Antibacterial Peptide Microcin J25 (MCCJ25) Inhibits Transcription by Binding within, and Obstructing, the RNA Polymerase Secondary Channel

MccJ25 inhibits transcription at the level of NTP uptake or NTP binding by RNAP

MccJ25 requires an extensive determinant within the RNAP secondary channel

MccJ25 binds within the RNAP secondary channel

MccJ25 obstructs the RNAP secondary channel

MccJ25 is a 21-residue “lariat protoknot,” consisting of an 8-residue cyclic segment followed by a 13-residue linear segment that loops back and threads through the cyclic segment (Fig. 4A; Bayro et al., 2003; Rosengren et al., 2003; Wilson et al., 2003). MccJ25 has dimensions of ∼27 Å × ∼14 Å and is roughly comma-shaped, with the cycle and threaded segment (residues 1-8 and 18-21) corresponding to the head of the comma, and with the connector segment (residues 9-17) corresponding to the tail of the comma (Fig. 4A).

MccJ25 exhibits structural complementarity, in both size and shape, to the RNAP secondary channel. In cross-section, the RNAP secondary channel has dimensions of ∼28 Å × ∼14 Å and is roughly comma-shaped, with the secondary-channel sub-region bounded by the β′ D/E helix, E helix, F-helix, and G-helix and loop corresponding to the head of the comma, and with the secondary-channel sub-region bounded by β′ residues 498-504, 732-733, 922-926, and 1244-1248 corresponding to the tail of the comma.

We have used this structural complementarity to dock the structure of MccJ25 into the structure of RNAP in order to construct a model for the structure of the MccJ25-RNAP complex (Fig. 4B,C). In the resulting model, the cycle and threaded segment of MccJ25 (the head of the comma) is placed in the secondary-channel sub-region bounded by the β′ D/E helix, E helix, F-helix, and G-helix and G-loop; and the connector segment of MccJ25 (the tail of the comma) is placed in the secondary-channel sub-region bounded by β′ residues 498-504, 732-733, 922-926, and 1244-1248 (Fig. 4B,C). The resulting model is supported by four observations. First, the model is sterically acceptable (Fig. 4B,C). Second, the model accounts for genetic data defining critical determinants of RNAP (Fig. 2; Tables 1,2)--placing MccJ25 in contact with, or adjacent to, the majority of sites of substitutions conferring MccJ25-resistance (red and pink residues in Fig. 4B,C), and, in particular, placing the cycle and threaded segment of MccJ25 in contact with, or adjacent to, the majority of sites of substitutions conferring highest-level MccJ25-resistance (labelled residues in Fig. 4C). Third, the model accounts for genetic and biochemical data defining critical determinants of MccJ25 (J.M., E.S., and R.H.E, unpublished data)--placing essential residues of MccJ25 on the face of MccJ25 directed toward RNAP, and placing non-essential residues of MccJ25 on the face of MccJ25 directed toward solvent. Fourth, the model accounts for systematic-FRET/distance-restrained-docking data defining possible locations of the Cy3 probe of Cy3-MccJ25 (Fig. 3E)--placing the attachment site of Cy3 on the face of MccJ25 directed toward the external mouth of the secondary channel, at a position of MccJ25 close to the external mouth of the secondary channel.

The most striking feature of the model is that it suggests that MccJ25 essentially completely seals the RNAP secondary channel--like a “cork in a bottle” (Fig. 4B,C). The model suggests that binding of MccJ25 would block passage of molecules the size of NTPs and possibly also would block passage of smaller molecules. It has been proposed that NTPs pass through the RNAP secondary channel to enter the RNAP active center (Zhang et al., 1999; Cramer et al., 2000; Korzheva et al., 2000; Gnatt et al., 2001; see however, Nedialkov et al., 2003), and that abortive RNA fragments, edited RNA fragments, and backtracked RNA segments pass through the secondary channel to exit the active center (Cramer et al., 2000; Korzheva et al., 2000; Gnatt et al., 2001). The model suggests that binding of MccJ25 would block these transactions. It also has been proposed that pyrophosphate may pass through the RNAP secondary channel to exit the RNAP active center. The model suggests that binding of MccJ25 might block this transaction. Finally, it has been shown that the transcript-cleavage factors GreA, GreB, and TFIIS must enter the secondary channel to access the active center (Opalka et al., 2003; Laptenko et al., 2003; Sosunova et al., 2003; Kettenberger et al., 2003). The model suggests that binding of MccJ25 would block function of transcript-cleavage factors, consistent with the observed binding competition between Cy3-MccJ25 and GreB (Fig. 3C).

