On the structure of the folded chromosome of Escherichia coli

Abstract

After careful lysis of exponentially growing Escherichia coli cells, the DNA is released in a highly-folded conformation (Stonington & Pettijohn, 1971), sedimenting as an heterogeneous population of structures with sedimentation coefficients ranging from 1300 s to 2200 s. Chromosomes arrested at initiation by amino-acid starvation sediment as an homogeneous 1300 s species. The gradual bimodal decrease in sedimentation rate seen after amino-acid starvation suggests that the faster sedimenting structures in exponentially growing cells represent the folded chromosomes at more advanced stages in their replication cycle. The folded chromosomes are supercoiled, as evidenced by the biphasic response of their sedimentation rates to increasing concentrations of ethidium bromide. The 1300 s folded chromosome lined up at initiation, as well as the 1300 s to 2200 s replicating chromosomes, have about the same concentration of superhelices as previously observed for intracellular λ and SV40 DNA's, namely, about one negative superhelical turn per 400 base pairs. Kicking with DNase releases these superhelical twists and relaxes the folded chromosome to slower sedimenting forms. However, somewhere between 6 and 40 nicks per DNA strand are required before a fully “relaxed complex” is obtained; this complex sediments 40% slower than the original non-DNase-treated chromosome, but still five times faster than the corresponding unfolded DNA. It appears to have lost all of its superhelical character, since it behaves exactly like linear DNA upon ethidium bromide intercalation. A preliminary model for the folded bacterial chromosome has the DNA looped around a core, probably an RNA species. The RNA-DNA interactions stabilize the folded structure and topologically divide the chromosome into a limited number of loops. There are somewhere between 12 and 80 loops per chromosome, each with the same superhelical concentration. A single-strand nick with DNase allows free rotation of the DNA chains within a loop, eliminating the superhelices of that particular loop without affecting the superhelical content of the rest of the chromosome. The bacterial chromosome can then be seen in three distinct, well-defined conformations, (a) Compact, folded and supercoiled. (b) Relaxed and folded, with no superhelices but retaining its RNA core and DNA loops. This relaxed complex is observed after extensive nicking with DNase, or after eliminating the super-helices by ethidium bromide intercalation, (c) Unfolded chromosome à la Cairns, observed after breaking the UNA core or otherwise disrupting the stabilizing interactions.