A bacteriophage transcription regulator inhibitsbacterial transcription initiation by p-factordisplacementBing Liu1, Andrey Shadrin1, Carol Sheppard1, Vladimir Mekler2, Yingqi Xu1,

Konstantin Severinov2, Steve Matthews1,* and Sivaramesh Wigneshweraraj1,*

1MRC Centre for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, UK,2Waksman Institute for Microbiology and Department of Molecular Biology and Biochemistry, Rutgers, TheState University of New Jersey, Piscataway, NJ USA

Received November 8, 2013; Revised December 4, 2013; Accepted December 30, 2013

ABSTRACT

Bacteriophages (phages) appropriate essentialprocesses of bacterial hosts to benefit their owndevelopment. The multisubunit bacterial RNApolymerase (RNAp) enzyme, which catalyses DNAtranscription, is targeted by phage-encodedtranscription regulators that selectively modulateits activity. Here, we describe the structural andmechanistic basis for the inhibition of bacterialRNAp by the transcription regulator P7 encoded byXanthomonas oryzae phage Xp10. We reveal that P7uses a two-step mechanism to simultaneouslyinteract with the catalytic b and b’ subunits of thebacterial RNAp and inhibits transcription initiationby inducing the displacement of the p70-factor oninitial engagement of RNAp with promoter DNA.The new mode of interaction with and inhibitionmechanism of bacterial RNAp by P7 underscorethe remarkable variety of mechanisms evolved byphages to interfere with host transcription.

INTRODUCTION

Bacteriophages (phages) use an impressive array ofmechanisms to inactivate or repurpose bacterial processesfor their own developmental needs (1,2). Many phagegenomes encode small proteins that specifically affect themultisubunit RNA polymerase (RNAp) of the bacterialhost, inhibiting bacterial DNA transcription whilepromoting regulated phage DNA transcription (1,2).Illuminating the functional mechanisms of phage-encoded RNAp inhibitors at the molecular and structural

level uncovers new ways by which bacterial transcriptionis thwarted by non-bacterial regulators of bacterialtranscription.

Bacterial transcription begins with the association of as-factor with the catalytic ‘core’ of the RNAp (subunitcomposition a2bb’o; E) resulting in the formation of theRNAp holoenzyme (Es). The s-factor confers promoterspecificity on the RNAp, and in the case of the primary s-factor, called s70 in Escherichia coli (Ec) and relatedbacteria, allows the specific recognition of the consensussequence elements located around positions �35 and �10(with respect to the transcription start site at+1) of bac-terial promoters in front of essential genes. Primary sfactors have four evolutionarily conserved regions (1–4),with regions 2 and 4 being the major determinants ofrecognition of the �10 and �35 promoter elements, re-spectively (3). On its own, s70 of Ec, the best-studiedprotein of its class, cannot recognize promoters. Onbinding to the core RNAp, s70 undergoes large conform-ational changes (4). In particular, s70 region 4 interactswith the structurally conserved and flexible ‘flap’ domainof the b subunit (specifically with the b flap domain helix),and this interaction leads to physical movement thatpositions s70 region 4 such that simultaneous interactionswith consensus �10 and �35 promoter elements becomepossible (5). The initial engagement of Es70 with thepromoter yields a closed promoter complex (RPc), whichisomerizes into the transcription-initiation competentopen promoter complex (RPo) [reviewed in (6)].

Phages have evolved mechanisms to exploit the s70

region 4/b flap interaction to repurpose the bacterialRNAp for transcription of phage genes (2). Knownphage-encoded transcription regulators specifically inter-fere with the s70 region 4/b flap interaction through direct

*To whom correspondence should be addressed. Tel: +44 207 594 1867; Fax: +44 207 594 3055; Email: s.r.wig@imperial.ac.ukCorrespondence may also be addressed to Steve Matthews. Tel: +44 20 7594 5315; Fax: +44 207 594 3057; Email: s.j.matthews@imperial.ac.uk

The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.

