Effect of aminoacyl transfer RNA synthetases on H-5 exchange of specific pyrimidines in transfer RNAs

Schoemaker, H.J.; Schimmel, Paul

doi:10.1021/bi00644a009

Cited by 39 publications

(20 citation statements)

References 43 publications

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“…The unique sequences, which have been highly conserved during evolution (K. Randerath and E. Randerath, unpublished) may conceivably be involved in interactions with the cognate aminoacyl-tRNA synthetase or other proteins. It is interesting to note that the shaded sequences G5-U-U and C7'-U-U-A are located next to U-8, a constant nucleotide, which has been shown to participate in synthetase binding [51].…”

Section: Discussionmentioning

confidence: 99%

Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Randerath

Gupta

Chia

et al. 1979

European Journal of Biochemistry

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A second major species of leucine tRNA, t R N A e G (formerly designated t R N A e A ) was purified from baker's yeast in a three-step procedure entailing BD-cellulose chromatography in the presence and absence of Mg2+ and Sephadex G-100 gel filtration. Results of aminoacylation and partial RNase TI digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNAscAA (tRNAP").The primary structure of baker's yeast tRNA?lG was elucidated by application of sensitive radioactive isotope derivative ('postlabeling') methods. Complete RNaseTl and A and partial RNase Uz fragments, prepared from non-radioactive tRNA and 5'-half and 3'-half molecules, were separated by two-dimensional polyethyleneimine-cellulose anion-exchange thin-layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis. Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods. Molar ratios of the fragments were determined from the radioactivity of 3H-labeled nucleoside trialcohols in combination with base analysis. 2I-O-Methylated guanosine was characterized using the [y-32P]ATP/polynucleotide kinase reaction. The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the RNA. A total of about 20 A260 units of the RNA was used for analysis, i.e. considerably less material than required for conventional spectrophotometric analysis.A different sequencing approach, consisting of a combination of 'readout sequencing' with tritium sequencing of complete RNase TI and A fragments, was applied to the 3'-half molecule. The 3'-half molecule was labeled with 32P at its 5' terminus, partially degraded with RNases TI, UZ, and Phy, and with alkali, and subjected to polyacrylamide gel electrophoresis. The sequence was read off the gel on the basis of cleavage patterns and size of the fragments. While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides. The readout sequencing procedure was found to require less than 0.01 unit of RNA and the analysis of the complete fragments about 6 A260 units.Interesting structural features of tRNAfLG are (a) the location of unique, leucine tRNA isoacceptor-specific sequences next to U-8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1-methyladenosine in the T loop, a modification not present in the structurally related tRNA3cA,, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.

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Section: Discussionmentioning

confidence: 99%

Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Randerath

Gupta

Chia

et al. 1979

European Journal of Biochemistry

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“…Peak I has tentatively been assigned to FU8. These results agree with the interaction between VRS and position 8 of the tRNA molecule reported with other tRNA-synthetase complexes (Schoemaker and Schimmel, 1977).…”

Section: Recognition Of Trna^®^ By Valyl-trna Synthetasesupporting

confidence: 92%

“…^^F NMR spectra of fFUra)tRNA^^^ mutated at position 8 5-Fluorouracil at position 8 of (FUra)tRNA^^^ was replaced by G, C and A, to assign FU8 and to investigate the role of uracil 8 in the recognition of tRNA by aminoacyl-tRNA synthetase (Schoemaker and Schimmel, 1977). Figure 23 shows the ^^F NMR spectra of (FUra)tRNA^®^ mutants A8, C8 and G8.…”

Section: Assignment Of Fu59 In the T-loopmentioning

confidence: 99%

“…However, replacement of It was suggested that U8, which is conserved in all tRNA sequences determined to date (Sprinzl et al, 1978), is involved in the interaction of tRNA with aminoacyl-tRNA synthetase. Synthetase-catalyzed exchange of hydrogen for tritium at U8 (Schoemaker and Schimmel, 1977) and inactivation of synthetase by 5-bromouridine , suggested a covalent adduct between the uracil-8 ring and the active site of the synthetase. The requirement for a U at position 8 for the recognition of tRNA^^^ by VRS was examined in this study.…”

Section: Recognition Of Trna^®^ By Valyl-trna Synthetasementioning

confidence: 99%

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Recognition of valine transfer RNA by valyl-tRNA synthetase from E coli: 19F NMR and aminoacylation studies involving wild type and mutant forms of 5-fluorouracil-substituted tRNAVal

Chu

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“…While the mechanism of tRNA discrimination by synthetases is not known, there is evidence that synthetases form a transient covalent adduct through Michael addition of an enzyme nucleophile to the electrophilic C-6 position of the uracil ring at position 8 in the primary structure (4,5). [All prokaryote and cytoplasmic eukaryote tRNAs have a uridine or 4-thiouridine at position 8 (6)].…”

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confidence: 99%

Covalent enzyme-RNA complex: a tRNA modification that prevents a covalent enzyme interaction also prevents aminoacylation.

Starzyk¹,

Schoemaker²,

Schimmel³

1985

Proc. Natl. Acad. Sci. U.S.A.

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Previous work indicates that aminoacyltRNA synthetases make a transient covalent adduct with cognate tRNAs, through Michael addition of an enzyme nucleophile to the carbon-6 position of uridine 8. We report the selective reduction of the 5,6 double bond of 4-thiouridine at position 8 in Escherichia coli tyrosine tRNA, so as to prevent formation of the presumed covalent enzyme-nucleic acid adduct. The completely reduced tRNA molecules are inactivated for aminoacylation. With partial reduction, a mixed pool of active and inactive molecules is created and the degree of inactivation exactly matches the extent of 4-thiouridine reduction. The active molecules recovered from this mixed pool are specifically unaltered at position 8. The results are consistent with the view that the covalent enzyme-RNA adduct is an obligatory intermediate for aminoacylation of this tRNA. Na BH4

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Effect of aminoacyl transfer RNA synthetases on H-5 exchange of specific pyrimidines in transfer RNAs

Cited by 39 publications

References 43 publications

Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Recognition of valine transfer RNA by valyl-tRNA synthetase from E coli: 19F NMR and aminoacylation studies involving wild type and mutant forms of 5-fluorouracil-substituted tRNAVal

Covalent enzyme-RNA complex: a tRNA modification that prevents a covalent enzyme interaction also prevents aminoacylation.

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Effect of aminoacyl transfer RNA synthetases on H-5 exchange of specific pyrimidines in transfer RNAs

Cited by 39 publications

References 43 publications

Yeast tRNALeuUAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Yeast tRNALeuUAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Recognition of valine transfer RNA by valyl-tRNA synthetase from E coli: 19F NMR and aminoacylation studies involving wild type and mutant forms of 5-fluorouracil-substituted tRNAVal

Covalent enzyme-RNA complex: a tRNA modification that prevents a covalent enzyme interaction also prevents aminoacylation.

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Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods