Extended Data Fig. 2: RNA extension assays on HIV-1 nucleic acid scaffold.
From: Structure of paused transcription complex Pol II–DSIF–NELF
a, HIV-1 nucleic acid scaffold used for RNA extension assays. The sequence is slightly altered from that used for cryo-EM to allow extension for eight bases before pausing. Known pause and arrest sites are marked on the sequence. b–e, Pol II ECs (75 nM) were reconstituted on the HIV-1 transcription scaffold (50 nM). A single reaction was incubated with ATP, CTP and UTP (0.5 mM) for 5 min to indicate the pause site (far right lane). Buffer (b), DSIF (b), NELF (c), DSIF and NELF (c), NELF tentacle mutants (d), or DSIF and NELF tentacle mutants (e) (300 nM) were incubated with the Pol II EC. NTPs were added (0.5 mM) and aliquots were taken at specific time points. Only a fraction of the starting RNA is successfully elongated owing to incomplete EC formation(see Methods for more information).