Nucleic Acids Research, 2014, 1–12doi:10.1093/nar/gku080

� The Author(s) 2014. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), whichpermits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acids Research Advance Access published January 30, 2014 at Im

perial College L

ondon on February 12, 2014http://nar.oxfordjournals.org/

Dow

nloaded from

contact with s70 region 4, b flap or both, thereby alteringtranscription initiation and/or post-initiation activities ofthe bacterial RNAp (2,7–10). Lytic bacteriophage Xp10,which infects the bacterial phytopathogen Xanthomonasoryzae (Xo), the causal agent of rice blight, relies on thehost RNAp and a phage-encoded single-subunit RNApfor the temporal expression of its genome. The transcrip-tion programme of Xp10 is complex and the preciseregulatory basis for the temporal transcriptionalregulation of Xp10 genes is not fully understood. Thetranscription of Xp10 genes (called L and early R genes;Figure 1A) during the early stages of infection is driven byat least four Xo Es70 dependent promoters, whereas thetranscription of Xp10 genes during the late stages ofinfection (called late R genes; Figure 1A) is largelydriven by Xp10 RNAp (11,12). The presence of atranscription terminator sequence located between earlyR genes and late R genes (Figure 1A) prevents anyunwanted transcription of late R genes during the earlystages of infection (11,12). A 8 kDa Xp10 protein, calledP7, which is an L gene product and a strong inhibitor ofRPo formation by the Xo Es70, is believed to facilitate theswitching between host and phage RNAp for thetranscription of Xp10 genes (13). P7 can also bind totranscribing host RNAp and function as an anti-terminator and allows the host RNAp to bypass thetranscription terminator located between the early Rgenes and late R genes to increase transcription of lateR genes during the late stages of infection by transcribinghost RNAp (i.e. RNAp molecules that have initiated tran-scription before the production of P7 or have escaped P7inhibition) (11–13). The first 10 amino acid (aa) residues ofthe b’ subunit of Xo Es70 contain the major determinantfor P7 binding (14). Previous studies have shown that P7affects the obligatory change in the interdomain distancebetween s70 regions 2 and 4 that occurs during RNApholoenzyme formation (13). However, the precisemechanism by which P7 inhibits RPo formation by XoEs70 remains unknown. Here, we describe the structuraland molecular basis for the interaction and mode oftranscription inhibition by P7. We show that P7 uses atwo-step mechanism to simultaneously interact with thecatalytic b’ and b subunits of the bacterial RNAp andcause the displacement of s70 on engagement of theRNAp holoenzyme with promoter DNA. Thus, P7represents a distinct type of phage-encoded bacterialtranscription regulator that inhibits transcriptioninitiation of bacterial RNAp by s-factor displacement.

MATERIALS AND METHODS

Plasmids and proteins

Plasmids encoding P7 fused to aNTD (pBRa) and differentXo and Ec b and Xo b’ fragments (details below) fused tothe � CI protein (pAC�CI) and mutant versions thereofwere constructed using standard recombinant DNAmethods. Plasmids encoding amino-terminal heart-muscle-kinase–tagged (for labeling with g-32P) P7 and Ecs70 were constructed by cloning the relevant DNAsequences into plasmid pET33b+ (Novagen). Proteins

were 32P-labelled in reactions containing 50 ml of proteinkinase A (PKA) reaction buffer, 500 ng of protein, 1 ml ofg-32P-ATP (250Ci/mmol, Perkin Elmer) and 5 U of PKAmade up to 100 ml with ddH20. Reactions were incubated at37�C and stopped by rapid freezing in small aliquots inliquid nitrogen. The aliquots were stored at �80�C anddiscarded after thawing. Wild-type and P7-sensitive coreRNAp was purified from Ec 397 c cells containing eitherpRL663WT or pRL663 [which encodes the P7-sensitive b’subunit; (14)], respectively, according to published proto-cols (15). The Ec s70 was purified essentially as previouslydescribed (16). Details of all the oligonucleotide primersused for cloning and mutagenesis and plasmids used inthis work are provided in Supplementary Tables S1 andS2. For nuclear magnetic resonance (NMR) spectroscopyand structure calculation, full-length P7 was expressedin pET-46 with EK/LIC site (Novagen). b’ NTD(MKDLLNLFNQ), b flap tip (KVTPKGESQLTPEEKLLRAIFGEKASDVKDS) and a further b’ NTD(MKDLL*NL*FNQ) in which two L* were uniformly13C/15N-labelled were chemically synthesized (PeptideSynthetics, UK). P7 samples for NMR analyses wereproduced with uniform 13C/15N labelling by expressing inminimal medium supplemented with 15NH4Cl and

