Conversion of simian virus 40 DNA to ordered nucleoprotein structures by extracts that direct accurate initiation by eukaryotic RNA polymerase II.
Interaction of SV40 DNA with three different HeLa cell extracts capable of directing correct initiation of transcription leads to the formation of ordered nucleoprotein complexes that are structurally similar to SV40 minichromosomes and eukaryotic chromatin. These nucleoprotein complexes can be conveniently purified by band sedimentation or gel filtration. Their sedimentation and elution properties resemble those of SV40 minichromosomes. Electron microscopy of purified complexes shows beaded structures that are sensitive to proteases, resulting in recovery of naked, largely undegraded DNA. Contour lengths and compaction ratios of these nucleoprotein complexes are similar to those of authentic SV40 minichromosomes. Their digestion patterns with micrococcal nuclease and pancreatic DNase I resemble those of SV40 minichromosomes. Such nucleosome-like structures can also be obtained with linear SV40 DNA. Unlike nucleosomes, no histones could be detected in the purified nucleoprotein complexes. Non-histone chromosomal protein fractions (high mol. wt. and free of high mobility group proteins) prepared from the HeLa cell extracts can also generate similar ordered structures. We conclude that ordered nucleoprotein structures with certain common characteristics can be formed by interaction of DNA with non-histone chromosomal proteins as well as with histones. Only the former structures are generated in currently used cell-free transcription systems. It appears that only those purified nucleoprotein complexes containing the promoter can be actively transcribed in the presence of additional cell-free extract, suggesting that such structures and their protein components may be important in transcription.
D000818:Animals; D002460:Cell Line; D002472:Cell Transformation, Viral; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I; D003852:Deoxyribonucleoproteins / Q000235:genetics*; D004706:Endodeoxyribonucleases; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007668:Kidney; D008836:Micrococcal Nuclease; D009698:Nucleoproteins / Q000235:genetics*; D010179:Pancreas / Q000201:enzymology; D012319:RNA Polymerase II / Q000378:metabolism*; D013539:Simian virus 40 / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D004279:DNA, Viral; D003852:Deoxyribonucleoproteins; D009698:Nucleoproteins; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease