Supporting InformationHudson et al. 10.1073/pnas.0908782106SI TextPlasmids. Plasmid pEcABC-H6 encodes Escherichia coli RNAP� subunit, � subunit, and C-terminally-hexahistidine-tagged ��subunit under control of the bacteriophage T7 gene 10 promoter,has a ColE1 replication origin, and confers ampicillin resistance.The plasmid was constructed from pIA299 (1) by replacementof the GTG start codon of the gene encoding C-terminally-hexahistidine-tagged �� by an ATG start codon; this replacementresults in an �4-fold increase in protein yield. Plasmid pRSF-duet-sigma encodes E. coli �70 under control of the bacterio-phage T7 gene 10 promoter, has an RSF1030 replication origin,and confers kanamycin resistance. The plasmid was constructedby replacement of the NcoI-HindIII segment of pRSFduet(Novagen) by an NcoI-HindIII rpoD segment generated byadd-on-PCR using pHTT7f1NH� (2) as template. PlasmidpCDF-omega encodes E. coli � under control of the bacterio-phage T7 gene 10 promoter, has a CloDF13 replication origin,and confers streptomycin and spectinomycin resistance (3).

RNAP Holoenzyme. E. coli BL21(DE3) (Novagen) was trans-formed with plasmid pEcABC-H6, pCDF-omega, and pRSF-duet-sigma and was cultured at 37 °C in 6 L LB media containing100 �g/mL ampicillin, 50 �g/mL streptomycin, and 40 �g/mLkanamycin. Production of recombinant E. coli RNAP was in-duced by the addition of 1 mM IPTG. All subsequent steps wereperformed at 4 °C. Cells were harvested, resuspended in 100 mL50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5% glycerol, 2 mMEDTA, 2 mM DTT, 0.5 mM phenylmethylsulfonyl f luoride(PMSF), and 1� Complete Protease Inhibitor Mixture (Roche),treated with 1 mg/mL lysozyme, and lysed by sonication (Son-icator 3000; Misonix). The lysate was centrifuged for 30 min at39,000 � g, and the supernatant was precipitated with dropwiseaddition of 5% polyethylenimine (PEI) to a final concentrationof 0.5%. Following centrifugation for 30 min at 17,000 � g,RNAP was extracted from the pellet with 50 mL 20 mMTris-HCl, pH 8.0, 5% glycerol, 1 M NaCl, 1 mM DTT, and 1�Complete Protease Inhibitor Mixture. The extract was centri-fuged for 30 min at 39,000 � g to remove insoluble material. Theenzyme was precipitated from the supernatant with dropwiseaddition of an equal volume of saturated ammonium sulfate.

The ammonium sulfate precipitate was resuspended in 25 mLbuffer A [20 mM Tris-HCl, pH 8.0, 5% glycerol, 500 mM NaCl,1 mM �-mercaptoethanol, 1 mM tris(2-carboxyethyl)phosphine]containing 1� Complete Protease Inhibitor Mixture and ad-sorbed onto Ni-NTA-agarose (Qiagen). After washes with 50 mLbuffer A and 50 mL buffer A containing 10 mM imidazole, theenzyme was eluted in 25 mL buffer A containing 150 mMimidazole. The eluted material was re-precipitated with drop-wise addition of an equal volume of 50% saturated ammoniumsulfate and centrifuged for 30 min at 17,000 � g.

RNAP was resuspended in TG (20 mM Tris-HCl, pH 8.0, 5%glycerol) containing 0.1 mM EDTA and 1 mM DTT and purifiedby heparin affinity (20 CV, 0.1–0.8 M NaCl gradient, elution at0.5 M NaCl) using a 5-mL heparin HiTrap column (GE Health-care) followed by anion exchange (25 CV, 0.3–0.5 M NaClgradient, elution at 0.37 M) using a Tricorn Q column (GEHealthcare). The recombinant enzyme was then concentrated toa volume of �2 mL and purified on a Superdex 200 16/60 columnunder isocratic conditions (TG containing 150 mM NaCl, 0.1mM EDTA, and 1 mM DTT at 0.25 mL/min). RNAP eluted after�4 h and was concentrated to 50 �M (calculated �280 � 254,000M�1cm�1) in 10 mM Tris, pH 8.0, containing 100 mM NaCl.

