1. The Flap Domain Is Required for Pause RNA Hairpin Inhibition of Catalysis by RNA Polymerase and Can Modulate Intrinsic Termination 
Innokenti Toulokhonov, Robert Landick 
Molecular Cell 
Volume 12, Issue 5, Pages (November 2003)
DOI: /S (03)

2. Figure 1 Pause Hairpin Interaction with RNAP and Flap Domain
(A) View into RNAP's main channel and RNA exit channel in a model of the his paused TEC (see Experimental Procedures). RNAP transcribes rightward along the DNA (template strand, dark green; nontemplate strand, light green). DNA upstream from the RNA:DNA hybrid and nontemplate DNA in the transcription bubble are removed for clarity. The flap domain (cyan) and loops in the clamp domain that close the main channel and RNA exit channel (rudder, lid, zipper, and ZBD; pink) are shown as worms; Zn2+ in the ZBD is yellow. The two flap deletions are highlighted in dark blue (890–914) and green (900–909). The flap and clamp domains can move relative to each other and to the main body of the enzyme (Cramer et al., 2001; Darst et al., 2002; Gnatt et al., 2001).
(B) Close-up stereo view of the RNA exit channel, with part of clamp removed to reveal steric clash between the pause hairpin and the clamp domain (jagged yellow line indicates where clamp was cut off). To optimize the view into the RNA exit channel, the TEC is rotated 10° to the right relative to the view in (A). Features are depicted and colored as in (A). The hydrophobic patch residues L901, L902, I905, and F906 are semitransparent white CPK surfaces. Basic residues in the flap arms that may contact RNA phosphates are blue rods. The spacers between the pause hairpin and the RNA:DNA hybrid are labeled −11 and −10.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 Effect of Flap Domain on Hairpin-Stabilized Pausing
(A) Schematic of the his pause template (Supplemental Table S1).
(B) Pausing by wild-type, ΔFT, and ΔFTH RNAPs at 20 μM GTP. RNAs were labeled by incorporation of [32P]CMP during formation of A29 complexes and then sampled at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, and 8 min after addition of NTPs to 200 μM (except GTP, 20 μM) and again (C) 5 min after adjusting all four NTPs to >250 μM (Experimental Procedures; the same time points were used in all experiments). P, the his pause RNA (U71). RO, run-off RNA formed when RNAP reaches the end of the template. Half-lives and pause efficiencies estimated as described previously (Landick et al., 1996b) are listed below each set of lanes.
(C) Pausing by wild-type and ΔFT RNAPs at 5 μM GTP in the absence and presence of oligonucleotide A, which disrupts the formation of the his pause hairpin. Experimental protocol and layout as in (B) except that 5 μM GTP was used in place of 20 μM GTP.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 Effect of Flap Domain on Hairpin-Independent Pausing
(A) Schematic of the ops pause template (Supplemental Table S1).
(B) Pausing by wild-type and ΔFT RNAPs at 20 μM GTP, as described in Figure 2B legend.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
The Pause Hairpin Forms in ΔFT TECs without Stabilizing the Pause
(A) Diagram of his pause RNA with C44-G59 bp flipped to G44-C59 (boxed) and locations of RNase T1 digestion (arrows). Antisense oligonucleotide B pairs to the pause RNA from C25 to U42.
(B) RNAs formed after RNase T1 digestion of U71 TECs containing wild-type or ΔFT RNAP and a single, 3′-terminal [α-32P]UMP. U71 TECs were formed on a template encoding the his pause RNA depicted in (A), but otherwise like the template depicted in Figure 2A (Supplemental Table S1). M, purified U71 RNA untreated (−), treated with RNase T1 (+), or treated with 50 mM sodium carbonate (pH 9), 1 mM Na2EDTA for 2 min at 95°C (OH−); samples of U71 TECs were treated as depicted in the figure and described in Experimental Procedures.
(C) Densitometric tracings of RNase T1 digestion products formed in the presence and absence of oligonucleotide B after treatment of U71 TECs containing wild-type or ΔFT RNAP.
(D) Experimental scheme for delay at U71 prior to addition of GTP.
(E) Pausing by wild-type and ΔFT RNAPs at 20 μM GTP with 5 min delay at U71 prior to GTP addition, otherwise as described in Figure 2B legend.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
Effect of Flap Tip on Translocation Register and Pyrophosphorolysis
(A) Cleavage of U71 RNA by Fe2+-generated free radicals in U71 TECs containing wild-type or ΔFT RNAPs. U71 TECs were formed on the template depicted in Figure 2A with 32P label at C53 and C57. The product of free radical cleavage is C70 RNA carrying a 3′ phosphate, which migrates between C70 and G69 RNAs (carrying 3′ OH groups). See Supplemental Figure S1 for confirmation of cleavage mapping.
(B) Diagram of his pause RNA showing the locations of free radical cleavage.
(C) Pyrophosphorolysis of U71 TECs containing wild-type or ΔFT RNAPs. U71 TECs were formed on the template depicted in Figure 2A and treated with 1 mM PPi (Experimental Procedures).
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6 Effect of Flap-Tip Deletions on Intrinsic Termination
(A) Structures of intrinsic terminators with the ΔG of formation (in kcal/mol at 1 M NaCl and 37°C) for the terminator hairpin alone (red; Zuker, 2003) and the 8 bp hybrid (black; Sugimoto et al., 1995). Underlined nucleotides are the positions of TEC dissociation. Sequences upstream from the hairpin and downstream from the hybrid that can modulate TE are not shown.
(B) Relationship between TE and ΔΔG‡. ΔΔG‡ is the energy barrier to elongation past the termination site (bypass; ΔG‡b) minus the energy barrier termination (ΔG‡t). TE can be calculated as ΔΔG‡ = −RT·ln((1/TE)−1) (von Hippel and Yager, 1992). Comparing ΔΔG‡ values removes the bias in comparing low or high values of TE, which is a hyperbolic function of relative rates (see plot and Experimental Procedures). A mutant RNAP that increases the termination barrier will decrease TE and ΔΔG‡, resulting in a negative value of ΔΔΔG‡ (ΔΔG‡ mut − ΔΔG‡ wt). We depict the effects of flap-tip mutants on the termination barrier rather than the bypass barrier because the mutants did not affect the overall elongation rate, although this assumption does not affect ΔΔΔG‡.
(C) Intrinsic termination by wild-type (black), ΔFT (blue), and ΔFTH (green) RNAPs. TE was measured at 200 μM NTPs, 20 mM NaCl, 10 mM MgCl2, 20 mM Tris·Cl, pH 8 (Experimental Procedures). Hybrid terminators swap the hairpin and hybrid regions of the his and T3 terminators as indicated (e.g., his T3 contains the hairpin from the his terminator and the hybrid from the T3 terminator). Bar graphs depict TE (top graph) or ΔΔΔG‡FT and ΔΔΔG‡FTH (bottom graph). Estimated errors are given from two measurements that typically varied by <±5%.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7 Model of Hairpin Formation in Paused or Terminating TECs
The flap domain (light blue), clamp domain (pink), and important clamp features (purple; see legend to Figure 1) are depicted in a TEC schematic with DNA (black) and RNA (red). The flexible flap tip is transparent blue. Possible alternate termination pathways are shown (see text). When the 3′-proximal U-tract is perfect, pullout could occur by slippage of the transcript over the template DNA; if not, forward translocation of RNAP without nucleotide addition (Yarnell and Roberts, 1999) likely occurs in this pathway.
Molecular Cell  , DOI: ( /S (03) )