Multiple Nucleotide Cofactor Use by Yeast Ligase in tRNA Splicing

We have examined multiple cofactor usage by yeast tRNA ligase in splicing in vitro. The ligase mechanism of action requires expenditure of two molar equivalents of nucleotide cofactor per mole of tRNA product. Recent evidence (Westaway, S.K., Belford, H.G., Apostol, B.L., Abelson, J., and Greer, C.L. (1993) J. Biol. Chem. 268, 2435-2443) demonstrated that the ligase-associated kinase activity is more efficient with GTP as cofactor than with ATP. Employing a ligase fusion construct with dihydrofolate reductase (Apostol, B.L., Westaway, S.K., Abelson, J., and Greer, C.L. (1991) J. Biol. Chem. 266, 7445-7455) for purposes of enzyme purification, we performed joining assays demonstrating that ATP and GTP are the most effective combination of cofactors. ATP was essential to the joining reaction, while UTP, CTP, or ATP replaced GTP inefficiently. Specific and functionally independent binding sites were confirmed for ATP and GTP by direct binding measurement. A third site was implicated in UTP- and CTP-ligase interactions. Comparison of binding constants with Kapp values determined for nucleotide-dependent joining suggested both that nucleotide triphosphate binding may be limiting in tRNA joining and that tRNA ligation occurs most efficiently using GTP for the kinase reaction and ATP as the adenylylate synthetase cofactor

Novel activity of a yeast ligase deletion polypeptide. Evidence for GTP-dependent tRNA splicing.

Yeast tRNA ligase possesses multiple activities which are required for the joining of tRNA halves during the tRNA splicing process: cyclic phosphodiesterase, kinase, adenylylate synthetase, and ligase. A deletion polypeptide of a dihydrofolate reductase-ligase fusion protein, designated DAC, was previously shown to join tRNA halves although ATP-dependent kinase activity was not measurable in the assay used. We describe here a characterization of the mechanism of joining used by DAC and the structure of the tRNA product. DAC produces a joined tRNA and a splice junction with a structure identical to that produced by DAKC, the full-length dihydrofolate reductase-ligase fusion. Furthermore, DAC can use GTP as the sole cofactor in the joining reaction, in contrast to DAKC, which can only complete splicing in the presence of ATP. Both enzymes exhibit GTP-dependent kinase activity at 100-fold greater efficiency than with ATP. These results suggest that a potential function for the center domain of tRNA ligase (missing in DAC) is to provide structural integrity and aid in substrate interactions and specificity. They also support the hypothesis that ligase may prefer to use two different cofactors during tRNA splicing

Structural, Electronic, and Magnetic Properties of Quasi-1D Quantum Magnets [Ni(HF2)(pyz)(2)]X (pyz = pyrazine; X = PF6-, SbF6-) Exhibiting Ni-FHF-Ni and Ni-pyz-Ni Spin Interactions

[Ni(HF(2))(pyz)(2)]X {pyz = pyrazine; X = PF(6)(-) (1), SbF(6)(-) (2)} were structurally characterized by synchrotron X-ray powder diffraction and found to possess axially compressed NiN(4)F(2) octahedra. At 298 K, 1 is monoclinic (C2/c) with unit cell parameters, a = 9.9481(3), b = 9.9421(3), c = 12.5953(4) Å, and β = 81.610(3)° while 2 is tetragonal (P4/nmm) with a = b = 9.9359(3) and c = 6.4471(2) Å and is isomorphic with the Cu-analogue. Infinite one-dimensional (1D) Ni-FHF-Ni chains propagate along the c-axis which are linked via μ-pyz bridges in the ab-plane to afford three-dimensional polymeric frameworks with PF(6)(-) and SbF(6)(-) counterions occupying the interior sites. A major difference between 1 and 2 is that the Ni-F-H bonds are bent (∼157°) in 1 but are linear in 2. Ligand field calculations (LFT) based on an angular overlap model (AOM), with comparison to the electronic absorption spectra, indicate greater π-donation of the HF(2)(-) ligand in 1 owing to the bent Ni-F-H bonds. Magnetic susceptibility data for 1 and 2 exhibit broad maxima at 7.4 and 15 K, respectively, and λ-like peaks in dχT/dT at 6.2 and 12.2 K that are ascribed to transitions to long-range antiferromagnetic order (T(N)). Muon-spin relaxation and specific heat studies confirm these T(N)'s. A comparative analysis of χ vs T to various 1D Heisenberg/Ising models suggests moderate antiferromagnetic interactions, with the primary interaction strength determined to be 3.05/3.42 K (1) and 5.65/6.37 K (2). However, high critical fields of 19 and 37.4 T obtained from low temperature pulsed-field magnetization data indicate that a single exchange constant (J(1D)) alone is insufficient to explain the data and that residual terms in the spin Hamiltonian, which could include interchain magnetic couplings (J(⊥)), as mediated by Ni-pyz-Ni, and single-ion anisotropy (D), must be considered. While it is difficult to draw absolute conclusions regarding the magnitude (and sign) of J(⊥) and D based solely on powder data, further support offered by related Ni(II)-pyz compounds and our LFT and density-functional theory (DFT) results lead us to a consistent quasi-1D magnetic description for 1 and 2