13C6-Glucose and purified using Ni-NTA agarose and size ex-clusion chromatography. To prepare a saturated sample ofP7 bound to the b’ NTD peptide, the peptide was added toa dilute sample of purified P7 and incubated at room tem-perature overnight. The sample was concentrated by ultra-filtration, and the complex was obtained by size exclusionchromatography using a Superdex 75 column (GE LifeSciences).

NMR spectroscopy and structure calculation

The backbone assignments of P7 and P7-b’ NTD complexwere both completed by using standard double- andtriple-resonance assignment approach (17). The datawere processed and analysed in NMRView and ourin-house assignment routines (18). Ha and Hbassignments of both projects were obtained usingHBHA(CBCACO)NH. The side-chain assignments ofthe two proteins were completed using HCCH TOCSYand (H)CC(CO)NH TOCSY. Proton resonance assign-ment of b’ NTD was based on 12C/14N-edited 2Dhomonuclear TOCSY/NOESY experiments, and to aidunambiguous assignment a sample was prepared inwhich the b’ NTD peptide was chemically synthesizedwith two leucine residues that were uniformly 13C/15Nlabelled (MKDLL*NL*FNQ). The distance restraintswere provided by 3D 1H-15N/13C NOESY NOESY-Heteronuclear Multiple Quantum Coherence (HSQC)experiments (mixing time, 100ms at 800MHz).Intermolecular NOEs provide the distance restraint forthe complex and were acquired using filter 13C/15NNOESY experiment on hybrid-labelled sample. TheARIA protocol was used for completion of the NOEassignment and structure calculation (19). Dihedralangle restraints derived from TALOS were alsoincorporated in the calculation. The frequency windowtolerances for assigning NOEs were ±0.04 ppm for

2 Nucleic Acids Research, 2014

at Imperial C

ollege London on February 12, 2014

http://nar.oxfordjournals.org/D

ownloaded from

A

C D

N

N

N

N

B

L genes

Early Rgenes

Late Rgenes

P7

E

Gal

acto

sida

se a

ctiv

ity(M

iller

uni

ts)

((((((((((((((((((MMMMMMMMMMMMMillilli

ererereeeeeeeeeeeeeeeunununununnuuuuuuuuuuuuu

ittitttttttttttsssssssssssssss))))))))))))))))

α-P7 variant

WT F50A V51A- L67A

Figure 1. The solution structures of P7 and P7-b’ NTD complex. (A) The groups of Xp10 genes that belong to the different temporal classes areindicated on the Xp10 genome (shown in circular organization). The host (white arrows) and phage (black arrows) RNAp dependent promoters inthe intergenic region separating the L genes and early R genes are shown at the top. In the L genes, the approximate location of the gene-encoded P7is indicated and the putative transcription terminators that separate early and late R genes are indicated by a hairpin (see text for details). This figurehas been adapted from Djordjevic et al. (12). (B) Cartoon representation of the solution structure of apo P7 showing the juxtaposition of the a helix

Nucleic Acids Research, 2014 3

(continued)

at Imperial C

ollege London on February 12, 2014

http://nar.oxfordjournals.org/D

ownloaded from

direct proton dimensions and ±0.05 ppm for indirectproton dimensions, and ±0.5 ppm for nitrogendimensions and ±1.1 ppm for carbon dimensions. TheARIA parameters p, Tv and Nv were set to defaultvalues. The 20 lowest energy structures had no NOEviolations >0.5 A and no dihedral angle violations >5�.Dihedral angle restraints derived from TALOS were alsoimplemented (20).For NMR titration experiments, either 15N or 15N13C-

labelled P7 was prepared in the NMR buffer. Unlabelledpeptides (b’ NTD or b flap domain tip helix) in the samebuffer was introduced at several steps up to a saturatingmolar excess and 2D 1H-15N HSQC spectra were recordedat each stage under identical experimental conditions.For spin labelling the C-terminus of P7 we introduced

the spin label S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSL) to theC-terminus of P7 by mutating a V70 to cysteine. Theparamagnetic reagent MTSL shortens the transverserelaxation time of nearby nuclei and therefore leads to aloss of peak intensity in the spectrum (21,22).In the recently described crystal structure of the Ec Es70

