Figure 4.

Identification of the trans-acting arginine finger R146. A) Ribbon diagram of gp16; mechanistically important residues, including R146, are labelled. The AMP-PNP substrate is shown as a stick figure, and the Mg²⁺ ion as a green sphere. The walker A and putative DNA translocating loops are colored magenta and green, respectively; the α-helix connecting the DNA translocating loop to the arginine finger R146 is shown in orange. B) SDA page gels showing the DNA packaging activity of gp16 mutants targeting arginines near R146. Arginine residues that cluster near the putative arginine finger R146 were mutated to lysine and assayed for the ability to support DNA packaging. To assess packaging, the packaged DNA that was protected from DNase treatment was extracted from particles and analyzed by agarose gel electrophoresis. Lanes were rearranged, indicated by a space between lanes, to facilitate viewing. C) Complementation experiments demonstrating that R146 is a trans-acting arginine finger. ATPase activity was measured for proheads complexed with mixtures of the catalytically inactive D/E mutant and the arginine finger mutant R146A. When mixed together, complementation occurs since the D/E mutant can contribute its arginine finger in-trans to an adjacent R146A subunit (n=4, error bars are SD). In a 50% mixing ratio, perfect complementation between a D/E adjacent to an R146 subunit would be 25%. Observed complementation between the mutants was 13.7% ± 1.6%. Similar results are obtained with the R146K mutant.