Limits...
Trigger-helix folding pathway and SI3 mediate catalysis and hairpin-stabilized pausing by Escherichia coli RNA polymerase.

Windgassen TA, Mooney RA, Nayak D, Palangat M, Zhang J, Landick R - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.

Show MeSH

Related in: MedlinePlus

Uncoupling TH1a formation from TH1b promotes catalysis and prevents hairpin-stabilization of pausing. (A) Processivity and pausing of wild-type, PGPP and F773V RNAPs analyzed by promoter-initiated transcription on a long template containing the ops pause, his pause and his terminator (pIA349, Supplementary Table S1), reactions separated by 8% PAGE. Reactions proceed from halted complexes (HC) after addition of all four NTP at time 0. RO, run-off product. (B) The pause fraction and escape rate on the his pause for wild-type, PGPP and F773V RNAPs were quantified on reconstituted scaffoldPEC as in Figure 5 but starting reactions at U29 (pause) at time 0 and measuring pause escape. Data are means ± SD of experimental triplicates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4227799&req=5

Figure 6: Uncoupling TH1a formation from TH1b promotes catalysis and prevents hairpin-stabilization of pausing. (A) Processivity and pausing of wild-type, PGPP and F773V RNAPs analyzed by promoter-initiated transcription on a long template containing the ops pause, his pause and his terminator (pIA349, Supplementary Table S1), reactions separated by 8% PAGE. Reactions proceed from halted complexes (HC) after addition of all four NTP at time 0. RO, run-off product. (B) The pause fraction and escape rate on the his pause for wild-type, PGPP and F773V RNAPs were quantified on reconstituted scaffoldPEC as in Figure 5 but starting reactions at U29 (pause) at time 0 and measuring pause escape. Data are means ± SD of experimental triplicates.

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).


Trigger-helix folding pathway and SI3 mediate catalysis and hairpin-stabilized pausing by Escherichia coli RNA polymerase.

Windgassen TA, Mooney RA, Nayak D, Palangat M, Zhang J, Landick R - Nucleic Acids Res. (2014)

Uncoupling TH1a formation from TH1b promotes catalysis and prevents hairpin-stabilization of pausing. (A) Processivity and pausing of wild-type, PGPP and F773V RNAPs analyzed by promoter-initiated transcription on a long template containing the ops pause, his pause and his terminator (pIA349, Supplementary Table S1), reactions separated by 8% PAGE. Reactions proceed from halted complexes (HC) after addition of all four NTP at time 0. RO, run-off product. (B) The pause fraction and escape rate on the his pause for wild-type, PGPP and F773V RNAPs were quantified on reconstituted scaffoldPEC as in Figure 5 but starting reactions at U29 (pause) at time 0 and measuring pause escape. Data are means ± SD of experimental triplicates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4227799&req=5

Figure 6: Uncoupling TH1a formation from TH1b promotes catalysis and prevents hairpin-stabilization of pausing. (A) Processivity and pausing of wild-type, PGPP and F773V RNAPs analyzed by promoter-initiated transcription on a long template containing the ops pause, his pause and his terminator (pIA349, Supplementary Table S1), reactions separated by 8% PAGE. Reactions proceed from halted complexes (HC) after addition of all four NTP at time 0. RO, run-off product. (B) The pause fraction and escape rate on the his pause for wild-type, PGPP and F773V RNAPs were quantified on reconstituted scaffoldPEC as in Figure 5 but starting reactions at U29 (pause) at time 0 and measuring pause escape. Data are means ± SD of experimental triplicates.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.

Show MeSH
Related in: MedlinePlus