Structure of a Ternary Transcription Activation Complex Deepti Jain, Bryce E. Nickels, Li Sun, Ann Hochschild, Seth A. Darst  Molecular Cell  Volume 13, Issue 1, Pages 45-53 (January 2004) DOI: 10.1016/S1097-2765(03)00483-0

Figure 1 Formation of a Ternary Complex between λcI, Taq σ4, and DNA (A) Test promoter to detect cooperative binding of λcI and Taq σ4. The test promoter is a derivative of placCons-35C (Nickels et al., 2002) that bears a consensus −35 element and modified λOL1 operator centered at −45.5 and −55, respectively, upstream of the transcription start site of a modified lac promoter. The −35 element centered at position −45.5 serves as a binding site for the Taq σ4 moiety (residues 351–438) tethered to the α N-terminal domain and linker (residues 1–248). (B) Effect of λcI on transcription from test promoter in the presence of the α-σA chimera. Cells harboring the test promoter and a linked lacZ reporter gene on an F′ episome were transformed with compatible plasmids encoding either λcI (pACλcI4B2) or no λcI (pACΔcI) and either the α-σA chimera (pBRα-σA) or α (pBRα). Plasmid pBRα-σA directs the synthesis of the α-σA chimera under the control of an IPTG-inducible promoter, whereas plasmid pACλcI4B2 directs the synthesis of λcI under the control of a constitutive promoter. The cells were grown in the presence of 1 μM IPTG and assayed for β-galactosidase activity. (C) DMS protection assay. A 3′ end-labeled DNA restriction fragment bearing the modified OL1 operator and consensus −35 element was incubated with saturating concentrations of λcI alone (lane 3), Taq σ4 alone (lane 1), λcI and Taq σ4 (lane 2), or no protein (lane 4) and subjected to DMS treatment followed by piperidine cleavage essentially as described (Sauer et al., 1979). Samples were electrophoresed on a 6% denaturing polyacrylamide gel and the bands visualized by phosphorimaging. The λcI protected guanines at positions 4′, 6′, 7′, and 9′ (consensus [c] half) and enhanced the reactivity of the guanine at position 8′ (nonconsensus [n-c] half), as previously observed (Johnson, 1980). In addition, the guanine at position 3′ in the n-c half (which is not a guanine in the context of wild-type OL1) was protected. Taq σ4 protected a single guanine at position −31′ (bottom strand). Previous DMS protection experiments performed with the σ70-containing RNAP holoenzyme revealed strong protection of the guanine at promoter position −31′ (Siebenlist et al., 1980). Molecular Cell 2004 13, 45-53DOI: (10.1016/S1097-2765(03)00483-0)

Figure 2 Structure of λcI/σ4/DNA Ternary Complex (A) Synthetic 27-mer oligonucleotides used for crystallization. The black numbers above or below the sequence denote the DNA position with respect to the transcription start site at +1. The −35 element is colored yellow. The λcI operator is magenta (except for bases within the −35 element), with base pairs of the consensus half labeled 1–8 (small magenta numbers on top) and the nonconsensus half labeled 1′–8′ (bottom). The central base pair of the operator (at −42) is labeled (*). (B) Two views of the λcI/σ4/DNA ternary complex, related by a 90° rotation about the horizontal axis as shown. Proteins are shown as α-carbon backbone ribbons, with λcI monomer A (λcIA, consensus half) colored dark green, λcIB (nonconsensus half) light green, and σ4 orange. The DNA is color coded as in (A), with the central base pair of the operator marked by a red “*.” A Ca2+ ion is shown as a yellow sphere. The region of the λcIB/σ4 protein/protein interface is boxed in gray and magnified in (C). (C) Stereoview detailing λcIB/σ4 protein/protein interactions, and selected protein/DNA interactions in the ternary complex. Proteins are shown as in (B), along with interacting side chains. Carbon atoms of protein side chains and DNA are colored as in (B), nitrogen atoms are blue, oxygens red, and phosphates magenta. Potential hydrogen bonds are shown with gray, dashed lines. Selected water molecules mediating protein/DNA interactions are shown as pink spheres. The side chains of σ4 are labeled according to Taq σA numbering (Campbell et al., 2002). Corresponding E. coli σ70 numbering is (Taq[E. coli]): 410(585), 413(588), 417(592), 418(593), 421(596). Molecular Cell 2004 13, 45-53DOI: (10.1016/S1097-2765(03)00483-0)

