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Supplemental Data for Mekler et al., Cell 108, pp. 519-614


Supplemental Figure S1. Labelled RNAP core
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(A) Incorporation of probe into RNAP core. Left, intein-mediated C-terminal incorporation of fluorescein using Cys-F (Mukhopadhyay et al., 2001; see also Chong et al., 1996, 1997; Muir et al., 1998). Center, synthesis of Cys-F. Right, strategies for site-specific incorporation of fluorescein into RNAP core at b' 1377, b 643 or b 937, and aII235. Intein, S. cerevisiae VMA-I intein; inteinMxe, M. xenopi GyrA intein; CBD, chitin binding domain; F, fluorescein; H6, hexahistidine. (See Experimental Procedures.)
(B) Labelled RNAP core. SDS-PAGE of RNAP core derivatives labelled at b' 1377, b 643, b 937, or aII235, followed by x/y fluorescence scanning (fluorescein channel; excitation, 488 nm; emission, 530 ± 5 nm).


Supplemental Figure S2. Labelled RNAP-promoter open complex and RNAP holoenzyme
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(A) Labelled RPo (representative data). Nondenaturing PAGE of RPo derivatives containing fluorescein-labelled core(probe at b' 1377, b 643, b 937, or aII 235) and tetramethylrhodamine-labelled s70 (probe at residue 59, 396, 459, 517, or 578), followed by x/y fluorescence scanning for fluorescein (left, F; excitation, 488 nm; emission, 530 ± 5 nm) and for tetramethylrhodamine (right, TMR; excitation, 514 nm; emission, >610 nm).
(B) Labelled holoenzyme (representative data). Analysis as in (A).


Supplemental Figure S3. Structural organization of RNAP-promoter open complex: FRET-only, FRET+steric, and FRET+steric, closed-claw models
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(A) Model for the structural organization of RPo from distance-constrained-docking run employing only FRET constraints (FRET-only model). sR2 and sR4 are shown as yellow ribbons, with the a-helices that mediate recognition of the promoter -10 element (region-2.3/region-2.4 a-helix) and -35 element (region-4.2 a -helix) highlighted in light yellow. sR1.1, sR3.1, and sR3.2 are shown as yellow spheres. RNAP core subunits b', b, aI, aII, and w subunits are shown in orange, green, light blue, dark blue, and gray; DNA nontemplate and template strands are shown in pink and gray. Probe sites in RNAP core and DNA are indicated by, respectively, circles and diamonds (filled circles and diamonds where visible; open circles and diamonds where not visible).
(B) Model for the structural organization of RPo from distance-constrained-docking run employing both FRET constraints and steric constraints (FRET+steric model). [Positions of sR2 and sR4 are similar to those in A, but unfavorable steric interactions, in particular, interpenetrations of s70 region 1.2 and b and of s70 region 4.1 and b', are reduced.]
(C) Model for the structural organization of RPo from distance-constrained-docking run employing both FRET constraints and steric constraints, and employing structure of core having the b' pincer rotated 16° into the active-center cleft--as observed in the crystallographic structure of a eukaryotic RNAP II tailed-template elongation complex (Gnatt et al., 2001), and as proposed for bacterial RPo (Korzheva et al., 2000; Ebright, 2000) (FRET+steric, closed-claw model). [Positions of sR2 and sR4 are similar to those in A and B, but the calculated sR2-core interaction energy is significantly more favorable (differences = 1,000 kcal/mol and 700 kcal/mol; values calculated following all-atoms energy minimization).]


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