Figure 4.
Photobleaching step analysis to determine TerL stoichiometry in the HK97 DNA packaging assembly. (A) BOBO-3-stained 5′-biotinylated DNA was pre-incubated with procapsids, TerS and GFP-labelled TerL protein. ATP was added to initiate translocation and chased with ATP-γ-S. The complex was immobilized on a streptavidin-coated surface for visualization by total internal reflection fluorescence microscopy. GFP, λex/λem = 488 nm/509 nm. BOBO-3, λex/λem = 570 nm/602 nm. (B) GFP fluorescence trace of one protein spot that co-localized with DNA. (C) Number of photobleaching steps observed for protein spots co-localizing with DNA (n = 65), which indicated an upper limit of 5 TerL subunits.