guanosine-diphosphate and xanthosine-5--triphosphate

Binding of a pre-mRNA substrate triggers spliceosome activation, whereas the release of the mRNA product triggers spliceosome disassembly. The mechanisms that underlie the regulation of these rearrangements remain unclear. We find evidence that the GTPase Snu114p mediates the regulation of spliceosome activation and disassembly. Specifically, both unwinding of U4/U6, required for spliceosome activation, and disassembly of the postsplicing U2/U6.U5.intron complex are repressed by Snu114p bound to GDP and derepressed by Snu114p bound to GTP or nonhydrolyzable GTP analogs. Further, similar to U4/U6 unwinding, spliceosome disassembly requires the DExD/H box ATPase Brr2p. Together, our data define a common mechanism for regulating and executing spliceosome activation and disassembly. Although sequence similarity with EF-G suggests Snu114p functions as a molecular motor, our findings indicate that Snu114p functions as a classic regulatory G protein. We propose that Snu114p serves as a signal-dependent switch that transduces signals to Brr2p to control spliceosome dynamics.

Topics: Adenosine Triphosphate; DEAD-box RNA Helicases; Guanosine Diphosphate; Guanosine Triphosphate; Introns; Models, Biological; Mutation; Nucleic Acid Conformation; Repressor Proteins; Ribonucleoprotein, U5 Small Nuclear; Ribonucleotides; RNA Helicases; RNA Nucleotidyltransferases; RNA Precursors; RNA Splicing; RNA Splicing Factors; RNA, Small Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spliceosomes

Arabidopsis Toc33 (atToc33) is a GTP-binding protein of the chloroplast outer envelope membrane. We studied its nucleotide-binding properties in vitro, and found that it binds GTP, GDP and XTP, with similar efficiencies, but not ATP. We further demonstrated that atToc33 has intrinsic GTPase activity. Mutations within the putative G4 motif of the atToc33 nucleotide-binding domain (D217N, D219N and E220Q) had no effect on nucleotide specificity or GTPase activity. Similarly, a mutation in the newly assigned G5 motif (E208Q) did not affect nucleotide specificity or GTPase activity. Furthermore, the D217N and D219N mutations did not affect atToc33 functionality in vivo. The data demonstrate that atToc33 belongs to a novel class of GTPases with unusual nucleotide-binding properties.

Topics: Adenosine Triphosphate; Amino Acid Motifs; Amino Acid Sequence; Arabidopsis Proteins; Chloroplasts; Genetic Complementation Test; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Membrane Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Plant Proteins; Plants, Genetically Modified; Protein Binding; Protein Structure, Tertiary; Ribonucleotides; Sequence Homology, Amino Acid; Time Factors

We synthesized 27 GTP analogues with modification or substitution at positions C2, C6, C8 and ribose moiety to investigate their effect on microtubule (Mt) assembly. It was found that C2 and C6 are both functional for the analogues supporting Mt assembly. It was surprising to find that 2-amino- ATP (n2ATP) substantially supports assembly, and that the appearance of the assembled Mts was indistinguishable from those assembled in the standard GTP assembly buffer solution. Furthermore, 2-amino dATP and dGTP are even more potent than GTP in supporting assembly. The substitution of oxo group at C6 with reactive thiol largely reduced the activity of the analogue to support assembly. When free rotation of the glycosidic linkage of GTP was blocked by the introduction of sulfur atom between C8 and C2' of ribose moiety, it resulted in total suppression of assembly. Purine nucleoside triphosphate was found to support assembly better than GTP, and even more efficient was 2-amino purine nucleoside triphosphate. Interestingly, their deoxy-type analogues were totally inhibitory. Although 2-amino 8-hydroxy ATP and other analogues supported assembly much better than did GTP, their diphosphate analogues were totally incapable of supporting assembly. Finally, bulky fluorescent probes were introduced at C3' of ribose moiety (Mant-8-Br-GTP or Mant-GTP) to visualize the fluorescent signal in assembled Mts. Even in this case, the number of most protofilaments was found to be 14, consistent with that found in Mts assembled in GTP standard buffer solution.

Topics: Adenine Nucleotides; Animals; Brain; Deoxyguanine Nucleotides; Dimerization; Guanosine Diphosphate; Guanosine Triphosphate; Hydrogen Bonding; Inosine Triphosphate; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Polymers; Protein Conformation; Purines; Ribonucleotides; Ribose; Swine; Tubulin

The D119N mutation of p21ras was prepared by site-directed mutagenesis. Its nucleotide binding properties were investigated using fluorescently labelled guanosine and xanthosine nucleotides. Its affinity for guanosine nucleotides is severely reduced, with a concomitant increase in the affinity for xanthosine nucleotides, which leads to an almost complete reversal of base specificity. The protein is a GTPase as well as a XTPase and the hydrolysis reaction can be efficiently stimulated by GAP. Dissociation of XDP from the mutant is stimulated by the guanine nucleotide exchange factor Cdc25Mm in a similar manner to that of GDP from wildtype. The interaction of the mutant with the effector domain of c-Raf kinase or Ral-GEF is normal. In microinjection experiments in PC12 and NIH3T3 cells the protein behaves as an oncogenic mutant due to its high dissociation rate for GDP. However, when the protein is loaded with XDP before microinjection the onset of the oncogenic signal can be efficiently retarded. Thus, the protein behaves initially as wildtype and later as an oncogenic protein.

Topics: 3T3 Cells; Animals; Base Sequence; Cell Cycle Proteins; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Mice; Microinjections; Molecular Sequence Data; Mutation; Phosphoprotein Phosphatases; Proto-Oncogene Proteins p21(ras); ras-GRF1; Ribonucleotides; Sensitivity and Specificity

The role of GTP-binding proteins in exocytosis in bovine adrenal chromaffin cells was examined using patch-clamp capacitance measurement. Internal dialysis with the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate and xanthosine triphosphate (XTP) activated a capacitance increase. Exocytosis triggered by XTP was blocked by guanosine 5'-[beta-thio]diphosphate (GDP beta S) but Ca(2+)-induced exocytosis was unaffected. The capacitance increase due to XTP could not be explained by Ca2+ mobilisation since Ins(1,4,5)P3 and caffeine did not mimic the response. Chromaffin cells appear to possess a Ca(2+)-independent pathway for exocytosis that involves GTP-binding proteins. The magnitude of the response to XTP suggested that GTP analogues stimulate both exocytosis and recruitment of secretory granules.

Topics: Adenosine Triphosphate; Adrenal Glands; Animals; Calcium; Cattle; Chromaffin System; Electric Conductivity; Exocytosis; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Ribonucleotides; Thionucleotides