An immuno-electron microscopical analysis of transcribing multinucleosomal templates: what happens to the histones?
Immuno-electron microscopy was used to visualize the structure of reconstituted chromatin after in vitro transcription by purified T7 RNA polymerase. T7 RNA polymerase disrupts the nucleosomal structure in the transcribed region. This disruption is not influenced by the template, linear or supercoiled, and the presence or absence of nucleosomal positioning sequences in the transcribed region. In this study, we used monoclonal autoantibodies reacting with the nucleosome core particles and epitopes within several regions of the four different core histones. Some of the residues recognized by the autoantibodies are accessible on the surface of the nucleosomes and some are more internal and therefore less exposed at the surface. We show that the loss of the nucleosomal configuration during transcription is due to the loss of histone/DNA binding and that at least part of the histones are transferred to the nascent RNA chains. Consequently, after in vitro transcription by T7 RNA polymerase, the nucleosomal template does not conserve its original configuration, and no interaction of antigen/antibodies is observed anymore in the region that has been transcribed. Therefore, we conclude that in our in vitro transcription assay, nucleosomes are detached from the template, and not simply unfolded with histones remaining attached to the DNA.
ten Heggeler-Bordier
B
B
Institut de Biologie Animale, Lausanne, CH-1015, Switzerland.
Muller
S
S
Monestier
M
M
Wahli
W
W
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D006657:Histones / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure*; D051379:Mice; D016253:Microscopy, Immunoelectron; D008968:Molecular Conformation; D009707:Nucleosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D012341:RNA, Ribosomal, 5S / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D016601:RNA-Binding Proteins / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000235:genetics; D014764:Viral Proteins
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D000939:Epitopes; D006657:Histones; D009707:Nucleosomes; D012341:RNA, Ribosomal, 5S; D016601:RNA-Binding Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases