Assuntos
Escherichia coli/genética , Genes de Plantas , Nucleopoliedrovírus/genética , Proteínas de Plantas/genética , Animais , Linhagem Celular , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , Expressão Gênica , Técnicas Genéticas , Vetores Genéticos , Corpos de Inclusão/química , Proteínas de Plantas/isolamento & purificação , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Spodoptera , TransfecçãoRESUMO
The maize transposable element Activator (Ac) encodes a transposase (TPase) protein, whose DNA-binding domain is located in a basic region around aa 200. The N-terminal 102 aa of the TPase are not required for the transposition reaction. In transfected petunia protoplasts, we analyzed the protein levels of the N-terminally truncated TPase and mutants thereof and the corresponding transposition frequencies. The TPase protein forms large insoluble aggregates at high expression levels. There is no proportionality observed between TPase levels and transposition frequency. Twenty-one mutations (of 26), which are distributed over the whole length of the protein, inactivate the TPase completely. By coexpressing inactive mutant and active truncated TPase, it was found that several mutations have a trans-dominant inhibitory effect. Among those are two DNA-binding-deficient mutants, indicating that inhibition of the active TPase is not caused by competition for the binding sites on the transposon. Accordingly, Ac TPase acts as an oligo- or multimer formed by protein-protein interactions. Peculiarly, two mutants lacking 53 and 98 aa from the C terminus that are themselves transpositionally inactive lead to an increased excision frequency when they are coexpressed with the active truncated TPase.
Assuntos
Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Nucleotidiltransferases/genética , Zea mays/genética , Sequência de Aminoácidos , Análise Mutacional de DNA , Genes Dominantes , Genes de Plantas , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Deleção de Sequência , Relação Estrutura-Atividade , TransposasesRESUMO
Ac encodes the 807 amino acid ORFa protein which binds specifically to multiple AAACGG motifs that are subterminally located in both ends of Ac. The wild-type ORFa protein and a number of deletion and amino acid exchange mutants were expressed in Escherichia coli, renatured and used for mobility shift assays. At least 136 amino acids from the N-terminus and 537 C-terminal amino acids may be removed from the ORFa protein without destroying the DNA binding domain, whereas a protein starting at amino acid 189 is DNA binding deficient. Certain basic amino acids between positions 190 and 200 are essential for DNA binding, as their substitution with uncharged amino acids leads to the loss of this function. The DNA binding domain of ORFa protein has an overall basic character, but no obvious sequence homology to any other known DNA binding protein. The homologies to the major open reading frames of transposable elements Tam3 from Antirrhinum majus and Hobo from Drosophila are found between the C-terminal two thirds of the three proteins. The ORFa protein forms discrete complexes with target DNA that appear, depending on the protein concentration, as a 'ladder' of bands on gels, indicating the occupation of target DNA by multiple ORFa protein molecules.