Catabolite Activator Protein. CAP was expressed and purified bycAMP affinity chromatography as described in ref. 4 and furtherpurified on a Heparin 16/10 FF column (GE Healthcare). CAPwas then concentrated and buffer exchanged to 0.3 mM (dimer),as determined by Bradford assay using a BSA standard, in 10 mMTris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA.

DNA. Gel-purified DNA oligodeoxyribonucleotides for lac(I-CAP)UP-UV5-bubble (Fig. 1) were purchased from IntegratedDNA Technologies. Initial concentrations in deionized water weredetermined by UV absorption using �260 � 958,800 M�1cm�1 and946,300 M�1cm�1 for the top and bottom strands, respectively. Thetwo DNA strands (1.5 �M each in 75 mM Tris-HCl, pH 8.0, 500mM NaCl) were annealed by heating at 90 °C for 10 min in a 4-Lwaterbath and then slowly cooling over 24 h to room temperature.Following cooling, the annealing procedure was repeated. Afterannealing, 10 mM MgCl2 was added, and the DNA was ethanol-precipitated and washed (5). When dry, the annealed DNA wasresuspended in deionized water and quantified by UV absorptionusing �260 � 1,382,100 M�1cm�1 (calculated assuming 28 unpairedDNA bases and 84 DNA base pairs).

Complex Assembly. The Class I CAP-RNAP-promoter complexwas assembled in HMC buffer (25 mM HEPES, pH 8.0, 100 mMKCl, 10 mM MgCl2, 1 mM DTT, 200 �M cAMP). RNAP wasadded in slight molar excess (1.05:1) to 10.9 �M lac(ICAP)UP-UV5-bubble DNA (Fig. 1) and incubated at 37 °C for 15 min.Heparin-Sepharose CL-6B resin (Amersham Biosciences) sus-pended in HMC buffer was then added to a concentration of 10mg/mL and incubated at 37 °C for 15 min to remove unboundRNAP. The resin was removed by filtration using a 0.22-�mMillex-GV filter unit. CAP was then added in 2-fold molar excess(2:1) to the RNAP-DNA complex and incubated at 37 °C for 15min. Complexes were stored at 4 °C for up to 1 week.

Electrophoretic Mobility Shift Assays. Class I CAP-RNAP-promotercomplexes were assembled in HMC buffer in a total volume of 12�L and contained 5 pmol DNA. Upon the addition of RNAPand/or CAP, samples were incubated at 37 °C for 15 min. Imme-diately before loading, 3 �L 25% glycerol were added, and sampleswere gently mixed. Samples were loaded onto a 5% polyacrylamideBio-Rad Ready Gel (Bio-Rad Laboratories) and electrophoresedat room temperature in Tris-borate-EDTA running buffer at 90 Veither for 70 min to resolve bound vs. unbound DNA or for 4 h toresolve CAP-RNAP-DNA vs. RNAP-DNA. DNA and DNA-containing complexes were visualized using SYBR GOLD (Invitro-gen) or 1 �g/mL ethidium bromide.

Specimen Preparation. Negatively stained EM specimens wereprepared using a carbon sandwich technique with uranyl formatestain (6). Four hundred mesh copper 2.0 � 0.5 hole patternC-flat grids (Protochips) with a thin layer of continuous carbonfloated on top were glow-discharged for 10 s. Class I complexeswere diluted 20-fold in HMC buffer. Uranyl formate (50 �L 2%,wt/vol) was drawn up into a disposable pipet tip, and followinga small air gap, �5 �L sample were drawn into the same tip. Thesample and then stain were delivered in one continuous motiononto a grid held at a 45° angle. The final drop of stain was allowedto sit on the grid for 1 min. The grid was then submerged in stainsolution and brought up under thin carbon to create the uppersandwich layer. The specimen was gently blotted from the sidewith filter paper and air-dried for 10 min before imaging.