Plasmids

Plasmids used in this work are described in Supplementary Information: Plasmids.

MccJ25

MccJ25 was prepared essentially as in Blond et al. 1999 (details in Supplementary Information: MccJ25).

Cy3-MccJ25

Cy3-MccJ25 was prepared using Lys-specific chemical modification. Reaction mixtures contained (200 μl): 1 mM [Lys13]MccJ25 derivative (prepared as for MccJ25, but using plasmid pTUC202-13K), 2 mM Cy3 NHS-ester (Amersham-Pharmacia Biotech), and 0.5 % (v/v) triethylamine in dimethylsulfoxide. Following 15 h at 4°C, products were purified by reversed-phase HPLC on a C18, 5 μm, 300 Å column (Rainin), with solvent A = 0.1% trifluoroacetic acid, solvent B = 90% acetonitrile and 0.1% trifluoroacetic acid, and gradient = 25-40% solvent B in solvent A in 30 min, and flow rate = 1 ml/min. Fractions containing Cy3-MccJ25 (retention time ∼29 min; detected by UV absorbance at 276 nm) were pooled, lyophilized, re-dissolved in 500 μl 30% methanol, and stored in aliquots at -20°C.

RNAP

RNAP core and holoenzyme were prepared as in Niu et al., 1996. [Ile931]β′ RNAP core and holoenzyme were prepared from strain 397c [rpoC^ts397 argG thi lac (λcI₈₅₇h₈₀S_t68dlac⁺); Christie et al., 1996] transformed with a pRL663 derivative encoding [Ile931]β′-RNAP using analogous procedures. Yields typically were 6 mg. Purities typically were >95%.

DAC-σ⁷⁰ RNAP

DAC-σ⁷⁰ was prepared using Cys-specific chemical modification. Reaction mixtures contained (1 ml): 20 μM single-Cys σ⁷⁰ derivative [prepared as in Mukhopadhyay et al., 2001; subjected to solid-phase reduction on Reduce-Imm (Pierce) per manufacturer's instructions immediately before use], 200 μM N-(7-dimethylamino-4- methylcoumarin-3-yl)maleimide (Molecular Probes), 100 mM sodium phosphate (pH 8.0), and 1 mM EDTA. Following 1 h at 4°C, products were purified by gel-filtration chromatography on Bio-Gel P6DG (Bio-Rad), and stored in 20 mM Tris-HCl (pH 7.9), 100 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT, and 50% glycerol at -20°C. Efficiencies of labelling, determined by measurement of UV-absorbance, were ∼90%; site-specificities of labelling, determined by comparison to products of control reactions with wild-type σ⁷⁰, were ∼90%.

DAC-σ⁷⁰ RNAP holoenzyme and DAC-σ⁷⁰ [Ile931]β′ RNAP holoenzyme were prepared by incubation of 5 pmol unlabelled RNAP core and [Ile931]β′ RNAP core, respectively, with 4 pmol DAC-σ⁷⁰ in 20 μl TB for 20 min at 25°C.

DAC-ω RNAP

DAC-ω was prepared using Cys-specific chemical modification. Reaction mixtures contained (1 ml): 20 μM single-Cys ω derivative [prepared essentially as described for ω in Mekler et al., 2002; subjected to solid-phase reduction on Reduce-Imm (Pierce) per manufacturer's instructions immediately before use], 200 μM N-(7-dimethylamino-4- methylcoumarin-3-yl)maleimide (Molecular Probes), 100 mM sodium phosphate (pH 8.0), 1 mM EDTA, and 6 M guanidine hydrochloride. Following 1 h at 25°C, products were purified by gel-filtration chromatography on Bio-Gel P6DG (Bio-Rad,). Site-specificities of labelling, determined by comparison to products of control reactions with wild-type ω, were ∼90%. DAC-ω RNAP holoenzyme and DAC-ω [Ile931]β′ RNAP were prepared by reconstitution as described for unlabelled RNAP holoenzyme in Naryshkin et al., 2001, using 500 μg β′ or [Ile931]β′, 300 μg β, 30 μg hexahistidine-tagged α, and 250 μg σ⁷⁰, including 100 μg DAC-ω_n, and including a final incubation for 45 min at 30°C. Products were purified using metal-ion-affinity chromatography on Ni:NTA-agarose and ion-exchange chromatography on Mono-Q (methods essentially as in Naryshkin et al., 2001 and Niu et al., 1996); concentrated (methods essentially as in Naryshkin et al., 2001); and stored in 20 mM Tris-HCl (pH) 7.9, 100 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT, and 50% glycerol at -20°C.

