Fig. 1

Cleavage and extension activity of the purified recombinant full-length PAc protein in vitro. A Amino acid sequence alignment of the core catalytic RT motifs 4 and 5 [5] (underlined) and the selected EN catalytic motifs (underlined, with mutagenized residues marked by asterisks) for four representatives from the Penelope/Poseidon PLE clade (Repbase entries from A. carolinensis, Petromyzon marinus, Branchiostoma floridae, Drosophila virilis); numbering on the top corresponds to PAc ORF. B Locus map showing repetitive structure of the PAc element. The schematic shows three example repeats with domains highlighted in blue (nucleic acid binding), white (reverse transcriptase), and red (endonuclease). pLTR, pseudo long terminal repeat; RT, reverse transcriptase; ORF, open reading frame; HHR, hammerhead ribozyme motif. Read coverage for one copy of the PAc retroelement (pLTR-ORF-pLTR from the Repbase consensus, File S1) from publicly available A. carolinensis RNA sequencing data is shown below the locus map. The side panel shows the degree of PAc ORF purification (Methods). C Overview of the in vitro assay consisting of an end-labeled dsDNA target, RNA template, and PAc protein. The schematic shows the expected effect when adding protein and RNA (cleavage), and adding protein, RNA, and dNTPs (extension). Denaturing gel shown for one RNA template for both the bottom strand (Cy5 fluorophore) and top strand (fluorescein fluorophore). The expected significance of each band as it relates to the diagram is shown by the line drawing on the left-hand side of both gels. D The effect of truncating the RNA template from the 5’ end on cleavage and extension activity using the in vitro assay. The 3’ end of the template is held constant at + 235 while the 5’ end is tiled from -126 to + 200 where bases are labeled relative to the start of the ORF. E The effect of changing the RNA template from the 3’ end on cleavage and extension activity using the in vitro assay. The 5’ end of the template is held constant at -145 while the 3’ end is tiled from -63 to + 378 where bases are labeled relative to the start of the ORF. F Cleavage frequency for both the top and bottom strands on a dsDNA target corresponding to the PAc ORF-pLTR junction. Bars indicate the percent of total reads mapping to a cleaved product at that location. G Reverse transcription initiation sites for a dsDNA target corresponding to the PAc ORF-pLTR junction and an RNA template spanning the PAc pLTR-ORF junction. Bars indicate the percent of total reads mapping to an insertion product that begins at that location