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Fig. 3 NSpt5 interferes with the repressive but not the stimulatory activity of endogenous DSIF in a dominant manner in vitro.
(A) The elongation stimulation activity of DSIF was assayed using pSLG402 as a template, which generates short (promoter-proximal) and long (promoter-distal, dependent on the elongation stimulatory activity of DSIF) RNase T1-resistant products under the control of the adenovirus major-late promoter. Transcription initiation/elongation was allowed to proceed for the indicated times. Time-dependent increase of distal transcripts is observed in the control (first three lanes), while NSpt5 slightly enhanced transcription at 2X or 4X concentration but not at 8X concentration. (B) pTF3-6C2AT, which generates a 380-nt RNase T1-resistant product under the control of the adenovirus E4 promoter, was used as a template, and transcription was allowed to proceed for 10 minutes. This product is sensitive to the elongation repressive activity of DSIF (in the presence of the P-TEFb inhibitor DRB)(first two lanes). NSpt5 inhibits the repression activity of endogenous DSIF at 8X concentration.