Negative elongation factor (NELF) and 
5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole sensitivity-inducing factor 
(DSIF) are involved in pausing RNA Polymerase II (Pol II) in the 
promoter-proximal region of the hsp70 gene in Drosophila, before heat shock 
induction. Such blocks in elongation are widespread in the Drosophila genome. 
However, the mechanism by which DSIF and NELF participate in setting up the 
paused Pol II remains unclear. We analyzed the interactions among DSIF, NELF, 
and a reconstituted Drosophila Pol II elongation complex to gain insight into 
the mechanism of pausing. Our results show that DSIF and NELF require a nascent 
transcript longer than 18 nt to stably associate with the Pol II elongation 
complex. Protein-RNA cross-linking reveals that Spt5, the largest subunit of 
DSIF, contacts the nascent RNA as the RNA emerges from the elongation complex. 
Taken together, these results provide a possible model by which DSIF binds the 
elongation complex via association with the nascent transcript and subsequently 
recruits NELF. Although DSIF and NELF were both required for inhibition of 
transcription, we did not detect a NELF-RNA contact when the nascent transcript 
was between 22 and 31 nt long, which encompasses the region where 
promoter-proximal pausing occurs on many genes in Drosophila. This raises the 
possibility that RNA binding by NELF is not necessary in promoter-proximal 
pausing.