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Nucleic Acids Research, 1982, Vol. 10, No. 18 5533-5552
© 1982

MOLECULAR BIOLOGY

Conversion of simian virus 40 DNA to ordered nucleoprotein structures by extracts that direct accurate initiation by eukaryotic RNA polymeraseII*+

Sandip N. Sinha{dagger}, Randolph J Hellwig{ddagger}, David P. Allison and Salil Niyogi

University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and Biology Division, Oak Ridge National Laboratory Oak Ridge, TN 37830, USA {dagger}Postdoctoral Investigator supported by subcontract No.3322 from the Biology Division of Oak Ridge National Laboratory to the University of Tennessee. On leave from Biology Division, Indian Jute Industries' Research Association Calcutta 700088, India {ddagger}Predoctoral Investigator supported by National Institutes of Health Grant GM 7431

To whom requests for reprints should be sent

Received April 5, 1982. Revised August 19, 1982. Accepted August 19, 1982.

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 mini-chromosomes 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.

+Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation


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