NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in 
extracts from human cells. A multifaceted approach was taken to investigate the 
potential role of these factors in promoter proximal pausing on the hsp70 gene 
in Drosophila. Immunodepletion of DSIF from a Drosophila nuclear extract reduced 
the level of polymerase that paused in the promoter proximal region of hsp70. 
Depletion of one NELF subunit in salivary glands using RNA interference also 
reduced the level of paused polymerase. In vivo protein-DNA cross-linking showed 
that NELF and DSIF associate with the promoter region before heat shock. 
Immunofluorescence analysis of polytene chromosomes corroborated the 
cross-linking result and showed that NELF, DSIF, and RNA polymerase IIa 
colocalize at the hsp70 genes, small heat shock genes, and many other 
chromosomal locations. Finally, following heat shock induction, DSIF and 
polymerase but not NELF were strongly recruited to chromosomal puffs harboring 
the hsp70 genes. We propose that NELF and DSIF cause polymerase to pause in the 
promoter proximal region of hsp70. The transcriptional activator, HSF, might 
cause NELF to dissociate from the elongation complex. DSIF continues to 
associate with the elongation complex and could serve a positive role in 
elongation.