Volume 8, Issue 1, Pages 21-31 (July 2001) Binding of the Initiation Factor σ70 to Core RNA Polymerase Is a Multistep Process  Tanja M. Gruber, Dmitriy Markov, Meghan M. Sharp, Brian A. Young, Chi Zen Lu, Hong Ji Zhong, Irina Artsimovitch, Katherine M. Geszvain, Terrance M. Arthur, Richard R. Burgess, Robert Landick, Konstantin Severinov, Carol A. Gross  Molecular Cell  Volume 8, Issue 1, Pages 21-31 (July 2001) DOI: 10.1016/S1097-2765(01)00292-1

Figure 1 The Fragments of RNA Polymerase Utilized in This Study Linear diagrams of σ70 (A), β (B), and β′ (C) with the fragments analyzed are displayed. The endpoints of the four fragments of β (β1–β4; B), the three fragments of β′ (β'1–β'3, C), and the fragments of σ70 were based on proteolytic cleavage experiments, naturally occurring split sites, sequence homology, and functional studies (discussed in the text). Evolutionarily conserved regions of β, β′, and σ subunits are labeled above the bar (Lonetto et al., 1992; Severinov, 2000). The cloned amino acid residues of each fragment are indicated in parentheses. The position of three structural features, the flexible flap in β and the Zn++ finger and coiled-coil in β′, are shown. Mutational changes are indicated below the lines. For the flap, we either deleted its tip or introduced one of five different point mutations (see Figure 5B). For the Zn++ finger, we either deleted it or replaced the four Cys that chelate the Zn++ with Ala (see Experimental Procedures). For the coiled-coil in β′1, we introduced the point mutation A302D. For σ70, the single point mutations that were introduced into the full-length construct are indicated below the line representing the construct. A variety of mutants were used: (1) those exhibiting decreased binding when competing with wild-type σ70 for binding to core (Q406A, E407K/A, P504A, E555A, L598A; Sharp et al., 1999), (2) those inferred from the crystal structure to be involved in binding but showed no defect in a competitive binding assay (R374A, K392A; Malhotra et al., 1996; Sharp et al., 1999), and (3) a mutant that interferes with open complex formation but did not have a severe binding defect (I53A; Bowers and Dombroski, 1999). Mutational changes present in the fragments are depicted by a short vertical line Molecular Cell 2001 8, 21-31DOI: (10.1016/S1097-2765(01)00292-1)

Figure 2 GST Pull-Down Experiments of Full-Length σ70 and Its Point Mutants (Top panel) Relative interaction of each core fragment with σ70; 250 nM GST-tagged σ70 attached to glutathione agarose beads was incubated with 750 nM amounts of each His6-tagged core fragment. Following washing, the fragments were eluted and visualized on a Western blot with an anti-His6 antibody. The bands from a single blot are aligned next to each other for easier viewing. (Bottom panel) Relative interaction of wild-type and mutant σ70 with core fragments. Western blot analysis of GST pull-down results of wild-type or single-point mutant constructs of σ70 with the five most strongly interacting core fragments; 250 nM of σ70 constructs were incubated with 750 nM core fragments. Graphs on right show a graphical representation of the binding defects of point mutations that yielded consistent and reproducible defects with the particular core fragment. Each measurement represents at least three experiments, and error bars representing standard deviation from the average are indicated. GST alone never yielded a significant signal and is only shown in this figure Molecular Cell 2001 8, 21-31DOI: (10.1016/S1097-2765(01)00292-1)

