Efficient transcription of a compact nucleoprotein complex isolated from purified simian virus 40 virions.
Simian virus 40 (SV40) virions were dissociated in vitro by treatment with ethylene glycol-bis-N-N'-tetraacetic acid and dithiothreitol. The compact nucleo-protein core released as a result of the dissociation had a sedimentation value of 110 to 115S compared with a value of 240S for intact virions. The viral cores contained a fraction of the viral proteins VP(1) and VP(2) in addition to the proteins found associated with the viral minichromosome, i.e., VP(3) and histones H(2)A, H(2)B, H(3), and H(4). Our results suggest that the association of VP(1), VP(2), or both with the viral minichromosome, in addition to maintaining a highly compact structure, modifies the transcriptional properties of the nucleoprotein complex. In the presence of saturating amounts of Escherichia coli RNA polymerase, 95 to 100% of the SV40 nucleoprotein cores were able to form transcriptional complexes. Sedimentation analysis of the core transcriptional complex indicated that the initiation and elongation of nascent RNA chains occurred on the compact SV40 core. Cesium chloride density gradient analysis of the SV40 virion core before and after transcription indicated that no substantial loss of protein occurred during the process of transcription. RNA synthesized from SV40 cores was a fairly homogeneous 16 to 18S species with an average chain length of approximately 2,300 nucleotides. Hybridization analysis of this RNA indicated that specific recognition of RNA polymerase promoter sites was preserved, since transcription was asymmetric, occurring preferentially on the "early" SV40 DNA strand. The rate of incorporation of ribonucleoside triphosphates into acid-insoluble RNA with SV40 cores as the template was 70 to 95% of that obtained with supercoiled SV40 form I DNA. SV40 minichromosomes, under identical transcription assay conditions, had an incorporation rate which was 20% of that obtained with SV40 form I DNA. These results show that association of protein VP(1) or VP(2) or both enhances the transcriptional activity and suggest that these "late" viral proteins may play a role in the regulation of expression of the SV40 genome.
D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D004279:DNA, Viral; D003852:Deoxyribonucleoproteins / Q000378:metabolism*; D004229:Dithiothreitol; D004533:Egtazic Acid; D004587:Electrophoresis, Agar Gel; D005786:Gene Expression Regulation; D006657:Histones / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D009698:Nucleoproteins / Q000378:metabolism*; D012318:RNA Polymerase I / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
D004279:DNA, Viral; D003852:Deoxyribonucleoproteins; D006657:Histones; D009698:Nucleoproteins; D014764:Viral Proteins; D004533:Egtazic Acid; D012318:RNA Polymerase I; D004229:Dithiothreitol