guanosine-triphosphate and Chromosome-Deletion

The GTPase-activating protein (GAP) stimulates the GTPase reaction of p21 by 5 orders of magnitude such that the kcat of the reaction is increased to 19 s-1. Mutations of residues in loop L1 (Gly-12 and Gly-13), in loop L2 (Thr-35 and Asp-38), and in loop L4 (Gln-61 and Glu-63) influence the reaction in different ways, but all of these mutant p21 proteins still form complexes with GAP. The C-terminal domain of the human GAP gene product, GAP334, which comprises residues 714 to 1047, is 20 times less active than full-length GAP on a molar basis and has a fourfold lower affinity. This finding indicates that the N terminus of GAP containing the SH2 domains modifies the interaction between the catalytic domain and p21.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Baculoviridae; Binding Sites; Cell Line; Chromosome Deletion; Cloning, Molecular; Genetic Vectors; GTPase-Activating Proteins; Guanosine Triphosphate; Insecta; Kinetics; Models, Structural; Molecular Sequence Data; Mutagenesis; PC12 Cells; Protein Conformation; Proteins; Proto-Oncogene Proteins p21(ras); ras GTPase-Activating Proteins; Recombinant Proteins

G proteins are active as long as GTP is bound to the alpha subunit. Activation ends when GTP is cleaved to GDP that then stays bound to the active site. Agonist-liganded receptors allow formation of the active state by decreasing the affinity of alpha subunits for GDP allowing exchange of GDP for GTP. Since receptors interact with the C terminus of the alpha subunits, we tested whether deletion of the C terminus could mimic activation by receptors. Three deletions and one point mutation at the C terminus of alpha o were engineered in alpha o cDNA by the polymerase chain reaction, transcribed into RNA, and translated in a rabbit reticulocyte lysate. The ability of in vitro synthesized protein to bind guanine nucleotide was inferred from analysis of native tryptic cleavage patterns, while the ability of the proteins to associate with beta gamma was measured by sucrose density gradient centrifugation. Deletion of 14 amino acids, alpha oD[341], from the C terminus causes a large decrease in GDP affinity, with little or no change in guanosine 5'-3-O-(thio)triphosphate affinity. When GTP is present, alpha oD[341] remains in the activated conformation because exchange of GTP for GDP is rapid. Deletion of 10 amino acids, alpha oD[345], lowers GDP affinity, but less dramatically than in alpha oD[341]. Deletion of 5 amino acids, alpha oD[350], or mutation of Arg-349 to proline alpha oR[349P] has no detectable effects on GDP affinity. Deletion of up to 10 amino acids from the C terminus does not prevent formation of alpha beta gamma heterotrimers. We propose that the C terminus of the alpha subunit is a mobile region that blocks dissociation of GDP. Agonist-liganded receptors may move it aside to allow release of GDP, exchange for GTP, and activation of the alpha subunit.

Topics: Amino Acid Sequence; Animals; Centrifugation, Density Gradient; Chromosome Deletion; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Molecular Sequence Data; Mutagenesis; Peptide Fragments; Polymerase Chain Reaction; Protein Binding; Protein Biosynthesis; Protein Engineering; Rabbits; Rats; Reticulocytes; Trypsin

The P7 element of group I introns contains a semiconserved "bulged" nucleotide, a C in group IA introns (nt 870 in the td intron) and an A in group IB introns [Cech, T.R. (1988) Gene 73, 259-271]. Variants U870, G870, and A870, isolated by a combination of in vitro and in vivo genetic strategies, indicate that C and A at position 870 are consistent with splicing whereas U and G are not. Although mutants G870 and U870 could be activated in vitro by increasing the Mg2+ concentration, their Km for GTP at pH 7 was 20-100-fold elevated, and they were unable to undergo site-specific hydrolysis. The dependence of the mutants on high guanosine concentrations could be substantially overcome by an increase in pH, suggesting that a tautomeric change, which makes U and G mimic C and A, is responsible for restoring function. In contrast to the striking Km effect, Vmax for the mutants differed by less than a factor of 2 from the wild type. Furthermore, streptomycin, an aminoglycoside antibiotic that competes with guanosine for its binding site, inhibited splicing of the U870 and G870 constructs at least as well as of the C870 and A870 variants, indicating that the guanosine-binding site of the mutants is proficient at interacting with a guanidino group. While our experiments argue against a hydrogen-bonding interaction between the C6-O of the cofactor and C4-NH2 of the bulged nucleotide, they are consistent with other models in which the C4-NH2 and/or N3 groups of the bulged C are involved in establishing an active ribozyme.