Electrophoretic mobility shift assays

Reaction mixtures contained (20 μl): 100 nM RNAP holoenzyme, 20 nM DNA fragment lacUV5-12(Cy5,+26) (Mukhopadhyay et al., 2001), and 0-100 μM MccJ25 in TB [50 mM Tris-HCl (pH 8.0), 100 mM KCl, 10 mM MgCl₂, 1 mM dithiothreitol, 10 μg/ml bovine serum albumin, and 5% glycerol]. Following 15 min at 37°C, 0.5 μl 1 mg/ml heparin was added (to disrupt non-specific complexes), and, following a further 2 min at 37°C, reaction mixtures were applied to 5% polyacrylamide slab gels (30:1 acrylamide/bisacrylamide; 6 × 9 × 0.1 cm) and electrophoresed in 90 mM Tris-borate (pH 8.0) and 0.2 mM EDTA (20 V/cm; 30 min at 37°C) and analyzed using a fluorescence scanner (Storm 860; Molecular Dynamics). Identities of tri- and tetranucleotide abortive products were defined as in Borowiec and Gralla (1985).

Transcription assays

Reaction mixtures contained (18 μl): 100 nM RNAP holoenzyme, 20 nM DNA fragment lacUV5-12(Cy5,+26) (Mukhopadhyay et al., 2001), and 0-100 μM MccJ25 in TB. Following 15 min at 37°C, 0.5 μl 1 mg/ml heparin was added, and, following a further 2 min at 37°C, RNA synthesis was initiated by addition of 2 μl 5 mM ApA and 125 μM (or 250 μM, 500 μM, and 1 mM) each of [α-³²P]UTP (0.6 Bq/fmol), ATP, CTP, GTP. Following 5 min at 37°C, reactions were terminated by addition of 10 μl 80% formamide, 10 mM EDTA, 0.04% bromophenol blue, and 0.04% xylene cyanol. Products were heated 10 min at 90°C, resolved by urea-PAGE (Sambrook and Russel, 2001), and quantified using a storage-phosphor scanner (Storm 860; Molecular Dynamics). Data were fit to full-competitive, partial-competitive, full-noncompetitive, partial-noncompetitive, full-uncompetitive, partial-uncompetitive, full-mixed, and partial-mixed models of inhibition using the Fit-To-Model feature of the SigmaPlot Enzyme Kinetics Module (SPSS).

Fluorescence-detected abortive initiation assays

Reaction mixtures contained (46.5 μl): 100 nM RNAP holoenzyme, 20 nM DNA fragment lacUV5-12 (Mukhopadhyay et al., 2001), and 0-100 μM MccJ25 in TB. Following 15 min at 37°C, 0.5 μl 1 mg/ml heparin was added, and, following a further 2 min at 37°C, 1 μl of 0.25-5 mM (γ-AmNS)UTP (Molecular Probes) was added, and reaction mixtures were transferred to sub-micro fluorometer cuvettes (Starna Cells). Following 2 min at 37°C, RNA synthesis was initiated by addition of 2.5 μl 10 mM A_pA, and fluorescence emission intensity was monitored 5 min at 37°C [excitation wavelength = 360 nm and emission wavelength = 500 nm; excitation and emission slit widths = 4 nm; QuantaMaster QM1 spectrofluorometer (PTI)]. The quantity of UMP incorporated into RNA was determined from the quantity of (γ-AmNS)UTP consumed, which, in turn, was calculated as follows (Schlageck et al. 1979):

where (γ-AmNS)UTP₀ is the quantity of (γ-AmNS)UTP at time 0, F₀ is the fluorescence emission intensity at time 0, and F_t is the fluorescence emission intensity at time t. Data were fit to full-competitive, partial-competitive, full-noncompetitive, partial-noncompetitive, full-uncompetitive, partial-uncompetitive, full-mixed, and partial-mixed models of inhibition as in the preceding section.