[PDB ID 4IGC; (23)] the N-terminal region aa residuesfrom 1 to 8 of the b’ subunit are absent. Furthermore, aaresidues 8–11 adopt an extended conformation rather thatthe helix seen the NMR structure presented. This togetherwith the observation that the fewer aa residues of the b’ N-terminus are modelled in other structures of the Ec RNApsuggests some degree of flexibility in this region (24,25).To construct a structural model for the P7-Ec Es70

complex for further analysis, the N-terminus of the b’subunit was truncated to residue 11 and then extendedwith our NMR structure of the P7-bound b’ aa residues1–10. The P7-b’ NTD moiety was rotated as a rigid-bodywhile steric clashes with Es70 were removed.

Western blotting

Western blotting experiments were conducted usingstandard laboratory protocols. Ec strain KS1 whole-cellextracts (corresponding to 2.5� 107 cells) prepared fromcells sampled at the end of the b-galactosidase assays wereanalysed by sodium dodecyl sulphate-polyacrylamide gelelectrophoresis gels and proteins were transferred to aHybond-ECL nitrocellulose membrane using a Trans-BlotSemi-Dry transfer cell (Bio-Rad). Anti-P7 polyclonalantibodies (Eurogentec) and horseradish peroxidaseconjugated anti-rabbit IgG (Sigma) in combination withthe ECL SuperSignal West Femto ChemiluminescentSubstrate kit (Pierce) was used to detect the a-P7 fusionprotein. Digital images of the blots were obtained using anLAS-3000 Fuji Imager.

In vitro transcription assays

In vitro transcription assays were conducted essentially aspreviously described (26). Reactions (10ml) were con-ducted using final concentrations of 100 nM Es70, 20 nMlacUV5 promoter DNA probe, 0.5mM dinucleotideprimer ApA, 0.05mM UTP and 3mCi of [a-32P]-UTP.P7 and Es70 (at a 4:1 molar ratio unless otherwiseindicated) were always pre-incubated before thepromoter DNA was added to the reaction. The reactionswere resolved on a 20% (w/v) urea-denaturing polyacryl-amide gel. The gel was visualized and quantified with theuse of an FLA-5000 PhosphorImager.

Native gel mobility assays

All native mobility shift assays were conducted essentiallyas described previously (26). Binding reactions (10ml) wereset up as described above for the in vitro transcriptionassays. The experiment shown in Supplementary FigureS4B was carried out at �4�C essentially as previouslydescribed (27).

Bacterial two-hybrid interaction assays

LacZ expression was determined from b-galactosidase assaysperformedwithmicrotiter plates and amicrotiter plate readeras previously described (28). Cultures of Ec strain KS1 weregrown for 16h at 30�C in the presence of ampicillin (100mg/ml), chloramphenicol (30mg/ml), kanamycin (25mg/ml) and0–100mM Isopropyl b-D-1-thiogalactopyranoside (IPTG).Experiments were conducted at least three times onseparate occasions with three biological replicates for eachexperiment. Similar results were obtained for all experiments.Values shown represent results from a single experiment andthe error bars represent standard deviation.

KMnO4 footprinting

Binding reactions were set up as described for the in vitrotranscription assays in the absence and presence of P7 butusing the T5 N25 promoter. The promoter probe for theKMnO4 footprinting experiments was formed by mixingequimolar amounts of 100 bp oligonucleotides represent-ing the template and non-template strand sequences of theT5 N25 promoter in a buffer containing 40mM Tris, pH7.9, 100mM NaCl, heating for 2min at 95�C, and slowlycooling down to 25�C; one of the oligonucleotides was 50-end-labelled with g32P-ATP to monitor strand-specific oxi-dation by KMnO4. Promoter complexes were treated with1mMKMnO4 at 37