Topics: Base Sequence; Chromosome Deletion; Escherichia coli; Genetic Variation; Guanosine Triphosphate; Introns; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleic Acid Conformation; Oligonucleotide Probes; Plasmids; RNA Precursors; RNA Splicing; RNA, Catalytic; T-Phages; Transcription, Genetic

Deletion mutants of the viral Harvey ras oncogene were generated by removing different lengths of the gene from either the amino or the carboxyl terminus. The deletion mutants, ras p21 expressed in Escherichia coli, yielded proteins of approximately 8, 10, 12, 14, 17, 18, 19, and 20 kDa. These proteins were utilized to identify epitopes recognized by a series of recently generated monoclonal antibodies as well as some previously reported monoclonal antibodies. Monoclonal antibodies that inhibited GTP binding, a major biochemical activity of the p21 protein, recognized two major regions of the protein. These regions were localized from amino acids 5 to 69 and 107 to 164, respectively, and were separated by another stretch from residues 70 to 106, whose antigenic determinants were not directly involved in GTP binding. Thus, the mapping of epitopes within the p21 molecule recognized by monoclonal antibodies has made it possible to localize important functional regions within the ras p21 molecule.

Topics: Animals; Antibodies, Monoclonal; Binding Sites; Chemical Precipitation; Chromosome Deletion; Epitopes; Genetic Engineering; GTP-Binding Proteins; Guanosine Triphosphate; Mice; Mutation; Oncogene Proteins, Viral; Oncogenes; Protein Conformation

Deletions of small sequences from the viral Harvey ras gene have been generated, and resulting ras p21 mutants have been expressed in Escherichia coli. Purification of each deleted protein allowed the in vitro characterization of GTP-binding, GTPase and autokinase activity of the proteins. Microinjection of the highly purified proteins into quiescent NIH/3T3 cells, as well as transfection experiments utilizing a long terminal repeat (LTR)-containing vector, were utilized to analyze the biological activity of the deleted proteins. Two small regions located at 6-23 and 152-165 residues are shown to be absolutely required for in vitro and in vivo activities of the ras product. By contrast, the variable region comprising amino acids 165-184 was shown not to be necessary for either in vitro or in vivo activities. Thus, we demonstrate that: (i) amino acid sequences at positions 5-23 and 152-165 of ras p21 protein are probably directly involved in the GTP-binding activity; (ii) GTP-binding is required for the transforming activity of ras p21 and by extension for the normal function of the proto-oncogene product; and (iii) the variable region at the C-terminal end of the ras p21 molecule from amino acids 165 to 184 is not required for transformation.

Topics: Animals; Cell Transformation, Neoplastic; Chromosome Deletion; DNA Restriction Enzymes; Escherichia coli; Genes, Viral; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Harvey murine sarcoma virus; Kinetics; Mice; Mutation; Neoplasm Proteins; Oncogene Protein p21(ras); Oncogenes; Phosphorylation; Plasmids; Protein Binding; Proto-Oncogenes; Sarcoma Viruses, Murine

The sequence of the cloned Tetrahymena ribosomal RNA intervening sequence (IVS) was altered at the site to which circularization normally occurs. The alterations caused circularization to shift to other sites, usually a nearby position which followed three pyrimidines. While a tripyrimidine sequence was the major determinant of a circularization site, both location of a sequence and local secondary structure may influence the use of that sequence. For some constructs circularization appeared to occur at the position following the 5' G, the nucleotide added to the IVS during its excision. Portions of the internal guide sequence (IGS), proposed to interact with the 3'exon were deleted without preventing exon ligation. Thus if the IGS-3'exon interaction exists, it is not essential for splicing in vitro.

Topics: Animals; Base Sequence; Chromosome Deletion; Guanosine Triphosphate; Nucleic Acid Conformation; Nucleic Acid Precursors; RNA Processing, Post-Transcriptional; RNA Splicing; RNA, Ribosomal; Tetrahymena