any reaction aimed at chemical discrimination of modified versus unmodified nucleotides has optimal conditions for selectivity that are ultimately anchored in relative reaction rates, whose ratio imposes intrinsic limits to selectivity. Here chemical reactivities of canonical and modified ribonucleosides are revisited as a basis for an understanding of the limits of selectivity achievable with chemical methods. Epitranscriptomic (RNA modification) analysis by high throughput approaches heavily relies on chemical reagents used for the detection, quantification, and localization of modifications. Such modified residues differ from their unmodified counterparts by reactivity toward specific chemicals. Here chemical reactivities of canonical and modified ribonucleosides are revisited as a basis for an understanding of the limits of selectivity achievable with chemical methods.
Article
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Purified yeast phenylalanyl-tRNA synthetase can aminoacylate (yeast) tRNAPhe, (wheat) tRNAPhe, and (Escherichia coli) tRNA1Val (1, 2). We now report that this synthetase can also aminoacylate (E. coli) tRNAPhe and (E. coli) tRNA1Ala. Highly purified (E. coli) tRNAPhe is heterologously aminoacylated to approximately 90% of the extent achieved with the homologous enzyme (crude E. coli phenylalanyl-tRNA synthetase). Pure (E. coli) tRNA1Ala (the major species) is heterologously aminoacylated to 70% of the extent achieved with the homologous synthetase (crude E. coli alanyl-tRNA synthetase). (E. coli) tRNAPhe is the fourth purified transfer RNA of known sequence to be shown to be an acceptable substrate for purified yeast phenylalanyl-tRNA synthetase. A comparison of these sequences shows that only one region is extremely similar in all four tRNAs. This region is located adjacent to the dihydrouridine loop, and consists of the nucleotides [Formula: see text] We conclude that this is the synthetase recognition site for yeast phenylalanyl-tRNA synthetase. This conclusion is further supported by partial fragment analysis of (E. coli) tRNA1Ala.
Article
When Escherichia coli MRE 600 or Bacillus subtilis W 23 are grown in glucose-salt medium supplemented with purines, thymidine and glycine, trimethoprim stops the synthesis of protein by causing a specific lack of methionyl-tRNA. The synthesis of RNA is simultaneously restricted by the stringent control mechanism. Guanosine tetraphosphate (ppGpp) largely accumulates. The addition of methionine abolishes the level of ppGpp and relieves the inhibition of RNA synthesis. The aminoacylation of methionine-specific tRNAs was found to be completely restored. The methionyl-tRNAfMet however does not become formylated. These results indicate that unformylated initiator tRNA is not a sufficient condition for the accumulation of ppGpp and the onset of stringent control.
Article
Three tRNAs specific for methionine, phenylalanine and tyrosine were isolated from the total tRNA of Bacillus subtilis by chromatographic procedures using BD-cellulose and reversed-phase (5) chromatography. The acceptor activities of the purified tRNAs are 1160, 1260 and 1320 pmoles per A260nm unit for tRNAMetf, tRNAPhe and tRNATyr2 respectively. In tRNAMetf and tRNAPhe ribothymidine, pseudouridine and dihydrouridine are present, in addition, in tRNAPhe 7-methyguanosine and a 2'-O-methylated nucleoside were found. The modified nucleosides of tRNATyr2 are ribothymidine, pseudouridine, dihydrouridine, 4-thiouridine and 1-methyladenosine. The results suggest the presence of 2-methylthio-N6(delta 2-isopentenyl)adenosine in tRNAPhe and tRNATyr2. The thermal denaturation profiles of the three tRAN species are presented.
Article
The major methylated bases of tRNA of Bacillus subtilis are 5-methyluracil (m5U), 7-methylguanine (m7G) and 1-methyladenine (m1A). In addition, tRNA from stationary phase cultures always contains considerable amounts of an unidentified methylated pyrimidine derivative, “P.” Trimethoprim, which inhibits tetrahydrofolate (FH4)-dependent transmethylation reactions, was used to accumulate m5U-deficient tRNA in B. subtilis. With Escherichia coli enzymes the methyl moiety of S-adenosylmethionine (SAM) can be incorporated into the tRNA, and 0.7 m5U residue is formed per one tRNA molecule. The FH4-dependent formation of m5U in tRNA of B. subtilis as well as SAM-dependent methylations are affected by antibiotics which interfere with ribosomal functions. tRNA from pactamycin- or chloramphenicol-treated cells serves as substrate for SAM-dependent m5U- and m7G-specific tRNA methyltransferases from E. coli. With homologous SAM-dependent tRNA methyltransferases considerably fewer, but specific, transmethylations were found. tRNA obtained from a methionine-requiring stringent mutant of B. subtilis starved for methionine in the presence of chloramphenicol accepts the methyl moiety from SAM with homologous enzymes. The pattern of the methylated bases upon in vitro methylation of this tRNA agrees well with the pattern of methioninederived methylated bases in total tRNA of B. subtilis. Significant alterations were found in the relative distribution of tRNA isoacceptors from pactamycin-treated B. subtilis, which may reflect an accumulation of premature tRNAs.