�C for 30 s. Reactions were terminatedby the addition of �40 mM of 300mM b-mercaptoethanolfollowed by ethanol precipitation and 20-min treatmentwith 10% piperidine at 95�C. Reaction products werenext treated with chloroform to remove piperidine,ethanol precipitated, dissolved in 8 ml of urea-formamide

Figure 1. Continuedand the b1 -b2-b3-b4 sheets. (C) Solution structure of P7 in complex with the first 10 aa residues of the b’ subunit from Xo RNAp (b’ NTD). P7 isshown in cyan and b’ NTD in orange. The position of N-termini for each chain is indicated. (D) Zoomed cartoon representation of the P7-b’ NTDcomplex showing residues located in the P7-b’ NTD binding site. Key interacting residues are labelled. (E) BTH interaction assay used to detectprotein–protein interaction between b’ NTD and mutants of P7. The diagram depicts how the interaction between b’ NTD, fused to the bacterio-phage � CI protein (�CI), and P7, fused to the a-NTD (a-P7), activates transcription of the lacZ gene. Results of the b-galactosidase assays expressedin Miller units are shown in the bar chart.

4 Nucleic Acids Research, 2014

at Imperial C

ollege London on February 12, 2014

http://nar.oxfordjournals.org/D

ownloaded from

loading buffer and resolved in 8% polyacrylamide ureadenaturing gel. The A/G ladder was prepared byincubating 40 pmoles of the template DNA probe in17 ml of ddH2O and 50 ml of formic acid for 5min atroom temperature. The DNA was then precipitated byadding 7 ml of 3M NaOAc and 900 ml of chilled 100%ethanol. The precipitated DNA was dissolved in 90 ml ofddH2O, 10 ml of piperidine was added and incubated at95�C for 10min. The reaction was chloroform/ethanolprecipitated twice; the DNA dissolved in urea-formamideloading buffer and 2–5 ml of this was used for gel loading.

RESULTS AND DISCUSSION

The solution structures of P7 and P7-b’ NTD complex

Previous work showed that aa residues 6–9 of the Xo b’subunit are important for the binding of P7 to the XoRNAp (14). To shed light on the mechanism bywhich P7 inhibits transcription initiation we determinedthe solution structures of apo P7 and a complexbetween P7 and Xo b’ residues 1–10 (hereafter referredto as the b’ NTD) using multidimensional NMR spectros-copy (Figure 1B-1D, Supplementary Figure S1A, B, TableS3). In the apo P7 structure, the P7 polypeptide folds intoa compact globular domain comprising an a-helix packedagainst a b sheet in a b1 -b2-b3-b4-a1 arrangement (Figure1B). In the P7-b’ NTD complex b’ NTD folds into a shorta-helix between aa residues 4–9 (Figure 1C and D) thatpositions hydrophobic aa residues L7 and F8 into anexposed hydrophobic cavity between P7 b1 and a1 andin close proximity to P7 residues F50 and V51 (Figure1C and D). Notably, the carboxyl-terminal ‘tail’ of P7(residues K65–R73) folds back and L67 also contactsthe b’ NTD (Figure 1B–D). Strikingly, in the structureof apo P7 the carboxyl-terminus exhibits multiple con-formations within the NMR ensemble that are distinctfrom the ordered conformation in the P7-b’ NTDcomplex (Supplementary Figure S1A, B). The rest of theP7 structure is similar in the b’ NTD complex and the freeprotein. In support of the large-scale conformationalchanges undergone by P7 on the binding to b’ NTD, weobserve dramatic chemical shift perturbations in the 2D1H-15N HSQC spectra of P7 saturated with b’ NTD(Supplementary Figure S1C). To provide furtherevidence for a conformational change in thecarboxyl-terminal ‘tail’ of P7 on b’ NTD binding, weintroduced the sulfhydryl-specific spin label MTSLto the carboxyl-terminus of P7 by mutating V70to cysteine. Paramagnetic MTSL shortens the transverserelaxation time of nearby nuclei and leads to a loss ofpeak intensity in the 1H-15N HSQC NMR spectrum(21,22). Consistent with the dynamic nature ofthe carboxyl-terminus of P7, a number of resonancesthat are widely distributed over the back face of thea1-helix and in the empty hydrophobic binding cavitydisappear in MTSL-labelled apo P7 (SupplementaryFigure S1D). On the binding to b’ NTD, the reductionsin peak intensities are restricted to amides in theimmediate vicinity of MTSL labelled position and alsoinclude the two labelled leucine amides in b’ NTD (L5

and L7; Supplementary Figure S1D; see ‘Materials andMethods’ section), thus confirming the ordering of thecarboxyl-terminus of P7.To verify the contributions of key aa residues in the P7-b’

