Separation and properties of two kinds of simian virus 40 late transcription complexes.
Simian virus 40 (SV40) transcription complexes were labeled in cells with 3-min pulses of [(3)H]uridine 48 h after infection and were extracted from nuclei in isotonic buffer or in a buffer containing Sarkosyl. In sucrose gradients, the labeled complexes sedimented faster than both free RNA and most SV40 nucleoproteins. Most of the pulse-labeled nascent RNA hybridized to the entire late region of SV40, remained bound to viral DNA in Cs(2)SO(4) gradients, and ranged in size from a few nucleotides to about 5,000 nucleotides, with a peak at about 700. In contrast, the SV40-associated RNA polymerase activity in the same preparations sedimented near the major peak of SV40 nucleoproteins and was clearly separated from the transcription complexes bearing pulse-labeled nascent RNA. The two kinds of transcription complexes were released from isolated nuclei at different rates. Complexes bearing pulse-labeled RNA were released immediately when the nuclei were agitated in a Dounce homogenizer in isotonic buffer, whereas most of the complexes bearing RNA polymerase active in vitro were released more slowly, during subsequent incubation of the nuclei at 0 degrees C. Since the complexes bearing pulse-labeled nascent RNA were virtually inactive in vitro, the blocked complexes described by Laub et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3297-3301, 1980) probably account for almost all the SV40-associated RNA polymerase activity studied previously by many investigators. New procedures must be developed to preserve the activity of the pulse-labeled complexes if the many advantages of the SV40 system for studying transcription by nucleoprotein complexes in vitro are to be realized fully.
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004262:DNA Restriction Enzymes; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification* / Q000378:metabolism; D007668:Kidney; D009698:Nucleoproteins / Q000302:isolation & purification*; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D013539:Simian virus 40 / Q000235:genetics*; D014158:Transcription, Genetic
D009698:Nucleoproteins; D012367:RNA, Viral; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes