Quantitative enzymatic hydrolysis of tRNAs

Gehrke, Charles W.; Kuo, Kenneth C.; McCune, Roy A.; Gerhardt, Klaus O.; Agris, Paul F.

doi:10.1016/s0378-4347(00)80479-x

Cited by 158 publications

(36 citation statements)

References 16 publications

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“…35 S-labeled tRNA was isolated as described (11,16). Nuclease digestion of tRNA was performed as described by Gehrke et al (33) with minor modifications. About 150 g of tRNA in 25 l of water was heated at 100°C for 2 min, cooled on ice, and then digested by incubation at 37°C for 4 h in a reaction mixture containing 0.65 mM ZnSO 4 , 6 units of P1 nuclease, and 0.16 mg͞ml of potato phosphatase in a total volume of 62 l. The mixture was heated at 100°C for 5 min and centrifuged (10,000 ϫ g).…”

Section: Methodsmentioning

confidence: 99%

TheiscSgene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H

Mihara

Kato

Lacourciere

et al. 2002

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

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Three NifS-like proteins, IscS, CSD, and CsdB, from Escherichia coli catalyze the removal of sulfur and selenium from L-cysteine and L-selenocysteine, respectively, to form L-alanine. These enzymes are proposed to function as sulfur-delivery proteins for iron-sulfur cluster, thiamin, 4-thiouridine, biotin, and molybdopterin. Recently, it was reported that selenium mobilized from free selenocysteine is incorporated specifically into a selenoprotein and tRNA in vivo, supporting the involvement of the NifS-like proteins in selenium metabolism. We here report evidence that a strain lacking IscS is incapable of synthesizing 5-methylaminomethyl-2-selenouridine and its precursor 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U) in tRNA, suggesting that the sulfur atom released from L-cysteine by the action of IscS is incorporated into mnm 5 s 2 U. In contrast, neither CSD nor CsdB was essential for production of mnm 5 s 2 U and 5-methylaminomethyl-2-selenouridine. The lack of IscS also caused a significant loss of the selenium-containing polypeptide of formate dehydrogenase H. Together, these results suggest a dual function of IscS in sulfur and selenium metabolism.

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Section: Methodsmentioning

confidence: 99%

TheiscSgene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H

Mihara

Kato

Lacourciere

et al. 2002

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

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“…All other nucleoside phosphoramidites were obtained from Glen Research (Sterling, VA). The oligomer was HPLC-purified as previously described (17) using a Nucleogen 60-7 DEAE (250 × 10 mm) column, and its nucleoside composition determined (18) (ii) Unimolecular Property of Samples. To demonstrate that the RNA was a monomer at the NMR concentrations of this study, we measured the translational diffusion coefficient of the unmodified and t 6 A 37 -modified ASLs, and compared them to that of other RNA sequences of various lengths.…”

Section: Methodsmentioning

confidence: 99%

Functional Anticodon Architecture of Human tRNA^Lys3 Includes Disruption of Intraloop Hydrogen Bonding by the Naturally Occurring Amino Acid Modification, t⁶A