Article
Highly purified tRNATrp from brewer's yeast prepared by countercurrent distribution followed by column chromatography on BD cellulose has been completely digested with pancreatic ribonuclease and T1 ribonuclease. The separation and the identification of the products are described. Analyses indicate that this tRNA is composed of 75 nucleotide residues including 17 minor nucleotides. This tRNA contains the G-T-Ψ-C sequence common to all tRNAs of known structure concerned in protein biosynthesis. The 5′terminal sequence is pG-A-A-G-Cp and the 3′terminal end is U-U-U-C-A-C-C-A. Only one endonucleotidic sequence, Cm-C-A, is able to form hydrogen bonds with the tryptophan codon U-G-G. The primary structure of this tRNA has been determined after partial digestion with pancreatic ribonuclease as stated in the following paper.
Article
It has been found that purified phenylalanyl- and valyl-tRNA synthetases from yeast catalyse the mischarging of numerous heterologous Escherichia coli and even of homologous yeast tRNAs when special aminoacylation conditions are used. For instance E. coli tRNAAla, tRNAVal, tRNALys and yeast tRNAAla, tRNAVal can be incorrectly aminoacylated by yeast phenylalanyl-tRNA synthetase, whereas E. coli tRNAAla, tRNAMet, tRNAIle, tRNAThr and yeast tRNAAla, tRNAPhe can be mischarged by yeast valyl-tRNA synthetase. The production of errors is highly dependant on the experimental conditions used, as their number and their level is increased when the Mg2+/ATP ratio or the enzyme concentration in the medium is increased or when the reactions are performed in the presence of dimethylsulfoxide. In the case of phenylalanyl-tRNA synthetase, conditions can be found where practically all E. coli tRNAs are wrongly aminoacylated, although at different levels. On the other hand, in the case of homologous systems some errors can only be detected when purified tRNAs are used, thus suggesting, when total tRNA is used, a competition between the cognate and non-cognate tRNAs which minimises the mischarging. The comparison of the sequences of the cognate and non cognate tRNAs which are aminoacylated either by the yeast phenylalanyl- or valyl-tRNA synthetase led us to ascribe some importance to several regions inside of these tRNAs, for instance (a) the dihydrouracil arm, (b) the terminal part of the amino-acid acceptor stem and (c) the extra-loop. These regions should be essentially necessary for the establisment of the correct tri-dimensional conformation necessary for the recognition by the aminoacyl-tRNA synthetases.
Article
Methionine-, valine-, phenylalanine- and tyrosine-specific tRNA were isolated from Escherichia coli by the use of two column chromatographic procedures: i.e., DEAE-Sephadex column chromatography and reverse-phase partition column chromatography. From the several criteria, it has been concluded that these amino acid-specific tRNA's are pure enough to use for structural study. Phenylalanine- and tyrosine-tRNA were clearly separated into two components by DEAE-Sephadex column chromatography.The base composition of methionine- and tyrosine-tRNA markedly deviated from that of unfractionated tRNA, but valine- and phenylalanine-tRNA had similar composition to unfractionated tRNA.The total increase in the absorbance of methionine-tRNA due to heating was 32 % which was considerably higher than that of other tRNA's, suggesting that methionine-tRNA formed a more rigid secondary structure than other tRNA's.The thermal denaturation curve of the amino acid-specific tRNA's was quite different from that of unfractionated tRNA. Especially the melting temperature (Tm) of methionine- and phenylalanine-tRNA in the absence of Mg2+ was 65° and 68° respectively, which was higher than that of unfractionated tRNA. Tm of methionine- and valine-tRNA in the presence of Mg2+ was 83°, which was considerably higher than the value of 75° given by unfractionated tRNA.
Article
Initiator methionine tRNA, tRNAfMet, was purified from Bacillus subtilis W168 by the use of two column chromatographies on DEAE-Sephadex A-50 and BD-cellulose. The nucleotide sequence was determined to be pC-G-C-G-G-G-G-U-G-G-A-G-C-A-G-U-U-C-G-G-D-A-G-C-U-C-G-U-C-G-G-G-C-U-C-A-U-A-A-C-C-C-G-A-A-G-G-U-C-G-C-A-G-G-T-psi-C-A-A-A-U-C-C-U-G-C-C-C-C-C-G-C-A-A-C-C-AOH. This TRNAfMet exhibited a melting temperature 8 degrees C lower than that of E. coli tRNAfMet in the presence of 0.01 M magnesium acetate.
  • D Kern
  • R Giege
  • J P Ebel
Kern, D., Giege, R. and Ebel, J.P. (1972) Eur. J. Biochem. 31, 148-155.
  • H H Arnold
  • R Raettig
  • G Keith
Arnold, H.H., Raettig, R. and Keith, G. (1977) FEBS Letts. 73, 210-214.
Postsynthetic Modification of Macromolecules pp
  • H Kersten
  • W Kersten
Kersten, H. and Kersten, W. (1975) in: FEBS Proc. 9th Meeting, Budapest 1974 Vol. 34, Postsynthetic Modification of Macromolecules pp. 99-llo, North-Holland, Amsterdam.
  • R H Doi
Doi, R.H. (1971) in: Methods in Molecular Biology (Laskia, A.J. and Last, J.A., eds.( Vol. 1, pp. 67-88, Marcel Dekker, New York.
  • C Geurrier-Takada
  • G Dirheimer
  • H Grosjean
  • G Keith
Geurrier-Takada, C., Dirheimer, G., Grosjean, H. and Keith, G. (1975) FEBS Letts. 60, 286-289.
  • S Nishimura
  • F Harada
  • U Narushima
  • T Seno
Nishimura, S., Harada, F., Narushima, U. and Seno, T. (1967) Biochim. Biophys. Acta 142, 133-148.