A UGU sequence in the anticodon loop is a minimum requirement for recognition by Escherichia coli tRNA-guanine transglycosylase.

Nakanishi, Saburo; Ueda, Takuya; Hori, Hiroyuki; Yamazaki, N; Okada, Norihiro; Watanabe, Kimitsuna

doi:10.1016/s0021-9258(18)31624-7

Cited by 61 publications

(17 citation statements)

References 20 publications

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“…Noncovalent MS analysis of the tRNA preparation alone revealed substantial heterogeneity with the detection of several entities of 27 kDa, which could be undoubtedly assigned to tRNA species differing in the degree of phosphorylation as well as in the presence or absence of particular nucleotides at the 5′ or 3′ end (Figure S6). Such heterogeneities are classically observed in tRNA analysis and do not affect the recognition of tRNA Tyr by the TGT enzyme. ,, We next performed titration experiments involving a fixed concentration of the QTRT1/QTRT2 dimer and increasing amounts of tRNA Tyr (Figure C). Noncovalent MS analysis of a 1:1:1 QTRT1:QTRT2:tRNA Tyr molar ratio reveals that the most abundant ion series corresponds to a 1:1:1 QTRT1:QTRT2:tRNA Tyr complex (119469 ± 15 Da).…”

Section: Resultsmentioning

confidence: 98%

“…Such heterogeneities are classically observed in tRNA analysis and do not affect the recognition of tRNA Tyr by the TGT enzyme. 17,34,55 We next performed titration experiments involving a fixed concentration of the QTRT1/QTRT2 dimer and increasing amounts of tRNA Tyr (Figure 1C). Noncovalent MS analysis of a 1:1:1 QTRT1:QTRT2:tRNA Tyr molar ratio reveals that the most abundant ion series corresponds to a 1:1:1 QTRT1:QTRT2:tRNA Tyr complex (119469 ± 15 Da).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Homodimer Architecture of QTRT2, the Noncatalytic Subunit of the Eukaryotic tRNA-Guanine Transglycosylase

et al. 2018

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The bacterial enzyme tRNA-guanine transglycosylase (TGT) is involved in the biosynthesis of queuosine, a modified nucleoside present in the anticodon wobble position of tRNA, tRNA, tRNA, and tRNA. Although it forms a stable homodimer endowed with two active sites, it is, for steric reasons, able to bind and convert only one tRNA molecule at a time. In contrast, its mammalian counterpart constitutes a heterodimer consisting of a catalytic and a noncatalytic subunit, termed QTRT1 and QTRT2, respectively. Both subunits are homologous to the bacterial enzyme, yet only QTRT1 possesses all the residues required for substrate binding and catalysis. In mice, genetic inactivation of the TGT results in the uncontrolled oxidation of tetrahydrobiopterin and, accordingly, phenylketonuria-like symptoms. For this reason and because of the recent finding that mammalian TGT may be utilized for the treatment of multiple sclerosis, this enzyme is of potential medical relevance, rendering detailed knowledge of its biochemistry and structural architecture highly desirable. In this study, we performed the kinetic characterization of the murine enzyme, investigated potential quaternary structures of QTRT1 and QTRT2 via noncovalent mass spectrometry, and, finally, determined the crystal structure of the murine noncatalytic TGT subunit, QTRT2. In the crystal, QTRT2 is clearly present as a homodimer that is strikingly similar to that formed by bacterial TGT. In particular, a cluster of four aromatic residues within the interface of the bacterial TGT, which constitutes a "hot spot" for dimer stability, is present in a similar constellation in QTRT2.

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Section: Resultsmentioning

confidence: 98%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Homodimer Architecture of QTRT2, the Noncatalytic Subunit of the Eukaryotic tRNA-Guanine Transglycosylase

et al. 2018

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“…1), 28,29 while related modifications (pre-Q 0 and pre-Q 1 ) are catalyze by similar enzymes in Archaea and Bacteria respectively. 30,31 In general, the sequence U 33 G 34 U 35 is essential for TGT recognition, 32 where the almost universal U 33 , which forms the U-turn in the anticodon loop in most tRNAs, is part of the recognition motif. It is thought that for activity, TGT requires an unpaired G 34 target.…”

Section: Tbrucei Encodes a Tgt Homologmentioning

confidence: 99%

Retrograde nuclear transport from the cytoplasm is required for tRNA^Tyrmaturation inT. brucei