et al. 2000

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The structure of the human tRNA Lys3 anticodon stem and loop domain (ASL Lys3 ) provides evidence of the physicochemical contributions of N6-threonylcarbamoyladenosine (t 6 A 37 ) to tRNA Lys3 functions. The t 6 A 37 -modified anticodon stem and loop domain of tRNA Lys3 UUU (ASL Lys3 UUU -t 6 A 37 ) with a UUU anticodon is bound by the appropriately programmed ribosomes, but the unmodified ASL Lys3 UUU is not [Yarian, C., Marszalek, M., Sochacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., and Agris, P. F., Biochemistry 39, 13390-13395]. The structure, determined to an average rmsd of 1.57 ( 0.33 Å (relative to the mean structure) by NMR spectroscopy and restrained molecular dynamics, is the first reported of an RNA in which a naturally occurring hypermodified nucleoside was introduced by automated chemical synthesis. The ASL Lys3 UUU -t 6 A 37 loop is significantly different than that of the unmodified ASL Lys3 UUU , although the five canonical base pairs of both ASL Lys3 UUU stems are in the standard A-form of helical RNA. t 6 A 37 , 3′-adjacent to the anticodon, adopts the form of a tricyclic nucleoside with an intraresidue H-bond and enhances base stacking on the 3′-side of the anticodon loop. Critically important to ribosome binding, incorporation of the modification negates formation of an intraloop U 33 ‚A 37 base pair that is observed in the unmodified ASL Lys3 UUU . The anticodon wobble position U 34 nucleobase in ASL Lys3 UUU -t 6 A 37 is significantly displaced from its position in the unmodified ASL and directed away from the codon-binding face of the loop resulting in only two anticodon bases for codon binding. This conformation is one explanation for ASL Lys3 UUU tendency to prematurely terminate translation and -1 frame shift. At the pH 5.6 conditions of our structure determination, A 38 is protonated and positively charged in ASL Lys3 UUU -t 6 A 37 and the unmodified ASL Lys3 UUU . The ionized carboxylic acid moiety of t 6 A 37 possibly neutralizes the positive charge of A + 38 . The protonated A + 38 can base pair with C 32 , but t 6 A 37 may weaken the interaction through steric interference. From these results, we conclude that ribosome binding cannot simply be an induced fit of the anticodon stem and loop, otherwise the unmodified ASL Lys3 UUU would bind as well as ASL Lys3 UUU -t 6 A 37 . t 6 A 37 and other position 37 modifications produce the open, structured loop required for ribosomal binding.Lysine tRNAs with UUU anticodons have a conventional role in ribosome-mediated protein synthesis. In addition, tRNA Lys UUU species facilitate -1 frameshifts for correct translation of the E. coli DNA polymerase γ subunit (1) and retroviral polymerases (2). Also, tRNA Lys UUU often misreads asparagine codons (3, 4) and peptidyl-tRNA Lys prematurely terminates translation more often than other tRNAs (5). In addition, reverse transcription of the HIV-1 genomic RNA is primed by the human tRNA Lys3 UUU . Formation of the viral replication initiation complex is enhanced in vitro by the pr...

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“…tRNA was extracted using NucleobondAX (Macherey-Nagel), following the manufacturer's instructions, eluted with 100 mM Tris/acetate, 15% ethanol, pH 6.3, and 0.6 M KCl, and digested (12 mg) with nuclease P1 (Roche) followed by bacterial alkaline phosphatase (Sigma) (Gehrke et al, 1982). After hydrolysis, samples were mixed with N 6 -(3-[1,1,1-2 H 3 ]methyl-[4,4,4-2 H 3 ]-but-2-enyl)adenosine ( 2 H 6 -i 6 A) (OlChemIm Ltd., Olomouc, Czech Republic) used as internal standard for LC-MS/MS analysis (50 ml, 95 pmol) and a 10-ml aliquot was injected into the HPLC column.…”

Section: Analysis Of I 6 a By Esi Lc-ms-msmentioning

confidence: 99%

Identification and functional characterization of the candidate tumor suppressor gene TRIT1 in human lung cancer

et al. 2005

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Quantitative enzymatic hydrolysis of tRNAs

Cited by 158 publications

References 16 publications

TheiscSgene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H

TheiscSgene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H

Functional Anticodon Architecture of Human tRNA^Lys3 Includes Disruption of Intraloop Hydrogen Bonding by the Naturally Occurring Amino Acid Modification, t⁶A

Identification and functional characterization of the candidate tumor suppressor gene TRIT1 in human lung cancer

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Quantitative enzymatic hydrolysis of tRNAs

Cited by 158 publications

References 16 publications

TheiscSgene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H

TheiscSgene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H

Functional Anticodon Architecture of Human tRNALys3 Includes Disruption of Intraloop Hydrogen Bonding by the Naturally Occurring Amino Acid Modification, t6A

Identification and functional characterization of the candidate tumor suppressor gene TRIT1 in human lung cancer

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Functional Anticodon Architecture of Human tRNA^Lys3 Includes Disruption of Intraloop Hydrogen Bonding by the Naturally Occurring Amino Acid Modification, t⁶A