The transcription elongation factor 
5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing 
factor (DSIF) regulates RNA polymerase II (RNAPII) processivity by promoting, in 
concert with negative elongation factor (NELF), promoter-proximal pausing of 
RNAPII. DSIF is also reportedly involved in transcriptional activation. However, 
the role of DSIF in transcriptional activation by DNA-binding activators is 
unclear. Here we show that DSIF acts cooperatively with a DNA-binding activator, 
Gal4-VP16, to promote transcriptional activation. In the absence of DSIF, 
Gal4-VP16-activated transcription resulted in frequent pausing of RNAPII during 
elongation in vitro. The presence of DSIF reduced pausing, thereby supporting 
Gal4-VP16-mediated activation. We found that DSIF exerts its positive effects 
within a short time-frame from initiation to elongation, and that NELF does not 
affect the positive regulatory function of DSIF. Knockdown of the gene encoding 
the large subunit of DSIF, human Spt5 (hSpt5), in HeLa cells reduced 
Gal4-VP16-mediated activation of a reporter gene, but had no effect on 
expression in the absence of activator. Together, these results provide evidence 
that higher-level transcription has a stronger requirement for DSIF, and that 
DSIF contributes to efficient transcriptional activation by preventing RNAPII 
pausing during transcription elongation.