RESUMO
Pogo is a transposable element with short terminal inverted repeats. It contains two open reading frames that are joined by splicing and code for the putative pogo transposase, the sequence of which indicates that it is related to the transposases of members of the Tc1/mariner family as well as proteins that have no known transposase activity including the centromere binding protein CENP-B. We have shown that the N-terminal region of pogo transposase binds in a sequence-specific manner to the ends of pogo and have identified residues essential for this. The results are consistent with a prediction that DNA binding is due to a helix-turn-helix motif within this region. The transposase recognises a 12 bp sequence, two copies of which are present at each end of pogo DNA. The outer two copies occur as inverted repeats 14 nucleotides from each end of the element, and contain a single base mismatch and indicate the inverted repeats of pogo are 26 nucleotides long. The inner copies occur as direct repeats, also with a single mismatch.
Assuntos
Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/genética , Sequências Hélice-Volta-Hélice , Sequências Repetidas Terminais , Transposases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/enzimologia , Escherichia coli/genética , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transposases/química , Transposases/genéticaRESUMO
I factors are members of the LINE-like family of transposable elements and move by reverse transcription of an RNA intermediate. Complete I factors contain two open reading frames. The amino acid sequence encoded by the first of these, ORF1, includes the motif CX2CX4HX4C that is characteristic of the nucleocapsid domain of retroviral gag polypeptides followed by a copy of the slightly different sequences CX2CX4HX6C and CX2CX9HX6C. The function of this protein is unknown. We have expressed this protein in Escherichia coli and Spodoptera frugiperda cells and have shown that it binds both DNA and RNA but without any evidence for sequence specificity. The properties of deletion derivatives of the protein indicate that more than one region is responsible for DNA binding and that the CCHC motif is not essential for this. The ORF1 protein expressed in either E. coli or Spodoptera cells forms high molecular weight structures that require the region of the protein including the CCHC motif for their formation. This protein can also accelerate the annealing of complementary single-stranded oligonucleotides. These results suggest that this protein may associate with the RNA transposition intermediates of the I factor to form particles that enter the nucleus during transposition and that it may stimulate both the priming of reverse transcription and integration. This may be generally true for the product of the first open reading frame of LINE-like elements.
