Visualization of a Spinach Plastid Transcriptionally Active DNA-Protein Complex in a Highly Condensed Structure

Abstract

A transcriptionally active DNA-protein complex isolated from spinach Spinacia oleracea plastids is visualized by electron microscopy in different conditions. This structure, after glutaraldehyde fixation, is highly condensed. DNA is supertwisted with proteins bound to it producing a beaded substructure. When glutaraldehyde fixation is omitted this structure is less condensed and DNA fibrils come out from a proteinous central body. The DNA-protein complex can be separated into two populations by CsCl centrifugation: one with a buoyant density of 1.570 grams per cubic centimeter and the other of 1.610 grams per cubic centimeter. By visualization of these two populations, it is concluded that proteins are either firmly bound to DNA in the central body, or more loosely bound to the DNA fibrils. These latter proteins could play a role in enzymic functions and/or in the supercoiling of DNA.The DNA from the DNA-protein complex possesses all fragments that belong to pure circular chloroplast DNA hydrolyzed by two restriction enzymes: Bam HI and Eco RI. Some molecules observed in a supercondensed form with a beaded substructure probably contain entire chloroplast DNA molecules.A hydrolysis test with microccocal nuclease gives no indication of the presence of ;nucleosome-like' structures. Thirty-six polypeptides with molecular weights ranging from 12,000 to 180,000 are present in the complex, and seven of them are highly soluble in 0.4 n H(2)SO(4); their molecular weights range from 14,000 to 46,000 as shown by two-dimensional gel electrophoresis.No linolenic acid can be detected in the preparation, indicating the absence of chloroplast membranes.