Cryo-EM structure of E. coli core RNAP. Shown in blue is a 200-Å thick cross section through the reconstructed tube, contoured at 1.1 σ. The noisy, inner circle of density is caused by the cylindrical lipid bilayer. Arranged around the outside of the lipid tube are the E. coli core RNAP molecules. The density caused by a single E. coli core RNAP molecule is highlighted in purple. The density for a single E. coli core RNAP molecule, extracted from the whole reconstruction and magnified, is shown in red in the middle of the cross section. Scale bars are for the whole reconstruction (blue) and magnified molecule (red).

Conformational change of the Taq core RNAP x-ray structure. Shown is the α-carbon backbone of the Taq core RNAP x-ray structure (blue ribbon). Superimposed on every fifth α-carbon is a vector (red) denoting the magnitude and direction of displacement to the same α-carbon of the flexed structure. The flexed structure (not shown) is available for download at ftp://ftp.scripps.edu/pub/wriggers/dars02. Figure was generated with the program vmd (37).

Sequence comparison between E. coli and Taq core RNAP subunits. The black bars represent the primary sequences of the core RNAP subunits (β′, β, α, and ω). The gray boxes indicate evolutionarily conserved regions among all prokaryotic, chloroplast, archaebacterial, and eukaryotic sequences, labeled A–H for β′ (7, 34) and A–I for β (7, 35). For α, motifs 1 and 2 are shown (2, 36). For ω, conserved regions 1 and 3 are shown (3). Insertions (>15 aa) in β′ and β are shown as white bars above (for E. coli insertions) or below (for Taq insertions). To the right of each subunit, the sequence identity (%)/sequence similarity (%) between the E. coli and Taq subunit is shown, calculated by ignoring the large, nonconserved inserts.

Fit of the Taq x-ray structure (αI, yellow; αII, green; β, cyan; β′, pink; ω, white) into the E. coli cryo-EM map (blue net). (a) One view of a single E. coli core RNAP molecule extracted from the cryo-EM map, with (Left) the cryo-EM map alone, (Center) the original (not flexed) Taq core RNAP x-ray structure (6 7) superimposed, showing the less than ideal fit of the β′ subunit, and (Right) the flexed Taq x-ray structure superimposed. (b) View (oriented with respect to a as indicated) of the E. coli core RNAP cryo-EM map with the flexed Taq core RNAP x-ray structure superimposed. (c) Stereo view (oriented with respect to b as indicated) of the E. coli core RNAP cryo-EM map with the flexed Taq core RNAP x-ray structure superimposed. The difference densities attributed to E. coli βDR1 and βDR2 (labeled DR1′ and DR2′ in all of the views) are shown in magenta. The red * in the lower-right view denotes the position of the positive difference peak determined by cryo-EM analysis of a mutant E. coli core RNAP containing a 30-kDa domain inserted in the middle of βDR2 (13). In all of the views, the active site Mg²⁺ is denoted by a magenta sphere. Differences between the E. coli cryo-EM map and the flexed Taq x-ray structure are labeled. These are E. coli βDR1 and βDR2, located near their insertion points with respect to Taq β208–233 (colored red and labeled a) and Taq β803–806 (colored red and labeled b). The red atoms labeled c denote a gap in the Taq β′ chain (from Taq β′ 32–68) that includes the Zn²⁺-binding motif universally conserved among prokaryotes. The red atoms labeled NCD1′ denote the gap in the Taq β′ chain (Taq β′ 156–451) caused by β′NCD1 (6, 7). The red atoms labeled NCD2 denote a gap in the Taq β′ chain (Taq β′ 1242–1249) where E. coli β′NCD2 is inserted (see Fig. 1). The ω subunit is also labeled. Figure was generated by using the program o (33).