Figure 3 Protein/DNA Interactions in the Ternary Complex (A) Schematic representation of protein/DNA interactions, plus λcIB/σ4 protein/protein interactions, in the ternary complex. The DNA is color coded as in Figure 2A. Colored boxes denote protein residues (dark green, λcIA; light green, λcIB; orange, σ4). Connecting black solid lines indicate hydrogen bonds (< 3.2 Å) or salt bridges (< 4 Å) between protein and DNA. The red dashed lines indicate hydrogen bonds and/or salt bridges between λcIB and σ4. Thick solid lines indicate more than one hydrogen bond with the same residue. Water molecules are shown as pink spheres. The λcI residues that show symmetric protein/DNA interactions in both monomers of the ternary complex are labeled with an “*.” (B) Comparison of binary and ternary complexes. The ternary complex DNA is shown with the same color coding and in the same orientation as Figure 2A, but as a phosphate backbone worm with base pairs shown as sticks. Proteins are shown as α-carbon backbone worms. The λcI dimer from the ternary complex is colored green, σ4 orange. The λcI dimer from the λcI/OL1 binary complex (Beamer and Pabo, 1992), and the structural core of σ4 from the σ4/−35 element binary complex, each superimposed according to overlapping C1′ atoms of the DNA, are shown in blue and cyan, respectively. Relative movements of the λcI and σ4 monomers from the binary to ternary complexes are denoted by the thick arrows. (C) The ternary complex DNA is shown with the same color coding as Figure 2A but in a different orientation. The path of the DNA helical axis, calculated using CURVES (Lavery and Sklenar, 1988), is shown for the λcI/σ4/DNA ternary complex (green), the λcI/OL1 binary complex (blue), and the σ4/−35 element binary complex (orange), superimposed on the ternary complex according to overlapping protein α-carbon backbones. The positions of the HTH motifs of λcI (light green) and σ4 (orange) are shown. Molecular Cell 2004 13, 45-53DOI: (10.1016/S1097-2765(03)00483-0)

Figure 4 Ternary Complex Models with Taq RNAP Holoenzyme (A) A productive complex (in which λcI and σ4 make favorable protein-protein interactions) and a hypothetical nonproductive complex (in which λcI and σ4 are misaligned, as is predicted to occur in the closed complex) are shown. RNAP holoenzyme is shown as a molecular surface (except σ4 is shown as a backbone worm), color coded as follows: αI, αII, ω, gray; β, cyan; β′, pink; σ, orange. The DNA is shown as phosphate backbone worms. For the productive complex, the DNA template strand is dark green, nontemplate strand light green, except the −35 element is yellow and the λcI operator is magenta. For reference, every fifth base pair between −15 and −60 (with respect to the transcription start site at +1) is shown schematically, and the positions are labeled in the scale above. The λcI dimer is shown as a green backbone worm. For the nonproductive complex, the DNA and λcI dimer are shown as blue backbone worms, and the DNA is shown only for the −35 element and upstream. The green and blue arrows indicate the upstream path of the DNA for the productive and nonproductive complexes, respectively. (B) Details of the λcIB/σ4 protein/protein interactions, and selected protein/DNA interactions in the ternary complex (same view as Figure 2C). Proteins are shown as α-carbon backbone worms, with σ4 orange, λcIB from the productive complex green, and λcIB from the nonproductive complex blue. Side chains involved in the interactions are shown and colored as in Figure 1C, as are hydrogen bonds seen in the productive complex (λcI/σ4/DNA ternary complex). Molecular Cell 2004 13, 45-53DOI: (10.1016/S1097-2765(03)00483-0)