The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA

Abstract

tRNA pseudouridine synthase I (PsiSI) catalyzes the conversion of uridine to Psi at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. PsiSI forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNA(Phe) containing FUra in place of Ura) to form a putative analog of a steady-state intermediate in the normal reaction pathway. Previously, we proposed that a conserved aspartate of the enzyme serves as a nucleophilic catalyst in both the normal enzyme reaction and in the formation of a covalent complex with FUra-tRNA. The covalent adduct between FUra-tRNA and PsiSI was isolated and disrupted by hydrolysis and the FUra-tRNA was recovered. The target FU39 of the recovered FUra-tRNA was modified by the addition of water across the 5,6-double bond of the pyrimidine base to form 5,6-dihydro-6-hydroxy-5-fluorouridine. We deduced that the conserved aspartate of the enzyme adds to the 6-position of the target FUra to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage to form the observed product. Assuming that an analogous covalent complex is formed in the normal reaction, we have deduced a complete mechanism for PsiS.

Scheme 1

Proposed mechanism I for ΨSI.

Scheme 2

Proposed mechanism II for ΨSI.

Figure 1

Denaturing PAGE of [³²P]FUra-tRNAs and complexes with ΨSI. (A) SDS/PAGE of FUra-tRNA (lane 1), ΨSI-FUra-tRNA covalent complex (lane 2), and RNase A digest of ΨSI-FUra-tRNA covalent complex from lane 2 (lane 3). (B) 7 M urea-15% PAGE of unmodified (lane 1), ΨSI-modified FUra-tRNA (lane 2), and the oligo containing FU39 after hydrolysis of the RNase A-digested ΨSI-FUra-tRNA complex from A, lane 3.

Figure 2

7 M urea-PAGE of partial RNase A digests of unmodified (lane 2) and ΨSI-modified FUra-tRNA (lane 1). FUra-tRNA was labeled with [³²P]pCp, and the location of the oligo containing modified FU39 (arrow) was determined by comparison to sequence ladders from RNase T1 and T2 digests (data not shown).

Figure 3

3′ End-group analysis of RNase A-digested ΨSI-modified FUra-tRNA. (A) 7 M urea-25% PAGE after complete RNase A digests of [5′-³²P]CMP-FUra-tRNA (lane 1) and ΨSI-modified [5′-³²P]CMP-FUra-tRNA (lane 2). (B) Two-dimensional TLC of band 3, lane 2 of A after nuclease P1 digestion to 5′-NMPs. (C) Two-dimensional TLC of band 3, lane 2 of A after RNase T2 digestion to 3′-NMPs. (D) Two-dimensional TLC of RNase T2 digest of ΨSI-modified [5′-³²P]CMP-FUra-tRNA. ³²P bands and spots were located by autoradiography.

Figure 4

HPLC analysis of modified FUrd. The nuclease P1 and phosphatase digest of ΨSI-modified [6-³H]FUra-tRNA was cochromatographed with FUrd and its photoproducts 1 and 2. The eluent was monitored by absorbance at 220 nm (line trace) and ³H counts (bar graph).

Figure 5

TLC of standards and radioactive nucleosides derived from digestion of ΨSI-modified [6-³H]FUra-tRNA. (Lane 1) [6-³H]5-FUrd; (lane 2) photoproducts of [6-³H]5-FUrd; (lane 3) HPLC-purified photoproduct 1 of [6-³H]5-FUrd; (lane 4) HPLC-purified photoproduct 2 of [6-³H]5-FUrd; (lane 5) nuclease P1 and alkaline phosphatase digests of ΨSI-modified [6-³H]FUra-tRNA; (lane 6) nuclease P1 and alkaline phosphatase digests of [6-³H]FUra-tRNA. Lanes 5 and 6 migrate aberrantly because of the high salt concentration; for clarity, the components of each lane are segregated by vertical lines.

Scheme 3

Hydrolysis of the ester linkage from ΨSI-FUra-tRNA complex.