GSE202749
The pausing zone and control of RNA polymerase II elongation by Spt5: implications for the pause-release model
The pause-release model of transcription proposes that pol II pauses 40-100 bases from the start site resulting in a pile-up that is relieved by subsequent release into productive elongation.  Pause release is facilitated by PTEFb phosphorylation of the pol II elongation factor, Spt5.  We mapped paused polymerases by eNETseq and found frequent pausing in zones that extend ~0.3-3kb into genes, even when PTEFb is inhibited. The fraction of paused polymerases or “pausiness” declines gradually over several kb, and not abruptly as predicted for a discrete pause release event. Spt5 depletion extends pausing zones suggesting that it promotes maturation of elongation complexes to a low-pausing state.  Expression of mutants after Spt5 depletion showed that phosphomimetic substitutions in the Spt5 CTR1 domain diminished pausing throughout genes. In contrast mutants that prevent phosphorylation of the Spt5 RNA-binding domain strengthened pausing. Thus distinct Spt5 phospho-isoforms set the balance between pausing and elongation. Overall design: anti-pol II mNET-seq & ChIP-seq