The Folded Genome of Escherichia coli Isolated in a Protein-DNA-RNA Complex

Abstract

The DNA of Escherichia coli has been isolated in a compact structure containing small amounts of protein and RNA and having a sedimentation coefficient of approximately 3200 S. The molecular weight of the DNA in the complex is very large (probably higher than 109); the protein is predominantly core RNA polymerase; the RNA is chiefly nascent messenger and ribosomal chains. Solutions containing the complex have low vicosities; this plus its sedimentation rate suggest that the DNA is in a tightly folded conformation. The DNA unfolds after exposure to RNase or heat; this indicates that an RNA component of the complex is involved in stabilizing the structure..The DNA in bacterial cells as seen under the electron microscope is confined in regions of the cell called "nuclear bodies," but no nuclear membrane is observed (see, for example, refs. 1 and 2). Yet these chromosome-like structures have defined boundaries, which reorganize during the nuclear segregation that accompanies cell division (3). This implies that DNA folding within the nuclear body is maintained-at least partly--by internal interactions between the elements or regions of the nuclear body. The tertiary conformation of DNA within this body is unknown; however, studies of the rates at which different operons can be transcribed and regulated suggest that a considerable portion (perhaps all) of the genome is available for free interaction with other macromolecules (for examples see refs. 4-7). The packaging of the DNA must also be compatible with the requirements of semiconservative DNA replication and the eventual segregation of daughter double-helices (8, 9). These considerations imply that the-, tertiary structure of DNA within the cell is organized; however, this structure and the interactions that stabilize it are still to be determined.We describe here the isolation from E. coli of a DNA complex in which the DNA genome remains folded into a structure which is compact and nearly homogeneous in sedimentation. The complex is approximately 80% DNA by weight; it includes a small amount of protein and also contains nascent RNA. At least some of the RNA of the complex is essential for stabilizing the packaged structure, since the DNA unfolds as a result of RNase treatment. MATERIALS AND METHODSThe E. coli strain D-10 (RNase I-, ref. 10) was used in these studies. The bacteria were grown into the log phase in M9 media supplemented with 0.2% casamino acids.Viscosities of solutions of the DNA complex were measured at 250C in a falling-ball viscometer.Proteins were obtained from a DNA complex that had been purified by a scaled-up modification of the procedure described in the legend to Fig. 1 When E. coli cells are gently opened in solutions of high ionic strength, DNA is released from the cells but the solution does not become viscous. A DNA complex, having a nearly homogeneous sedimentation profile, can be isolated from the bulk of the ultraviolet-absorbing material of the lysate ( Fig. 1; see also ref. 12). This complex contains: nearly all of the DN...

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Rate zonal density gradient ultracentrifugation analysis of repair of radiation damage to the folded chromosome of Escherichia coli

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