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�CTD-�R4-DNA Crystal Structure. Crystals of a transcription acti-vation subassembly composed of �CTD (� subunit residues,246–329), �R4 (Thermus aquaticus � residues 366–438 with424–429, KYHESR, replaced with E. coli sequence residues,RHPSR), and duplex DNA (stand 1: 5�-TGGAAAAAAG-TACTTGACATGG-3�, strand 2: 5�-CCATGTCAAG-

TACTTTTTTCC-3�) were obtained in 28% (wt/vol) PEG 4000,0.2 M ammonium acetate, 0.01 M sarcosine, 0.1 M sodiumcitrate, pH 5.6. X-ray diffraction data were collected to 3.3 Å atthe National Synchrotron Light Source beamline X25, and thestructure was determined using molecular replacement.

1. Artsimovitch I, Svetlov V, Murakami KS, Landick R (2003) Co-overexpression of Esche-richia coli RNA polymerase subunits allows isolation and analysis of mutant enzymeslacking lineage-specific sequence insertions. J Biol Chem 278:12344–12355.

2. Chen H, Tang H, Ebright RH (2003) Functional interaction between RNA polymerase �

subunit C-terminal domain and �70 in UP-element- and activator-dependent transcrip-tion. Mol Cell 11:1621–1633.

3. Vrentas CE, Gaal T, Ross W, Ebright RH, Gourse RL (2005) Response of RNA polymeraseto ppGpp: Requirement for the omega subunit and relief of this requirement by DksA.Genes Dev 19:2378–2387.

4. Zhang XP, Gunasekera A, Ebright YW, Ebright RH (1991) Derivatives of CAP having nosolvent-accessible cysteine residues, or having a unique solvent-accessible cysteineresidue at amino acid 2 of the helix-turn-helix motif. J Biomol Struct Dyn 9:463–473.

5. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

6. Ohi M, Li Y, Cheng Y, Walz T (2004) Negative staining and image classification—powerful tools in modern electron microscopy. Biol Proced Online 6:23–34.

7. Mukhopadhyay J, et al. (2008) The RNA polymerase ‘‘switch region’’ is a target forinhibitors. Cell 135:295–307.

8. Lawson CL, et al. (2004) Catabolite activator protein: DNA binding and transcriptionactivation. Curr Opin Struct Biol 14:10–20.

9. Chlenov M, et al. (2005) Structure and function of lineage-specific sequence insertionsin the bacterial RNA polymerase �� subunit. J Mol Biol 353:138–154.

10. Tuske S, et al. (2005) Inhibition of bacterial RNA polymerase by streptolydigin: Stabi-lization of a straight-bridge-helix active-center conformation. Cell 122:541–552.

11. Malhotra A, Severinova E, Darst SA (1996) Crystal structure of a �70 subunit fragmentfrom E. coli RNA polymerase. Cell 87:127–136.

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Fig. S1. Electrophoretic-mobility-shift assay of Class I CAP-RNAP-promoter complex: (1) RNAP � DNA, (2) CAP � RNAP � DNA.

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Fig. S2. T. thermophilus RNAP structure and sequence comparison with E. coli RNAP. (A) Crystal structure of T. thermophilus RNAP (PDB id 3dxj) (7). RNAPsubunit colors are: �I, yellow; �II, green; �, cyan; ��, pink; �, gray; �, orange. (B) Gaussian-blurred volume derived from the T. thermophilus RNAP crystal structureused as starting point for projection matching in reconstruction of E. coli Class I CAP-RNAP-promoter complex. (C) Schematic sequence alignments of T.thermophilus and E. coli � (cyan), �� (pink), and �70 (orange) subunits illustrating relative positions of E. coli-specific sequence inserts ��GNCD, �DR1, �DR2, �

residues 1126–1179, and �70NCR.

