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. Author manuscript; available in PMC: 2013 Nov 20.
Published in final edited form as: Biochemistry. 2012 Nov 7;51(46):9342–9353. doi: 10.1021/bi300890y

Figure 2. Purification and Packaging Activity of the Lambda Terminase Protomer.

Figure 2

Panel A. Sedimentation velocity data for the terminase mix (blue) and isolated protomer (red) were analyzed using the van Holde-Weischet method as described in Experimental Procedures. The mix clearly contains both the protomer and assembled species while the isolated protomer is homogenous. Inset – c(s) analysis of the same data using Sedfit. Panel B. Fidelity of DNA Packaging by the Mix and by the Isolated Terminase Protomer. The DNase protection assay was performed as described in Experimental Procedures using 100 nM terminase protomer or terminase mix and 5 nM mature lambda DNA (λ-DNA) or pCT-λ, as indicated. The position of mature λ DNA, uncleaved pCT-λ, and the DL and DR products resulting from cos-cleavage of pCT-λ are indicated at left. There is no difference between the protomer and the mix when packaging a mature lambda genome (left). The mix packages full-length pCT-λ and both nuclease products in a linked maturation/packaging reaction. In contrast, the protomer packages the matured DL genome end with high fidelity. Panel C. The observed rate of ATP hydrolysis by the terminase protomer was determined in the presence of 50 μM (grey bars) and 1 mM (white bars) ATP. Duplex DNA (pCT-λ) was added to the reaction mixture as indicated. Each bar represents the average of at least three separate experiments with standard deviations indicated.