Structure of in-vivo transcribing chromatin as studied in simian virus 40 minichromosomes

Abstract

In order to study the structure of chromatin during transcription, individual in-vivo transcribing simian virus 40 (SV40) minichromosomes were analyzed in the electron microscope after crosslinking the nascent RNA strands with different psoralen derivatives to the template DNA. Since psoralen crosslinks the DNA between nucleosomes, spreading of the crosslinked DNA and DNA-RNA complexes reveals single-stranded bubbles at positions where nucleosomes were located. We found that the transcribing SV40 minichromosomes contained a similar number of nucleosomes as did the minichromosomes without crosslinked nascent RNA. The nascent RNA was crosslinked in about equal proportions either in single-stranded bubbles of nucleosomal length or in continuously crosslinked regions between bubbles, in contrast with control experiments with ribosomal chromatin of Dictyostelium. Treatment of SV40 minichromosomes with 1.2 M-NaCl before and during photocrosslinking with psoralen led to the disappearance of the single-stranded bubbles. Since no bubbles could be detected at the attachment sites of the RNA molecules when the nucleosomes were disrupted in high salt, and since in about half of the molecules the RNA was attached to fully crosslinked linker DNA, we assume that the single-stranded bubbles with crosslinked RNA are not due to protection by the elongating RNA polymerase II complex, but are rather due to nucleosome-like structures. At the resolution level of single nucleosomes, these results imply for the first time that nucleosome-like structures (perhaps modified compared with "normal" nucleosomes) on SV40 minichromosomes do not prevent transcription elongation by RNA polymerase II.