1. Volume 9, Issue 3, Pages 527-539 (March 2002)
Structure of the Bacterial RNA Polymerase Promoter Specificity σ Subunit 
Elizabeth A. Campbell, Oriana Muzzin, Mark Chlenov, Jing L. Sun, C.Anders Olson, Oren Weinman, Michelle L. Trester-Zedlitz, Seth A. Darst 
Molecular Cell 
Volume 9, Issue 3, Pages (March 2002)
DOI: /S (02)
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2. Figure 1
Conserved Regions of the σ70 Family, In Situ Proteolysis, and Domain Structures
(A) The thick bar represents the 438 aa Taq σA primary sequence with amino acid numbering below. Evolutionarily conserved regions are labeled and color coded (Lonetto et al., 1992) but with region 2.5 (Barne et al., 1997) renamed 3.0. The histogram immediately above the bar represents the level of sequence identity within the conserved regions of 53 group 1 σ's (see supplemental data at Gruber and Bryant, 1997) as follows: 100% sequence identity, tall red bar; 20%, small blue bar; intermediate levels, orange, light green, and light blue bars. The thin, horizontal black bars represent limited proteolysis results. The trypsin-resistant fragment of E. coli σ70 (Severinova et al., 1996; Malhotra et al., 1996) is illustrated on top (includes a 175 aa insert between conserved regions 1.2 and 2.1 compared with Taq σA). Below, the in situ proteolysis results of Taq σA in crystallization drops is schematically illustrated. The crystal structures of fragments c and e3 (highlighted with pink shading) were determined.
(B) Taq σA (49.8 kDa) was incubated for several months in hanging drop crystallization trials and then analyzed by SDS-PAGE and Coomassie blue staining. Lane 1: protein directly from the supernatant of a centrifuged crystallization drop. The major, stable proteolytic fragments are labeled a–e. Lane 2: hexagonal crystals (crystal I) were analyzed, indicating that they contain predominantly fragment c. Lane 3: thin, hexagonal rod crystals (crystal II) were analyzed, indicating that they contain fragment e. The band labeled fragment e actually contained three polypeptides with slightly different N termini (e1, e2, and e3; Table1); only e3 was found in the washed, dissolved crystals (Table 1).
(C) Backbone ribbons of Taq σA proteolytic fragments c and e3. The conserved regions of the σ70 family (Barne et al., 1997; Lonetto et al., 1992) are color coded as in Figure 1A. Depth cueing causes parts of the structure far from the viewer to fade into the white background. The two fragments fold into three distinct structural domains, labeled σ2, σ3, and σ4.
(D) Schematic drawing showing the knot formed by the σ2 and σ3 domains of two noncrystallographically related σ2-3 molecules (green and yellow). The yellow molecule corresponds to the orientation of the σ2-3 molecule in Figure 1C.
Molecular Cell 2002 9, DOI: ( /S (02) )
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3. Figure 2 Structure and Function of σ2-3
(A) Two views of the α carbon backbone of σ2, shown as a worm and color-coded as in Figure 1C. The nonconserved insert (gray) between conserved regions 1.2 and 2.1 is partially transparent so as not to obscure features behind it. Marked with spheres and labeled are the α carbon positions of residues shown to be important for critical functions of σ2: orange, core RNAP binding (Joo et al., 1997; Sharp et al., 1999); green, –10 element recognition (Daniels et al., 1990; Kenney et al., 1989; Siegele et al., 1989; Tatti et al., 1991; Waldburger et al., 1990; Zuber et al., 1989); cyan, universally conserved aromatic residues important for open complex formation (deHaseth and Helmann, 1995; Helmann and Chamberlin, 1988; Jones and Moran, 1992; Juang and Helmann, 1994, 1995); blue, universally conserved basic residues critical for DNA binding (Tomsic et al., 2001). The view on the left is similar to the view of Figure 1C; the view on the right reveals the exposed face on top containing all of the amino acid positions important for DNA interactions and the exposed face of the region 2.2 helix (orange backbone) containing core RNAP mutants. Amino acid labeling is according to Taq σA numbering. Corresponding E. coli σ70 numbering is (Taq/E. coli): L207/L384, V210/V387, L225/L402, D226/D403, Q229/Q406, E230/E407, N232/N409, I236/M413, R237/K414, K241/K418, F248/Y425, Y253/Y430, W256/W433, W257/W434, Q260/Q437, N263/T440, and R264/R441.
