Identification of residues in the mobile H3 region of NusAN required for transcription pause enhancement. (A) Model of NusAN (bronze) bound to RNAP (grey) over the β-FTH (FTH). RNA within the exit channel is shown in yellow and NusAN residues that form the basic patch are shown in magenta. Template DNA strand (orange), non-template strand (cyan). (B–D) Measurement of transcription pause half lives. (B) Transcription pause assays with a schematic of the template shown above the gel. P, his pause site; T, termination site; RO, run-off (when RNAP falls off the end of template DNA). Wedges above gel indicate the relative time points at which samples were taken from 0.5 to 10 min (0.5, 1, 1.5, 2, 5 and 10 min). (C) Determination of transcription pause half-life (t1/2) using data from (B). (D) Half lives for NusAN constructs with altered basic patch residues. Half lives were determined in triplicate and presented as% pause activity relative to wild-type ± SD. Δ Helix, H3 deleted; all Ala, residues K36, K37, R104, Q108, K111, Q112, Q116, R119 altered to alanine. Other amino acid changes are shown below their respective columns.