et al. 2017

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Retrograde transport of tRNAs from the cytoplasm to the nucleus was first described in Saccharomyces cerevisiae and most recently in mammalian systems. Although the function of retrograde transport is not completely clear, it plays a role in the cellular response to changes in nutrient availability. Under low nutrient conditions tRNAs are sent from the cytoplasm to nucleus and presumably remain in storage there until nutrient levels improve. However, in S. cerevisiae tRNA retrograde transport is constitutive and occurs even when nutrient levels are adequate. Constitutive transport is important, at least, for the proper maturation of tRNA, which undergoes cytoplasmic splicing, but requires the action of a nuclear modification enzyme that only acts on a spliced tRNA. A lingering question in retrograde tRNA transport is whether it is relegated to S. cerevisiae and multicellular eukaryotes or alternatively, is a pathway with deeper evolutionary roots. In the early branching eukaryote Trypanosoma brucei, tRNA splicing, like in yeast, occurs in the cytoplasm. In the present report, we have used a combination of cell fractionation and molecular approaches that show the presence of significant amounts of spliced tRNA in the nucleus of T. brucei. Notably, the modification enzyme tRNA-guanine transglycosylase (TGT) localizes to the nucleus and, as shown here, is not able to add queuosine (Q) to an intron-containing tRNA. We suggest that retrograde transport is partly the result of the differential intracellular localization of the splicing machinery (cytoplasmic) and a modification enzyme, TGT (nuclear). These findings expand the evolutionary distribution of retrograde transport mechanisms to include early diverging eukaryotes, while highlighting its importance for queuosine biosynthesis.

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“…This precursor is further elaborated to queuine by two subsequent enzymic reactions (Slany & Kersten, 1994). We have discovered that the TGT from E. coli is a zinc metalloprotein (Chong et al, 1995), and some of the aspects of tRNA recognition by the E. coli TGT have been elucidated (Cumow & Garcia, 1994 Cumow et al, 1993; Mueller & Slany, 1995;Nakanishi et al, 1994). Studies on a series of synthetic 5and 6-substituted 2-aminopyrrolo [2,3-d]pyrimidin-4(3//)-ones have revealed that the E. coli TGT tolerates a wide diversity of substituents (isosteric, or nearly so, to the aminomethyl group of preQi) at the 5 position and that 7-methylated analogues are competitive inhibitors, with respect to guanine, of TGT catalyzed exchange of radiolabeled guanine into tRNA (Hoops et al, 1995).…”

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confidence: 99%

Mechanism-Based Inactivation of tRNA-Guanine Transglycosylase from Escherichia coli by 2-Amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one

Townsend¹,

Garcia²

1995

Biochemistry

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In Escherichia coli, tRNA-guanine transglycosylase (TGT) catalyzes the incorporation of the queuine precursor preQ1 [2-amino-5-(aminomethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one] into tRNA. This precursor is further elaborated to queuine by two subsequent enzymic reactions [Slany, R. K., & Kersten, H. (1994) Biochimie 76, 1178-1182]. Our previous studies [Hoops, G. C., Townsend, L. B., & Garcia, G. A., (1995) Biochemistry (in press)] on a series of synthetic 5- and 6-substituted 2-aminopyrrolo[2,3-d]pyrimidin-4(3H) -ones have revealed that the E. coli TGT tolerates a wide diversity of substituents (isosteric, or nearly so, to the aminomethyl group of preQ1) at the 5 position. We report here that 2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4 (3H)-one (FMPP) inactivates TGT in a time- and concentration-dependent manner with k(inact) = 0.074 min-1 and KI = 136 microM. A competitive inhibitor (7-methyl-preQ1), with respect to preQ1, of TGT [Hoops, G.C., Townsend, L.B., & Garcia, G.A. (1995) Biochemistry (in press)] protects the enzyme from inactivation by FMPP. FMPP also acts as a competitive inhibitor (KI = 114 microM) of TGT under initial velocity conditions. The rate of fluoride release from FMPP is slightly faster (0.064 min-1) than the k(inact) (0.053 min-1) at 300 microM FMPP, consistent with fluoride release preceding inactivation. FMPP appears to partition between "normal" turnover (kcat = 0.461 min-1 and Km = 152 microM), inactivation, and an alternative processing to an unidentified, fluoride-released product.(ABSTRACT TRUNCATED AT 250 WORDS)

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A UGU sequence in the anticodon loop is a minimum requirement for recognition by Escherichia coli tRNA-guanine transglycosylase.

Cited by 61 publications

References 20 publications

Homodimer Architecture of QTRT2, the Noncatalytic Subunit of the Eukaryotic tRNA-Guanine Transglycosylase

Homodimer Architecture of QTRT2, the Noncatalytic Subunit of the Eukaryotic tRNA-Guanine Transglycosylase

Retrograde nuclear transport from the cytoplasm is required for tRNA^Tyrmaturation inT. brucei

Mechanism-Based Inactivation of tRNA-Guanine Transglycosylase from Escherichia coli by 2-Amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one

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A UGU sequence in the anticodon loop is a minimum requirement for recognition by Escherichia coli tRNA-guanine transglycosylase.

Cited by 61 publications

References 20 publications

Homodimer Architecture of QTRT2, the Noncatalytic Subunit of the Eukaryotic tRNA-Guanine Transglycosylase

Homodimer Architecture of QTRT2, the Noncatalytic Subunit of the Eukaryotic tRNA-Guanine Transglycosylase

Retrograde nuclear transport from the cytoplasm is required for tRNATyrmaturation inT. brucei

Mechanism-Based Inactivation of tRNA-Guanine Transglycosylase from Escherichia coli by 2-Amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one

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Retrograde nuclear transport from the cytoplasm is required for tRNA^Tyrmaturation inT. brucei