Aminoacyl-tRNA synthetase-catalyzed cleavage of the glycosidic bond of 5-halogenated uridines.

Koontz, Stephen W.; Schimmel, Paul

doi:10.1016/s0021-9258(19)86307-x

Cited by 30 publications

(4 citation statements)

References 14 publications

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“…Direct contact of VRS with FU8 is consistent with proposals suggesting that a covalent bond between U8 of tRNA and aminoacyl-tRNA synthetases is a common intermediate in the aminoacylation reaction, at least in prokaryotes (Schoemaker & Schimmel, 1977; Koontz & Schimmel, 1979;Starzyk et al, 1982). Inactivation of synthetases by 5-bromouridine, and the synthetase-catalyzed exchange reaction between solvent and hydrogen at carbon-5 of uracil-8, suggested formation of a Michael adduct by nucleophilic attack of an enzyme SH group on the 6-position of U8 in tRNA.…”

Section: Discussionsupporting

confidence: 88%

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study

Chu¹,

Horowitz²

1991

Biochemistry

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Interactions of 5-fluorouracil-substituted Escherichia coli tRNAVal with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene [Heck, J.D., & Hatfield, G.W. (1988) J. Biol. Chem. 263, 868-877]. Apparent KM and Vmax values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNAVal. Binding of VRS to (FUra)tRNAVal induces structural perturbations that are reflected in selective changes in the 19F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNAVal along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNAVal, suggesting conformational changes in this part of the molecule. No 19F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNAVal that has been proposed as a common intermediate in the aminoacylation reaction.

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Section: Discussionsupporting

confidence: 88%

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study

Chu¹,

Horowitz²

1991

Biochemistry

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“…Thymidylate synthetase catalyzes the debromination of BrdUMP (Garrett et al, 1979), and tRNA synthetases catalyze deribosylation of BrU (Koontz & Schimmel, 1979). Coat protein was assayed under a variety of conditions for its ability to catalyze some conversion of BrU nucleosides.…”

Section: Resultsmentioning

confidence: 99%

Nucleoside and nucleotide inactivation of R17 coat protein: evidence for a transient covalent RNA-protein bond

Romaniuk¹,

Uhlenbeck²

1985

Biochemistry

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R17 coat protein forms a specific complex with a 21-nucleotide RNA hairpin containing the initiation site for the phage replicase gene. The RNA binding activity of the protein is inhibited by prior incubation with 5-bromouridine (BrU). The inactivation occurs with pseudo-first-order kinetics, and the inactive protein is stable to dilution. RNA binding activity of the BrU-inactivated protein is restored upon incubation with dithiothreitol. Inactivation of coat protein by N-ethylmaleimide or p-(chloromercuri)-benzenesulfonate indicates that a cysteine residue is located near the RNA binding site. Since 5-bromodeoxyuridine does not inactivate coat protein, a specific binding event appears to be required before inactivation can occur. Surprisingly, unmodified cytidine nucleotides also inactivate coat protein, with a specificity similar to the modified analogues. These results are discussed with regard to the formation of a transient covalent RNA-protein bond.

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“…Several studies have focused on characterizing the molecular elements that mediate the interaction between RNA binding proteins and their target RNA. The R17 bacteriophage coat protein (21±23), aminoacyl-tRNA synthetase (24,25) and iron-responsive factor (26,27) represent three well-characterized RNA binding proteins. In each instance, a free cysteine sulfhydryl group(s) on the RNA binding protein forms a covalent Michael adduct with the C-6 position on the corresponding uracil ring of the target RNA.…”

Section: Discussionmentioning

confidence: 99%

Role of cysteine amino acid residues on the RNA binding activity of human thymidylate synthase

Lin

Maley

et al. 2003

Nucleic Acids Research

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The role of cysteine sulfhydryl residues on the RNA binding activity of human thymidylate synthase (TS) was investigated by mutating each cysteine residue on human TS to a corresponding alanine residue. Enzymatic activities of TS:C43A and TS:C210A mutant proteins were nearly identical to wild-type TS, while TS:C180A and TS:C199A mutants expressed >80% of wild-type enzyme activity. In contrast, TS:C195A was completely inactive. Mutant proteins, TS:C195A, TS:C199A and TS:C210A, retained RNA binding activity to nearly the same degree as wild-type human TS. RNA binding activity of TS:C43A was reduced by 30% when compared to wild-type TS, while TS:C180A was completely devoid of RNA binding activity. In vitro translation studies confirmed that mutant proteins TS:C43A, TS:C195A, TS:C199A and TS:C210A, significantly repressed human TS mRNA translation, while TS:C180A was unable to do so. To confirm the in vivo significance of the cysteine sulfhydryl residue, mutant proteins TS:C180A and TS:C195A were each expressed in human colon cancer HCT-C18:TS(-) cells that expressed a functionally inactive TS. A recombinant luciferase reporter gene under the control of a TS-response element was co-transfected into these same cells, and luciferase activity increased in the presence of the TS:C195A mutant TS protein to a level similar to that observed upon expression of wild-type TS protein. In contrast, luciferase activity remained unchanged in cells expressing the TS:C180A mutant protein. Taken together, these findings identify Cys-180 as a critical residue for the in vitro and in vivo translational regulatory effects of human TS.

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Aminoacyl-tRNA synthetase-catalyzed cleavage of the glycosidic bond of 5-halogenated uridines.

Cited by 30 publications

References 14 publications

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study

Nucleoside and nucleotide inactivation of R17 coat protein: evidence for a transient covalent RNA-protein bond

Role of cysteine amino acid residues on the RNA binding activity of human thymidylate synthase

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