Figure 4 Relative Interaction of Wild-Type and Mutant σ70 Domains with Core Fragments (A) Region 1.1 interactions with core fragments. (B) Region 1.2–2.4 interactions with core fragments. (C) Region 3.1–4.2 interactions with core fragments. For each segment, the top panel indicates the relative interaction of each core fragment with the particular σ70 domain tested, following the procedure described in Figure 2 (top panel). For each segment, the bottom panel indicates the relative interaction of wild-type and mutant σ70 domains with core fragments, following the procedure described in Figure 2 (bottom panel). To obtain a better signal, when the interactions of σ70 fragment 1.2–2.4 and its mutants were compared, 1500 nM of β′1 were used (B, bottom panel); for 3.1–4.2 and its mutants 1500 nM of β′1 and β4 were used (C, bottom panel). Graphs on right show a graphical representation of consistent binding defects. The measurement represents three experiments, and error bars are indicated Molecular Cell 2001 8, 21-31DOI: (10.1016/S1097-2765(01)00292-1)

Figure 3 A Comparison between the Interactions of Full-Length σ70 and Its Fragments with Core Fragments His6-tagged core fragments are visualized on a Western blot with an anti-His6 antibody after incubation with GST-tagged σ70 fragments attached to glutathione agarose beads. Core fragments were present in a 3-fold molar ratio over GST-tagged σ70 Molecular Cell 2001 8, 21-31DOI: (10.1016/S1097-2765(01)00292-1)

Figure 5 Effects of Mutational Changes in Core on Interaction with σ70 (A) Relative binding of core fragment β′1 and its mutants to σ70. A302D represents a mutation within the β′ coiled-coil motif. ΔZn lacks the Zn++ finger region of this fragment (aa 35–107), whereas 4C4A denotes the construct which has the four Cys residues coordinating the Zn++ mutated to Ala. (B) Relative binding of σ70/Q406A and 3.1–4.2 fragments to core fragments β′1, β′1 ΔZn, and β′1 4C4A. (C) Relative binding of core fragment β3 and its mutants to σ70. “Δ” refers to the construct of β3 containing a deletion of the tip of the flap element (aa 900–909). The effects of five single mutational changes (K900E, L901R, I905N, F906A, and K909A) within this region are also shown. (D) Relative binding of region 1.1–2.4 and its point mutant I53A to core fragment β3. In all cases, binding was measured as describe in Figure 2B. Graphs on right show a graphical representation of the binding defects of point mutations that yielded consistent and reproducible defects with the particular core fragment. Each measurement represents at least three experiments, and error bars are indicated Molecular Cell 2001 8, 21-31DOI: (10.1016/S1097-2765(01)00292-1)

Figure 6 Structural Model of the Initial Interactions of σ70 with Core Surface representation of the three-dimensional structure of T. aquaticus RNA polymerase (Zhang et al., 1999), where the β′ subunit is shown in pink, the β subunit in light blue, and the two α subunits and the ω subunit in gray. The three initial interaction sites of σ70 with E. coli core are modeled onto the structure and indicated in darker colors. Region 1.1 (residue I53) of σ70 interacts with the tip of the flap domain of β (dark blue). Region 2.2 (residues Q406, E407) of σ70 interacts with the coiled-coil domain of β′ (red). Region 3.1 (residue P504) interacts with a region close to the active site which is contained within fragment β4 of β (magenta). The active site Mg2+ is depicted in yellow. For details, see text Molecular Cell 2001 8, 21-31DOI: (10.1016/S1097-2765(01)00292-1)

Figure 7 A Model of the New Contact Sites Formed at Each Stage of σ70-Core Interaction A model of interaction sites utilized in the initial interface (A) and the new interaction sites between σ70 and core in the holoenzyme interface are shown (B). Although not indicated in the figure, we believe that initial interface contacts are maintained in the holoenzyme interface (see text). The σ70 domains are placed further apart in (B), representing the conformational changes upon initial binding to core, as well as the reorientation of regions 1.1 and 3.1–4.2 by 20 Å relative to the region 1.2–2.4 domain upon core binding (Callaci et al., 1999). Rectangles represent σ70 fragments, and ovals represent core fragments. Specific residues implicated in interactions are indicated. Interactions indicated in gray did not satisfy as stringent conditions as the interactions displayed in black (see Discussion) Molecular Cell 2001 8, 21-31DOI: (10.1016/S1097-2765(01)00292-1)