1. RNA Displacement and Resolution of the Transcription Bubble during Transcription by T7 RNA Polymerase 
Manli Jiang, Na Ma, Dmitry G. Vassylyev, William T. McAllister 
Molecular Cell 
Volume 15, Issue 5, Pages (September 2004)
DOI: /j.molcel

2. Figure 1 Formation of the RNA Exit Pore during the Transition to an EC
Peptides that are involved in the crosslink to the RNA nucleotide at −14 (723–743, 751–783, 290–315) and at −9 (744–750), as well as other important regions, are mapped onto the structure of T7 RNAP in the IC (upper panels) and EC (lower panels) (Cheetham and Steitz, 1999; Tahirov et al., 2002; Yin and Steitz, 2002). The T and NT strands of the DNA are in red and blue, respectively. The view is into the RNA exit pore; the displaced RNA (yellow) is observed emerging toward the viewer (lower panel). The side chains of residues K302 and K303 are in ball-and-stick representation. A surface view indicating electrostatic potential (positive charge, blue; negative, red; neutral, white) is shown in the right portion of each panel (same orientation).
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Effects of Changes in Template Topology and Alterations in the RNA Exit Pore
(A) Templates were constructed by annealing together synthetic oligomers of DNA as indicated. In the ds template (template A), the T and NT strands are complementary over their entire length; in the “bubble” template (template B), the NT strand is not complementary from −4 to +5.
(B and C) The templates above were transcribed by wild-type (wt) T7 RNAP or by a mutant (M) RNAP (K302E/ K303E). The products were labeled by incorporation of [γ-32P]GTP and resolved by electrophoresis in 20% polyacrylamide gels (B). The relative abundance of transcripts of the sizes indicated is given in (C).
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Changes in the RNA Displacement Loop Affect Transcript Extension beyond 7 nt
(A) A ds template (template A in Figure 2) was transcribed by wt RNAP or by mutant RNAPs in which hydrophilic residues at the tip of the displacement loop (Q58, E63, and D66) were substituted with alanine (3A), arginine (3R), or methionine (3M). The products were labeled by incorporation of [γ-32P]GTP and resolved by electrophoresis in 20% polyacrylamide gels. The relative abundance of transcripts of the sizes indicated is given in the chart. A ladder of polyG products (which arise by transcript slippage) is shown in the left lane.
(B) A bubble template (template B in Figure 2) was transcribed by wt RNAP in the presence of 50 μM GTP (control) or in the presence of a 20-fold (1 mM) excess of guanosine (Gua), GMP, GDP, or GTP. Transcripts were labeled commencing at 4 nt by incorporation of [α-32P]ATP and resolved by electrophoresis in 20% polyacrylamide gels; due to differences in the initiating nucleotide, products below 6 nt migrate anomalously and have not been identified. A densitometric scan of each lane is shown at the right; the chart shows the ratio of products 6–8 nt versus 11–13 nt for each reaction.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 Role of the NT Strand in the Transition to an EC
(A) Templates were assembled by annealing appropriate DNA oligomers, as indicated, and transcribed by wt RNAP. The products were labeled by incorporation of [γ-32P]GTP and resolved as in Figure 2. In template A, the NT strand is complementary to the T strand over its full length (−24 to +34; initiation occurs at +1). In templates B and C, the NT strand is not complementary to the T strand from −4 to +5 or −4 to +9, respectively. Templates D–G, in which the NT strand is interrupted or gapped, were formed by annealing two NT strand oligomers as indicated; the upstream and downstream boundaries of the interruptions are: template D, −5/−4; template E, −4/−1; template F, −4/+5; template G, −4/+9. The templates were transcribed by wt RNAP, and the products were resolved as in Figure 2.
(B) T and NT strands are depicted as in (A). In templates D–G, the NT strand is mismatched at +1; +1 and +2; +1, +2, +3; or −1; respectively.
(C) T and NT strands are depicted as in (A). In templates C–H, the downstream boundary of the NT strand extended to −5, −1, +6, +10, +15, and +22, respectively
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
RNA Displacement and Termination at Class I and Class II Signals
(A) Effects of disruptions in the NT strand on termination at a class II terminator. Templates having a class II (PTH) termination signal (shaded box) 27 nt downstream from the promoter start site were constructed by annealing together T and NT strand oligomers, as indicated, and transcribed by wt RNAP using [α-32P]ATP as the labeled nucleotide. There are two sites of termination at the PTH signal (double underline) giving rise to termination products of 37 and 41 nt and a runoff product of 56 nt.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Organization of the Upstream and Downstream Edges of the Transcription Bubble
(A) The upstream boundary of the transcription bubble (Tahirov et al., 2002; Yin and Steitz, 2002). DNA and RNA are colored as in Figure 1. The view of the transcription complex relative to that of Figure 1 has been rotated ∼90° around the vertical axis such that the displaced RNA is directed toward the left rear of the viewing plane. The RNA displacement loop (residues 56–71) is green, and the side chains of Q58, E63, and D66 are highlighted. The flap domain (residues 152–204) is in magenta, and the side chains of K172 and R173 are highlighted. The tip of the specificity loop (residues 749–753) is in cyan. A portion of the thumb domain (residues 384–402) that makes contacts with the RNA:DNA hybrid is in orange; the side chain of Y385 (substitution of which results in failure to terminate at a class II termination signal [Brieba et al., 2000]) is highlighted.
(B) The downstream edge of the bubble. The complex has been rotated such that the displaced RNA exits to the rear and upper right of the viewing plane. Note that upstream elements of the flap domain (magenta; K163, K164) approach closely to the point of separation of the T and NT strands at the leading edge of the bubble in a similar manner as K172 and R173 at the trailing edge of the bubble (left panel). The intervening portion of the flap domain forms part of the NT strand binding channel.
Molecular Cell  , DOI: ( /j.molcel )