(B) The α carbon backbone of σ3, shown as a worm and color-coded as in Figure 1C. Marked with spheres and labeled are the α carbon positions of residues important for σ3 functions: orange, core RNAP binding (Hernandez and Cashel, 1995; Joo et al., 1998; Sharp et al., 1999); magenta, extended –10 recognition (Barne et al., 1997). Amino acid labeling is according to Taq σA numbering. Corresponding E. coli σ70 numbering is (Taq/E. coli): H278/H455, E281/E458, M310/M487, S331/S506, and P329/P504.
(C) Abortive initiation assays (McClure et al., 1978) on a –10/–35 promoter (T7 A1) and an extended –10 promoter (gal P). Transcription complexes were formed with E. coli (lanes 1, 2, 8, and 9) or Taq (lanes 3–7 and 10–14) core RNAP, the indicated σ factor or Taq σA fragment, 0.1 mM initiator dinucleotide CpA, and promoter DNA. Transcription was initiated by the addition of [α-P32]UTP. The reaction product CpApU, where bold type denotes the radioactive label, was separated by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography.
(D) Effect of initiator dinucleotide (CpA) concentration on abortive initiation from gal P. Transcription complexes were formed with Taq core RNAP and the indicated Taq σA fragment, promoter DNA, and the indicated concentration of CpA and assayed as above. The relative activities determined from the quantitated bands, normalized to 100 for full-length Taq σA (lane 2), are shown below the gel.
Molecular Cell 2002 9, DOI: ( /S (02) )
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4. Figure 3
Superposition of σ4 Structures, Crystallization DNA, and Overview of the σ4-DNA Complex
(A) The α carbon backbones of the three independently determined σ4 structures, shown as worms (blue, σ4; orange, σ4-DNA molecule A; yellow, σ4-DNA molecule B) aligned over the structural core (Taq , corresponding to Ec ).
(B) Synthetic 11-mer oligonucleotides used for crystallization with –35 element denoted by yellow shading.
(C) The contents of two asymmetric units from the σ4-DNA crystal are shown. The σ4 molecules are shown as α carbon backbone worms. On the right, σ4 molecule A (specifically bound to the –35 element DNA) is orange, and molecule B, which does not make specific interactions with the DNA, is yellow. The DNA nontemplate strand is light green, and the template strand is dark green. The bases of the –35 element are yellow. The sequence of the –35 element is denoted for the nontemplate strand. On the left, the asymmetric unit related by crystallographic symmetry is shaded gray. The path of the DNA helix axis, calculated using CURVES (Lavery and Sklenar, 1988), is denoted by a pink line.
Molecular Cell 2002 9, DOI: ( /S (02) )
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5. Figure 4 Protein-DNA Contacts
(A) Schematic representation of σ4-DNA contacts, all from the major groove. The nontemplate strand is light green, and the template strand is dark green. The bases of the –35 element are yellow. The upstream DNA from a symmetry-related DNA molecule is gray. Colored boxes denote σ4 residues making DNA contact (tan, region 4.1; brown, region 4.2). Connecting solid lines indicate hydrogen bonds (<3.2 Å) or salt bridges (<4.0 Å) with the magenta lines (from Taq-L398 and E399, corresponding to Ec-L573 and E574) denoting main chain –NH contacts. The thick solid lines (from Taq-R379 and R409, corresponding to Ec-R554 and R584) indicate two hydrogen bonds from the same residue. Intervening water molecules are shown as pink circles. Dashed blue lines indicate potential van der Waal's (hydrophobic) contacts (<4.0 Å). Bridging phosphates with red dots (–35 to –38, and –31′) interfere with RNAP binding when ethylated (Siebenlist and Gilbert, 1980; Siebenlist et al., 1980). Dimethyl sulfate methylation of N7 on –31′G (underlined in red) strongly inhibits RNAP binding (Siebenlist and Gilbert, 1980; Siebenlist et al., 1980). Binding of RNAP also strongly protects this position against methylation (Johnsrud, 1978; Ross et al., 2001; Siebenlist and Gilbert, 1980; Siebenlist et al., 1980). Amino acid labeling is according to Taq σA numbering. Corresponding E. coli σ70 numbering is (Taq/E. coli): R379/R554, R387/R562, L398/L573, E399/E574, T408/T583, R409/R584, E410/E585, R411/R586, R413/R588, Q414/Q589, and K418/K593.
