Single-event analysis of the packaging of bacteriophage T7 DNA concatemers in vitro.
Bacteriophage T7 packages its double-stranded DNA genome in a preformed protein capsid (procapsid). The DNA substrate for packaging is a head-to-tail multimer (concatemer) of the mature 40-kilobase pair genome. Mature genomes are cleaved from the concatemer during packaging. In the present study, fluorescence microscopy is used to observe T7 concatemeric DNA packaging at the level of a single (microscopic) event. Metabolism-dependent cleavage to form several fragments is observed when T7 concatemers are incubated in an extract of T7-infected Escherichia coli (in vitro). The following observations indicate that the fragment-producing metabolic event is DNA packaging: 1) most fragments have the hydrodynamic radius (R(H)) of bacteriophage particles (+/-3%) when R(H) is determined by analysis of Brownian motion; 2) the fragments also have the fluorescence intensity (I) of bacteriophage particles (+/-6%); 3) as a fragment forms, a progressive decrease occurs in both R(H) and I. The decrease in I follows a pattern expected for intracapsid steric restriction of 4',6-diamidino-2-phenylindole (DAPI) binding to packaged DNA. The observed in vitro packaging of a concatemer's genomes always occurs in a synchronized cluster. Therefore, the following hypothesis is proposed: the observed packaging of concatemer-associated T7 genomes is cooperative.
Sun
M
M
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78284-7760 USA.
Louie
D
D
Serwer
P
P
D017123:Bacteriophage T7 / Q000235:genetics*; D002213:Capsid / Q000737:chemistry / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000821:virology; D016679:Genome, Viral; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D013816:Thermodynamics; D014783:Viscosity
D004279:DNA, Viral