The expression of many metazoan genes is regulated through controlled release of 
RNA polymerase II (Pol II) that has paused during early transcription 
elongation. Pausing is highly enriched at genes in stimulus-responsive pathways, 
where it has been proposed to poise downstream targets for rapid gene 
activation. However, whether this represents the major function of pausing in 
these pathways remains to be determined. To address this question, we analyzed 
pausing within several stimulus-responsive networks in Drosophila and discovered 
that paused Pol II is much more prevalent at genes encoding components and 
regulators of signal transduction cascades than at inducible downstream targets. 
Within immune-responsive pathways, we found that pausing maintains basal 
expression of critical network hubs, including the key NF-κB transcription 
factor that triggers gene activation. Accordingly, loss of pausing through 
knockdown of the pause-inducing factor NELF leads to broadly attenuated immune 
gene activation. Investigation of murine embryonic stem cells revealed that 
pausing is similarly widespread at genes encoding signaling components that 
regulate self-renewal, particularly within the MAPK/ERK pathway. We conclude 
that the role of pausing goes well beyond poising-inducible genes for activation 
and propose that the primary function of paused Pol II is to establish basal 
activity of signal-responsive networks.