(B) Stereo view showing σ4-DNA interactions in the major groove of the –35 element DNA. The α carbon backbone of σ4 is shown as a worm, with region 4.1 colored tan, region 4.2 colored brown, and the rest colored gray. Side chains and main chain nitrogens that contact the DNA are shown (as illustrated schematically in Figure 4A). Potential hydrogen bonds (<3.2 Å) are shown as gray, dashed lines. Carbon atoms of the side chains are colored as the backbone, nitrogen atoms are blue, and oxygen atoms are red. Water molecules mediating protein-DNA contacts are shown in pink. For the DNA, the nontemplate strand is light green, and the template strand is dark green. The bases of the –35 element are yellow. DNA atoms that contact the protein are colored blue (nitrogens), red (oxygens), or cyan (van der Waal's contacts). DNA from the upstream, symmetry-related molecule is shaded gray. Amino acid labeling is according to Taq σA numbering. Corresponding E. coli σ70 numbering is listed in the legend for Figure 2A.
Molecular Cell 2002 9, DOI: ( /S (02) )
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6. Figure 5 Surface Properties of σ4
(A) Orthogonal views of σ4-DNA, showing surfaces involved in DNA binding and core RNAP binding. The α carbon backbone (at 50% scale) is shown in the same orientation to the lower left of each surface diagram. The DNA nontemplate strand is light green, and the template strand is dark green. The –35 element bases are yellow. The protein surface is gray except for the following color-coding as denoted in the color-wheel (left): residues in the DNA binding interface (<4.0 Å; Figure 4), blue, green, or magenta; hydrophobic residues, yellow, green, or orange; core RNAP binding mutants (Sharp et al., 1999), red, orange, or magenta. The core RNAP binding mutants are labeled as well. The concave pocket coated with hydrophobic residues is indicated. Amino acid labeling is according to Taq σA numbering. Corresponding E. coli σ70 numbering is (Taq/E. coli): R387/R562, K388/F563, L390/I565, and L423/598.
(B) Same views of σ4-DNA as Figure 5A, showing the two clusters of positive control mutants. The DNA is color coded as in Figure 5A, except the backbone of the DNA where the downstream subunits of the dimeric activators catabolite activator protein (CAP), FNR, and phage λcI bind in the major groove is red. The protein surface is gray, except selected positive control mutants are color coded as follows: blue, basic residues; red, acidic; cyan, polar; yellow, hydrophobic. The positive control mutants are also labeled; the lowercase black letters indicate which activator function is disrupted by the particular mutant: c, CAP (Lonetto et al., 1998); f, FNR (Lonetto et al., 1998); l, λcI (Kuldell and Hochschild, 1994; Li et al., 1994); p, PhoB (Kim et al., 1995). Amino acid labeling is according to Taq σA numbering. Corresponding E. coli σ70 numbering is (Taq/E. coli): E395/D570, H396/Y571, T397/T572, L398/L573, E400/E575, A403/K578, E416/E591, K418/K593, R421/R596, K422/K597, K424/R599, H426/H600, and R429/R603.
Molecular Cell 2002 9, DOI: ( /S (02) )
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