RNA-induced changes in the activity of the endonuclease encoded by the R2 retrotransposable element.
R2 is a non-long terminal repeat retrotransposable element that inserts itself site specifically in the 28S rRNA genes of arthropods. The 120-kDa protein encoded by R2 has been shown to cleave one strand of the 28S gene at the target site and to use the 3' hydroxyl group generated from this nick to prime reverse transcription of its own RNA. This reaction has been termed target-primed reverse transcription (TPRT). Cleavage of the second DNA strand can occur in the presence or absence of reverse transcription but requires RNA. In this study, more sensitive in vitro assays have enabled further characterization of these reactions. R2 protein is capable of only a single round of TPRT because, once bound to the target DNA, it does not dissociate at physiological ionic strengths. Analysis of the role of RNA in the DNA cleavage reaction has revealed that the binding of RNA induces the R2 protein to form a multimeric complex. While larger complexes may form, the active component appears to be a dimer based on sedimentation studies and the change in stoichiometry of the cleavage reaction from a 1:1 ratio of protein subunit to target DNA in the absence of RNA to a 2:1 ratio of subunit to DNA target in the presence of RNA. Nonspecific RNA can also induce formation of this RNA-protein (RNP) complex, but the association of the protein with R2 RNA is stronger as revealed by its stability in 0.4 M NaCl. Finally, formation of the RNP complex gives rise to a 150-fold increase in the ability of the R2 endonuclease to find the target site. The specificity of this RNP complex is sufficiently great that it can find the 28S gene target site and conduct the TPRT reaction with total genomic DNA.
Yang
J
J
Department of Biology, University of Rochester, Rochester, New York 14627, USA.
Eickbush
T H
TH
D000818:Animals; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004331:Drosophila melanogaster; D004926:Escherichia coli; D019476:Insect Proteins; D007700:Kinetics; D009994:Osmolar Concentration; D012313:RNA / Q000378:metabolism*; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism
D019476:Insect Proteins; D012339:RNA, Ribosomal, 28S; D012261:Ribonucleoproteins; D012313:RNA; D004247:DNA; C101001:R2 reverse transcriptase, Bombyx mori; D012194:RNA-Directed DNA Polymerase