Trigger-helix folding pathway and SI3 mediate catalysis and hairpin-stabilized pausing by Escherichia coli RNA polymerase.
Bottom Line: Conversely, a substitution that disrupts the TH folding pathway and uncouples establishment of key TH-NTP contacts from complete TH formation and clamp movement allowed rapid catalysis and eliminated hairpin-stabilized pausing.The effect of SI3 depends on the jaw domain, but not on downstream duplex DNA.Our results support the view that both SI3 and the pause hairpin modulate TL folding in a constrained pathway of intermediate states.
Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.Show MeSH
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Mentions: CPR crosslinking assays (Figures 3, 6 and 7) were performed as described previously (17). Nucleic acid scaffolds were prepared by annealing 10 μM RNA, 12 μM template DNA (T-DNA) and 15 μM non-template DNA (NT-DNA) in reconstitution buffer (20 mM Tris-HCl pH 8, 20 mM NaCl and 1 mM EDTA). ECs (elongation complexes) and paused elongation complexes (PECs) were formed by incubating 1 μM RNAP and 2 μM scaffold in buffer A (50 mM Tris-HCl pH 8, 20 mM NaCl, 10 mM MgCl2, 1 mM EDTA and 2.5 ug/ml acetylated bovine serum albumin (BSA)) for 15 min at room temperature (RT). For crosslinking reactions with NTP, ECs were incubated for 15 min at RT with 10 mM GTP. Free RNAP, EC, or PEC (final RNAP 0.8 uM and scaffold 1.6 uM) were incubated for 60 min with 2.5 mM CSSC and 0.05–20 mM DTT (E = –0.140 to –0.414 V; 0.1 mM CSSC and 0.002–0.8 mM DTT was used for U937–1139 RNAP) and stopped with 50 mM iodoacetamide. Samples were separated by native PAGE to verify reconstitution efficiency and by sodium dodecyl sulphate-PAGE (SDS-PAGE) 7.5% GE Healthcare PhastGel (10–15% PhastGel for any U937–1139 RNAP) to quantify formation of crosslinks. Gels were silverstained or Coomassie brilliant blue R-250 stained, image digitized (FluorChem CCD; Protein Simple, Inc.) and quantified (ImageJ).
Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.