FIGURE 1.

Y-DNA packaging substrates with a 3.7-kb leader sequence complexed with gp17 terminase-ATPase stall in proximity to the prohead portal. A, diagram of the N-terminal GFP portal-containing procapsid with anchored, stalled 3.7-kb TxY-DNA. B, the structure of the 90-bp TxY-DNA and its sequence. YIL and YIR represent the two strands of the Y-DNA. C, construction of the 3.7-kb leader TxY-DNA. D, schematic of different Y-DNA modifications with the same basic sequence used in various experiments without any fluorophore: (panel i) with Alexa 488 at the junction (panel ii), with Cy3 dye and Alexa 488 in the complementary strand at 11-bp distance (panel iii), with Cy3 dye and Alexa 488 in the complementary strand at 16-bp distance (panel iv). Ra, Rb, and Rc oligonucleotides together (panel iv), form the complement to the YIL strand of the double dye-labeled Y-DNAs. The Y-DNA portal interaction studies were carried out by assaying packaging activity by FCS and nuclease assay using either the wild type or GFP-gp20 proheads and 90-base-long Y-DNA ligated to a 3.7-kb leader sequence as substrate in the reaction mixture.