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Fig. S3. Class I CAP-RNAP-promoter complex reconstruction. (A) Fourier shell correlation plotted vs. resolution. (B) Representative 2D class average (Left) withcorresponding 2D projection of the 3D map (Center) and fitted EM coordinate model in the same orientation (Right). CAP upstream and downstream subunitsare in blue and light blue, respectively; DNA is in red; RNAP subunits are colored as in Fig. S2. Labels correspond to readily identifiable features in the 2D average.(C) Additional class averages (Center) and corresponding map projections (Bottom) matched with reference-free class averages (Top). The reference-freeaverages were calculated using EMAN refine2d.py. (D) Orientation distribution of particles displayed in polar projection [plot center is � � 0° (zenith), � � 0°(azimuth); horizontal line spans � � 0°–90° for � � 0°; outer circle spans � � 0°–360° for � � 90°; �� direction is clockwise]. The number of particles for each classaverage is indicated by color-coding with lowest values in red and the highest values in green (see legend). Classes shown in B and C are indicated on the plotby labels and thick black circles.

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Fig. S4. Structure of the Class I CAP-RNAP-promoter complex: comparison of the EM reconstruction fitted model (Top) and the Lawson et al. 2004 publishedmodel (Bottom; stereo panels). The view orientation of RNAP is identical in each panel. The view orientation of the published model is as in ref. 8. CAP upstreamand downstream subunits are in blue and light blue, respectively; cAMP ligands bound to CAP are in red; other colors are as in Fig. S3.

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Fig. S5. Class I CAP-RNAP-promoter complex EM reconstruction and fitted model: E. coli-specific �� trigger loop nonconserved domain (��GNCD; stereo pairs;density contoured at 2.6�). (A) Fitted crystal structures of ��GNCD [�� residues 944-1129; PDBid 2auk (9); SBHMa domain in orange and SBMHb domain in yellow]and T. thermophilus RNAP with �� trigger loop in open conformational state [PDBid 1zyr (10); �� trigger loop in green; �� jaw in blue; other colors as in Fig. S3].(B) Fitted EM model. Connection points between ��GNCD and the trigger loop are indicated by black spheres.

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Fig. S6. Class I CAP-RNAP-promoter complex EM reconstruction and fitted model: regions containing E. coli-specific � insertions: �DR1, �DR2, and � residues1–1179 (stereo pairs; contour level 2.6�). (A) Region containing �DR1. (B) Region containing �DR2. (C) Region containing � residues 1126–1179 (as well as �

N-terminal residues 1–9). Within each panel, insertion starting and ending points are indicated by black spheres. RNAP subunits and DNA are colored as in Fig.S3.

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Fig. S7. Class I CAP-RNAP-promoter complex EM reconstruction and fitted model: region containing the E. coli-specific �70NCR (stereo pairs; contour level 2.6�).(A) Fitted crystal structure of E. coli �70 fragment comprising �70 region 1.2, �70NCR, and �70 region 2 (�70 residues 114–448; PDBid 1sig) (11). (B) Fitted EM model.�70NCR (�70 residues 138–351) is in green; other colors are as in Fig. S3. Starting and ending points for the �70NCR insertion are indicated by black spheres. Inboth panels, the approximate location of a 20-residue negatively charged loop that is disordered in the �70 fragment crystal structure is indicated as a dottedblack line.

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Table S1. Fitting of Crystal Structures to the Class I EM map

Fitted componentAverage map value at

fitted atoms* Shift, ņ Rotation, °†

CAP dimer (1lb2) 3.3 1.4 2.9T. thermophilus RNAP (1dxj) 6.0 0.1 0.3RNAP �2NTD dimer (1bdf) 7.0 0.2 2.8RNAP �70 region 1.2–2 (1sig) 5.8 0.9 6.7RNAP �� GNCD (2auk)‡ 3.3 n.a. n.a.

*Fitting was performed using the UCSF Chimera �fit in map� function unless otherwise noted.†Difference in real-space fitted component position for EMAN �eotest� even vs. odd maps (each map indepen-dently reconstructed with one-half of the image set).

‡Component was fitted manually.

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