Takeshi Seno

Purification of methionine-, valine-, phenylalanine- and tyrosine-specific tRNA from Escherichia coli.

Abstract 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 tRNAs 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 tRNAs, suggesting that methionine-tRNA formed a more rigid secondary structure than other tRNAs. The thermal denaturation curve of the amino acid-specific tRNAs 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.

Purification of Escherichia coli methionine tRNAF and methionine tRNAM and studies on their biophysical and biochemical properties

Abstract Purification of methionine tRNAF, methionine tRNAM1 and methionine tRNAM2 from Escherichia coli on a large scale has been achieved by the combination of three column chromatographic procedures; (1) DEAE-Sephadex A-50 column chromatography with an NaCl gradient elution, (2) rechromatography by DEAE-Sephadex A-50 eluted simply with a buffer containing 0.375 M NaCl, and (3) resolution of pure methionine tRNA into three multi-components by BD-cellulose column chromatography or by ‘Freon’ column chromatography. In comparison with methionine tRNAM1 and with methionine tRNAM2, methionine tRNAF exhibited both markedly deviating thermal denaturation patterns, with higher Tm values when measured either at 260 mμ or at 280 mμ, and an unusual increase in absorbance at 280 mμ. Base analysis of the tRNAs gave results to support the above observations: methionine tRNAF possessed an unusually high G+C content, thus contributing to a more rigid conformation, than those of the two methionine tRNAMs. As for minor base components, molar yields of 7-methyl guanosine and 4-thiouridine were identified in each of the three tRNAs. Molar yields of 2′-O-methylcytidylyl (3′ → 5′) uridine 3′-phosphate and cytidine-3′,5′-diphosphate were found in methionine tRNAF.

Characteristic behavior of 4-thiouridine region of individual amino acid-specific Escherichia coli tRNA's upon heat denaturation

Abstract With the use of ultraviolet absorbance at around 335 mμ, which is specific for 4-thiouracil residue in native Escherichia coli tRNA, the behavior of the 4-thiouridine region in response to heat denaturation has been investigated in comparison with that of the whole region by using seven individual amino acid-specific tRNAs. The data indicated that the melting out temperature of the 4-thiouridine region depended upon the amino acid-specific tRNA species used. In addition, the 4-thiouridine region in many of the tRNA species melted out in bi-modal fashion. Further study of the outstanding bi-modal melting of the 4-thiouridine region in tRNAMetF has indicated that a local rearrangement in the tRNA molecule occurred at around 70°, which was between the two phases. The discrepancy between the bi-modal behavior of 4-thiouridine and the fact that only one 4-thiouridine residue per tRNA molecule is present could be interpreted to suggest that the two-dimensional structure of tRNA was further folded to construct a more complicated form.

Involvement of the anticodon region of Escherichia coli tRNAGln and tRNAGlu in the specific interaction with cognate aminoacyl-tRNA synthetase: Alteration of the 2-thiouridine derivatives located in the anticodon of the tRNAs by BrCN or sulfur deprivation

Abstract Treatment of unfractionated Escherichia coli tRNA with BrCN produces a decrease in glutamine and glutamate acceptance. This decrease may be explained by the specific cyanation of the 2-thiouridine derivative, located at the 5′-terminal of the anticodon of the tRNAs corresponding to these amino acids; the modification could prevent the accurate fit between the tRNAs and their cognate aminoacyl-tRNA synthetases. The activity of tRNA was measured in two ways, by aminoacylation and by ATP—PP i exchange in the presence of tRNA. Transfer RNA Glu and tRNA Gln 1 showed a decrease in affinity for their cognate synthetases following BrCN treatment, indicated by a 10-fold increase in the apparent K m . tRNA Gln 2 and other tRNAs not possessing a BrCN-reactive group in the anticodon were not affected by BrCN treatment. Transfer RNA Glu or tRNA Gln prepared from a cysteine-requiring relaxed mutant of E. coli grown in sulfur-deficient medium, in which the usual complement of the 2-thiouridine derivative was lacking, could promote the ATP—PP i exchange reaction and be aminoacylated. This suggests that the presence of the 2-thiouridine derivative itself in the anticodon of the tRNA is not a necessary requirement of the tRNAs for the specific interaction with cognate aminoacyl-tRNA synthetase.

Recovery of transfer RNA functions by combining fragmented Escherichia coli formylmethionine transfer RNA

Abstract 1. Limited digestion of Escherichia coli tRNAfMet by ribonuclease T1 resulted mainly in a split at -G-D(dihydrouridine)-sequence in the D loop of the tRNA. The resulting 5′- and 3′-half molecules restored the methionine-acceptor activity and the transformylation ability only when mixed. No activities were recovered with either half alone. It was not necessary to preincubate the mixture of the two halves in any particular conditions for the restoration of the activities. 2. The saturation experiment showed that the active complex molecule was formed by a mutual one-to-one interaction of the 5′- and 3′-half molecules. Thermal denaturation experiment revealed that the 5′- and 3′-halves of the tRNA, when mixed, actually interacted to form a complex. However, another 3′-half molecule in which a CAACCA-OH sequence is missing from the 3′-end could not interact to form an active complex. 3. As for the code recognition ability, the reconstituted molecule showed abnormal binding to poly (U4, G) in the absence of ribosome. However, it maintained the ability to recognize the initiator triplets, ApUpG and GpUpG, in the presence of ribosome. As a conclusion, the intactness of the -G-D- sequence in the D loop is not required for the recognition of methionyl-RNA synthetase but is required for the formation of the correct ribosome-mRNA-tRNA complexes. 4. The data were discussed in reference to a possibility that the aminoacyl RNA synthetase recognizes the specificity of tertiary conformation of tRNA molecule rather than the specificity of primary nucleotide sequences. An important participation of the nucleotide sequence at the 3′-end of the tRNA molecule was also discussed on the basis of the present data.

Fragmented E. coli methionine tRNAf as methyl acceptor for rat liver tRNA methylase: Alteration of the site of methylation by the conformational change of tRNA structure resulting from fragmentation

Abstract In vitro methylation of E. coli tRNAfMet fragments or their reconstituted molecule by rat liver methylase showed that (1) the whole tRNA molecule was not necessary for recognition by tRNA methylase, and (2) the extent and the site of methylation of fragments differed depending upon the type of fragment used. This indicates that the conformational structure of the methyl acceptor molecule is important for recognition by the tRNA methylase.

Specific hybridization of amino acid-specific transfer RNA to DNA in Escherichia coli

Abstract The extent of heterogeneity of nucleotide sequences present among different types of Escherichia coli tRNA specific for individual amino acids has been examined by means of the DNA-[ 32 P]tRNA hybridization technique. The kinetic study revealed that methionine-specific [ 32 P]tRNA hybridized with about 5 % of the DNA region involved in the full saturation with unfractionated tRNA. Competition experiments with methionine-, valine-, phenylalanine- or tyrosine-specific tRNA as competitors showed that each type of tRNA is able to discriminate with respect to the others upon annealing with the corresponding sites on the DNA. The possible heterogeneity of nucleotide sequences among individual amino acid-specific tRNAs is discussed.