FRET binding assays

Assay mixtures (50 μl) contained 100 nM DAC-σ⁷⁰ RNAP holoenzyme (labelled at residue 517 of σ⁷⁰) and 0-20 μM Cy3-MccJ25 in TB at 25°C. Fluorescence emission intensities were measured before and 5 min after addition of 1 μl 2 mM unlabeled MccJ25 [excitation wavelength = 385 nm; emission wavelength = 465 nm; excitation and emission slit widths = 5 nm; QuantaMaster QM1 spectrofluorometer (PTI)]. Efficiencies of fluorescence resonance energy transfer (E) and fractional saturations (θ) were calculated as:

where F and F_x are emission intensities before and after addition of 40 μM unlabeled MccJ25, and where E_max is E at saturation. The equilibrium dissociation constant (K_d,Cy3-MccJ25) was extracted by non-linear regression using the equation:

where [Cy3-MccJ25] is the concentration of Cy3-MccJ25.

To assess kinetics of binding, using a stopped-flow instrument (SFM-4/QS; Bio-Logic), 100 μl 5 μM or 10 μM Cy3-MccJ25 in TB was mixed with 100 μl 100 nM DAC-σ⁷⁰ RNAP in TB, and fluorescence emission intensity was monitored as a function of time following mixing [excitation wavelength = 400 nm; emission wavelength = 455 nm; QuantaMaster QM1 spectrofluorometer (PTI); data from four shots averaged]. The observed rate of complex formation, k_obs, the association rate constant, k_on, and the dissociation rate constant, k_off, were calculated as:

where F is fluorescence emission intensity at time, t; F_o + a is the fluorescence emission intensity at t = 0; F_o is fluorescence emission intensity at t = ∞; and k_obs,1 and k_obs,2 are observed rates at [Cy3-MccJ25]₁ and [Cy3-MccJ25]₂.

FRET competition assays

Assay mixtures (50 μl) contained 100 nM DAC-σ⁷⁰ RNAP holoenzyme (labelled at residue 517 of σ⁷⁰), 5 μM Cy3-MccJ25, and 0-20 μM competitor [MccJ25 or GreB (prepared as in Koulich et al., 1997)] in TB at 25°C. Fluorescence emission intensities were measured before and 5 min after addition of 1 μl 2 mM unlabeled MccJ25, and E and θ were calculated as above. Equilibrium dissociation constants (K_d) were calculated as:

where IC₅₀ is the concentration of competitor that yields half-maximal competition.

FRET distance measurements

Assay mixtures (50 μl) contained 100 nM DAC-labelled RNAP holoenzyme derivative or corresponding DAC-labelled [Ile931]β′ RNAP holoenzyme derivative in 50 mM Tris-HCl (pH 8.0), 800 mM KCl, 10 mM MgCl₂, 1 mM dithiothreitol, 10 μg/ml bovine serum albumin, and 5% glycerol at 25°C. Fluorescence emission intensities were measured before and 5 min after addition of 2 μl 250 μM Cy3-MccJ25, and values of E were calculated as:

where F_o,R and F_R are emission intensities before and after addition of Cy3-MccJ25 to DAC-labelled RNAP holoenzyme derivative, and F_o,R′ and F_R′ are emission intensities before and after addition of Cy3-MccJ25 to the corresponding DAC-labelled [Ile931]β′ RNAP holoenzyme derivative.

Donor-acceptor distances (R) were calculated as:

where R_o is the Förster parameter (49.4 Å in this study, calculated essentially as in Mekler et al., 2002). Distance-restrained docking was performed essentially as in Mekler et al., 2002 (details in Supplementary Information: Distance-restrained docking).

