Separation and Properties of Two Kinds of Simian Virus 40 Late Transcription Complexes

Abstract

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°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.