Characterization of the XXXX DNA packaging reaction in vitro in a defined system.

@entity56197 @entity12865 @entity855
The @entity56197 DNA packaging system in vitro defined here is composed of purified proheads and two non-capsid proteins, the products of genes 18 and 19 (gp18 and gp19). In this system, a precursor complex (50 S complex) accumulates in the presence of @entity12865 ( @entity855 -gamma-S), a non-hydrolyzable analog of @entity855 . The 50 S complex is converted to a filled head in the presence of @entity855 . The conversion of the 50 S complex, formed by preincubation with @entity855 -gamma-S, to the mature head proceeds in a synchronous manner after the addition of @entity855 . The lag time for formation of mature heads from the 50 S complex is 1.8, 4.5 and 6.8 minutes at 30, 25 and 20 degrees C, respectively. DNA is translocated into the capsid at a constant rate of 5.7 x 10(3) base-pairs per minute at 20 degrees C. The conversion of the 50 S complex to the mature head exhibits a sigmoidal relationship with respect to the concentration of @entity855 , the concentration for half-maximal activity being about 20 microM. The transition of the prohead to the expanded capsid occurs at 20 degrees C at one minute 40 seconds after the initiation of DNA translocation, when one-fourth of the genome has been packaged into a prohead. At the same time, the capsid-DNA complex becomes stable to high concentrations of salt. When DNA translocation is interrupted by the addition of @entity855 -gamma-S, packaged DNA exists at 0 degrees C as well as at 20 degrees C but the exit of DNA stops after one-third of the genome is inside the capsid. After exit, DNA is retranslocated into the expanded capsid by the addition of @entity855 at a rate of about 5.7 x 10(3) base-pairs per minute at 20 degrees C. The decrease in concentration of @entity855 interrupts DNA translocation into the capsid but does not induce DNA exit. Interrupted DNA translocation may be reinitiated by the addition of @entity855 . DNA exit is not induced by the addition of @entity855 -gamma-S to mature heads or partially filled heads pretreated with DNase.
