Chromatin structure of the ribosomal RNA genes of Tetrahymena thermophila as analyzed by trimethylpsoralen crosslinking in vivo

Abstract

Rapidly growing Tetrahymena thermophila were treated with 4,5′,8-trimethylpsoralen plus various doses of 360 nm radiation to crosslink their DNA in vivo. The DNA was then purified, the palindromic, extrachromosomal ribosomal RNA genes (rDNA) were isolated, and the positions of crosslinks were mapped by electron microscopy of the DNA under totally denaturing conditions. The bulk DNA contained crosslinks separated by intervals of 200 and 400 base-pairs, indicative of a nucleosome chromatin structure. The distribution of distances between adjacent crosslinks in the rDNA differed in transcribed and non-transcribed regions. At subsaturating levels of photoreaction, the transcribed regions showed a novel pattern with adjacent crosslinks separated by intervals of 215, 335, and 460 basepairs. The distribution was consistent with an altered conformation of nucleosomes or with a chromatin structure in which proteins other than histones play a major role. The central spacer region of the rDNA showed a broad distribution of distances between crosslinks with no apparent unit of periodicity. Only the terminal spacer regions showed a pattern of crosslinkage that could be reconciled with a nucleosome structure. The frequency of crosslinking in vivo along the rDNA molecules was related to transcriptional activity: the spacer regions were protected from trimethylpsoralen crosslinking in vivo, while the gene regions were relatively accessible. The palindromic rDNA molecules from log phase cells were predominantly in the linear conformation after fixation of the secondary structure in vivo with trimethylpsoralen. Cruciform structures with enough intrastrand base-pairing to be recognized by electron microscopy were not observed.