Random mutagenesis

Random mutagenesis was performed by error-prone PCR amplification of the XbaI-SnaBI (codons 1-292), SnaBI-SphI (codons 292-546), SphI-SalI (codons 546-876), SalI-BspEI (codons 876-1213), and BspEI-XhoI (codons 1213-1408) rpoC segments of plasmid pRL663 (Wang et al., 1995)(methods as in Zhou et al. 1991). Mutagenized plasmid DNA was introduced by transformation into strain Stbl2 (Invitrogen), transformants (∼10⁴ cells) were applied to LB-agar plates (Sambrook and Russel, 2001) containing 1 μg/ml MccJ25 and 200 μg/ml ampicillin, and plates were incubated 24 h at 37°C followed by 0-48 h at 25°C. For each MccJ25^r clone (identified as a clone yielding a colony on the original selective plate and also yielding colonies when re-streaked to the same medium and incubated 16 h at 37°C), plasmid DNA was prepared, plasmid DNA was introduced by transformation into strain DH5α (Invitrogen), transformants (∼10⁴ cells) were applied to LB-agar plates containing 1 μg/ml MccJ25 and 200 μg/ml ampicillin and, in parallel, to LB-agar plates containing 200 μg/ml ampicillin, and plates were incubated 16 h at 32°C. For each plasmid-linked MccJ25^r clone (identified as a clone yielding comparable numbers of colonies on the plates with and without MccJ25), the nucleotide sequence of the mutagenized rpoC segment was determined by dideoxy nucleotide sequencing.

Saturation mutagenesis

A set of “doped” oligodeoxyribonucleotide primers corresponding to codons 425-437, 487-509, 592-608, 675-703, 723-743, 739-757, 772-793, 918-937, 1132-1141, and 1236-1251 of the rpoC gene of plasmid pRL663 (Wang et al., 1995), and codons 542-549 of the rpoB gene of plasmid pRL706 (Severinov et al., 1998), was synthesized on an AB392 automated synthesizer (Applied Biosystems) using solid-phase β-cyanoethylphosphoramidite chemistry (sequences in Supplementary Table 3). The level of “doping” (nucleotide misincorporation) was selected to yield an average of 0.4-1 substitution per molecule of oligodeoxyribonucleotide primer (equations in Hermes et al., 1989). Thus, the nucleotides corresponding to codons 429-433, 597-603, and 1136-1137 of rpoC were synthesized using phosphoramidite reservoirs containing 92% of the correct phosphoramidite and 8% of a 1:1:1:1 mix of dA, dC, dG, and dT phosphoramidites (i.e., 94% total correct phosphoramidite and 6% total incorrect phosphoramidite); the nucleotides corresponding to codons 492-504, 680-698, 726-740, 741-754, 775-790, 922-933, and 1239-1248 of rpoC, and codons 542-549 of rpoB, were synthesized using phosphoramidite reservoirs containing 98% of the correct phosphoramidite and 2% of a 1:1:1:1 mix of dA, dC, dG, and dT phosphoramidites (i.e., 98.5% total correct phosphoramidite and 1.5% total incorrect phosphoramidite); and all other nucleotides were synthesized using phosphoramidite reservoirs containing 100% of the correct phosphoramidite. Primer-extension mutagenesis reactions were performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), with a “doped” oligodeoxyribonucleotide primer, a complementary wild-type oligodeoxyribonucleotide primer, and pRL663 as template (primers at 75 nM; all other components at concentrations as specified by the manufacturer). Mutagenized plasmid DNA was introduced into cells, and plasmid-linked MccJ25^r clones were identified and characterized, as in the preceding section.

Complementation assays

Strain 397c [rpoC^ts397 argG thi lac (λcI₈₅₇h₈₀S_t68dlac⁺); Christie et al., 1996] was transformed with pRL663 or a pRL663 derivative, transformants (∼10⁴ cells) were applied to LB-agar plates containing 1 μg/ml MccJ25 and 200 μg/ml ampicillin, plates were incubated 16 h at 37°C, and bacterial growth was scored.

Minimum bacteriocidal concentration (MBC) assays

Strain DH5α (Invitrogen) was transformed with pRL663 or a pRL663 derivative, or pRL706 or a pRL706 derivative, transformants (10⁶ cells) were incubated 2 h at 37°C in 1 ml LB (Sambrook and Russel, 2001) containing 0.01, 0.05, 0.1, or 1 mg/ml MccJ25; aliquots (10 μl) were applied to LB-agar plates containing 200 μg/ml ampicillin; plates were incubated 16 h at 37°C; and colonies were counted. The MBC was defined as the lowest concentration of MccJ25 that yielded a colony count of <5.