1. Transcription Initiation in a Single-Subunit RNA Polymerase Proceeds through DNA Scrunching and Rotation of the N-Terminal Subdomains 
Guo-Qing Tang, Rahul Roy, Taekjip Ha, Smita S. Patel 
Molecular Cell 
Volume 30, Issue 5, Pages (June 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
Three Models of Initial Transcription: DNA Scrunching, RNAP Translation, and RNAP Rotation
(A) Modeled structure of the initiation complex (IC3) of T7 RNAP (rendered using PDB 1QLN using Accelrys DS ViewerPro 5.0). RNA transcript is not shown, and the DNA fragment upstream of −17 is reconstructed using a DNA model server (Munteanu et al., 1998). The C-terminal domain of T7 RNAP is gray, the N subdomain (aa 1−70) is yellow, the core subdomain (aa 72–151, 206–257) is pink, the flap-like domain (subdomain H, aa 152–205) is green, and the specificity loop (aa 738–773) is blue. The DNA template and nontemplate strands are in green and red, respectively. The donor dye was placed at −4 in the NT strand (−4NT) or at the upstream 5′ end (−22NT), and the acceptor dye was placed at downstream positions in the T strand.
(B) Cartoons show the three models of T7 RNAP transcription initiation pathway: DNA scrunching (top), RNAP translation (middle), and RNAP rotation (bottom). During transcription initiation, the downstream DNA moves toward the active center (thick black arrow), while the promoter remains bound to the N-terminal subdomain. In the pure DNA scrunching model, the promoter and the upstream edge of the bubble (−4 NT) remain unmoved, while the downstream template is compacted around the active center (dotted circle). In the translation model, the promoter moves away from the fixed active center in the C-terminal domain, accompanied by the translational detachment of the N-terminal domain. In the pure rotation model, the N terminal domain undergoes a series of conformational changes that result in the rotation of the promoter and final transition into the elongation conformation at 8/9 nt RNA synthesis.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 FRET between Dyes at −4 and +18 Supports DNA Scrunching
(A) The cartoons show T7 RNAP-DNA complexes from IC2 to IC7 and EC. The structures in the cartoons do not imply any mechanism of initiation. For example, the structures of the closed complex, IC7, and EC/IC10 are not known and speculative. The various transcriptional complexes were generated by mixing dye-labeled promoter (100 nM, seq5 or seq16), T7 RNAP (120 nM) with 3′-dGTP (IC2), GTP (IC3), GTP + 3′-dATP (IC4, seq5 and 16), GTP + ATP + 3′-dCTP (IC7, seq16), or GTP + ATP + CTP + 3′-dUTP (IC/EC10, seq16). The concentration of 3′-dGTP and GTP was 1 mM each and the rest of the NTPs was 0.5 mM each.
(B) FRET (y axis) was measured between dyes at −4NT(TAMRA) and +18T (Alexa 647) on the DNA at 25°C.
(C) The D-A distances (Rda) were calculated from the FRET values using Ro (Figure S2) and Equation 1.
Data with error bars (standard deviation) were averaged from multiple measurements.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Ensemble FRET Supports DNA Scrunching during Initial Transcription
(A) Predicted changes in distance between dyes at −4NT and N + m in the T7 RNAP-DNA according to the DNA scrunching mechanism (left) or the translation mechanism (right). The numbers within the spheres are schematic positions of the donor dye Cy3 (shaded sphere) and the 5′-end of the DNA (blank sphere) predicted by the translation model upon walking from +4 to +7. In the N/N + m walking experiments, complexes halted at position N contained the acceptor dye Cy5 at the N + m position (black hexagon). The DNA scrunching model predicts that the D-A spatial distances (RDA) between dyes at −4NT and N + m will remain constant from 4 to 7 nt RNA synthesis, whereas the translation model predicts that the distance will progressively increase.
(B) Average D-A distances between Cy3 and Cy5 in ICN complexes in the N/N + 2 and N/N + 3 experiments calculated from FRET values (Table S2).
(C) Average D-A distances in the N/N + 4 and N/N + 5 experiments. Data with error bars (standard deviation) were averaged from multiple measurements.
In panels (B) and (C), the D-A distances in EC halted at +14, +15, or +12 are also shown.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Single-Molecule FRET Supports DNA Scrunching
(A) The diagram shows the single-molecule FRET experimental scheme.
(B) FRET histograms of single-molecule T7 RNAP initial transcription. The x axis represents the ratios of IA/(ID + IA), termed apparent FRET (FRETapp), and the y axis represents the frequency of transcription complexes with respective FRET values. Peak FRET values as indicated by the dashed line were used for the estimation of D-A distances (RDA).
(C) RDA plotted as a function of walking position (N) for DNA-only and transcribing complexes calculated from the FRET values (Table S2). Data with error bars (standard deviation) are averaged from multiple measurements.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Movement of the Promoter during Transcription Initiation
(A) D-A distance changes between TAMRA at −22NT and Alexa 647 at N + m downstream of +5 during 4–7 nt synthesis, as predicted by DNA scrunching (top, constant distances) and scrunching + rotation (bottom, increased distances). The numbers in the spheres are schematic positions of TAMRA predicted by the scrunching and rotation mechanism as the downstream DNA moves toward the active center.
(B) D-A distances in ICN in the N/N + 2 and N/N + 3 experiments (as in Figure 3) calculated from FRET values (Table S2).
(C) D-A distances in ICN in the N + 4 or N + 5 experiments. Data with error bars (standard deviation) were averaged from multiple measurements.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Additional FRET Studies Support DNA Scrunching/Promoter Rotation Mechanism
(A) Predicted D-A distances between TAMRA at −22 (shaded sphere) and Alexa 647 (black hexagon) at +18 in ICN and between TAMRA at −22 and Alexa 647 at +(18 − N) in IC2 according to the pure DNA scrunching, translation, or scrunching/rotation model. The D-A distance in ICN is predicted to be equal (pure DNA scrunching model), greater (pure translation model), or shorter (scrunching/rotation model) than in the IC2.
(B) Mean D-A distances between dyes at −22NT and +18 in ICN compared to those between dyes at −22 NT and +(18 − N) in IC2.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Degree of Rotation of the Promoter during Initial Transcription
(A) A simplified geometry of the promoter DNA bound to T7 RNAP. RDA1 is the D-A distance from the upstream edge of the transcription bubble (−4NT) (red sphere) to the downstream edge of the bubble, 2–5 nt (N + m) (blue hexagon) beyond the active site (green), RDA2 is the D-A distance from the promoter end (−22) to the downstream edge of the bubble, and RDA3 is the D-A distance from the upstream end (−22) to −4. FRET studies indicated that RDA1 and RDA3 remain constant from IC4 to IC7, whereas RDA2 increases to R∗DA2. In a given ICN complex, RDA1 and the angle, α, between RDA1 and RDA3 may vary as m varies, and the increase in RDA2 to R∗DA2 can be explained by the rotation of the promoter around a hinge point at −4 toward the downstream DNA. Based on the experimental D-A distances, the maximal rotation of promoter is estimated to be 15°−22° by 7 nt synthesis.
(B) Proposed rotation of the promoter around the hinge at −4 (discontinuous arrow) and scrunching of the downstream DNA in the active site pocket (dotted circle) during synthesis of 3–4 to 7 nt RNA. Corresponding rotary movements of the N-terminal subdomains and the specificity loop (colored as in Figure 1A, in the discontinuous rectangle) against the C-terminal subdomains (gray) are illustrated by the semitransparent model as the RNA is extended from 3 to 7 nt the promoter.
Molecular Cell  , DOI: ( /j.molcel )
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