NTD interface to the binding of P7 to the b’ subunit of theRNAp, a bacterial-two hybrid (BTH) interaction assay wasused. In this assay, a contact between P7 fused to a com-ponent of RNAp (here, the a subunit) and b’ NTD fused toa DNA-binding protein (here, the CI protein of bacterio-phage �) activates transcription of a lacZ reporter geneunder the control of a promoter bearing an upstream �operator, a recognition site for the CI protein fusion (29).Alanine substitutions at P7 residues F50 and V51 thatshould interfere with the interaction with b’ NTD basedon structural analysis, decreased test promoter activity(Figure 1E). In contrast, the L67A substitution had noeffect. Because the intracellular levels of all mutant a-P7fusion proteins are similar to wild-type a-P7 fusion proteinlevel (Supplementary Figure S1E) the results suggest thataa residues F50 and V51 in P7 are the major determinantsfor interaction with b’ subunit of the RNAp while thecontact with L67 is less important.

P7 simultaneously interacts with the RNAp b and b’subunit of the RNAp

As two functionally important structural elements ofthe bacterial RNAp, the Zn-finger (Xo b’ aa 63–95) andthe b’ zipper (Xo b’ aa 38–59) are adjacent to the b’ NTD(30,31), we used the BTH interaction assay to assesswhether these additional regions of Xo b’ contribute toP7 binding. Accordingly, we measured if P7 can interactwith different fragments of Xo b’ comprising aa residues1–85 (contains the b’ NTD and the b’ zipper), 1–95 (b’NTD, the b’ zipper domain, and the Zn-finger), 11–95(contains only the b’ zipper domain and the Zn-fingerelement) and 63–95 (contains only the Zn-finger). TheBTH construct containing the b’ NTD served as apositive control. The results show that the b’ NTD is suffi-cient for the binding of P7 to the RNAp (Figure 2A).Because the interaction between P7 and the b’ NTDalone is stronger than the interaction between P7 andthe b’ fragment containing the b’ zipper and Zn-fingerdomains (fragment comprising aa residues 1–95), wesuggest that these domains do not contribute significantlyto the binding of P7 to the RNAp. Consistent with thisview, no interaction between fragments comprising aaresidues 63–95 (Zn-finger only) and 11–95 (zipper andZn-finger only) is detected. We then derived a structuralmodel for the P7-Es70 complex by extending the b’subunit of the recently described crystal structure of theEc Es70 (23) with our P7-b’ NTD solution structure (see‘Materials and Methods’ section). In the model, P7 isadjacent to the b flap domain and region 4 of s70

(Figure 2B), suggesting that P7 could interact with the bflap domain and/or region 4 of s70. We reasoned that thisinteraction, if present, should occur with the Ec RNAp, asa hybrid Ec-based RNAp (containing the b’ NTD, i.e.aa residues 1–10 of the Ec b’ subunit substituted withcorresponding aa residues of the Xo b’ subunit; hereafterreferred to as P7SEs70) binds P7 and is readily inhibited

Nucleic Acids Research, 2014 5

at Imperial C

ollege London on February 12, 2014

http://nar.oxfordjournals.org/D

ownloaded from

A

0

1000

2000

3000

4000

5000

6000

7000

8000

0 20 40 60 80 100

λCI-β’ NTD (aa1-10) + α-P7

λCI-β’ (aa1-85: β’ NTD+zipper) + α-P7

λCI-β’ (aa1-95: β’ NTD+zipper+Zn-finger) + α-P7

λCI-β’ (aa63-95: Zn-finger only) + α-P7

λCI-β’ (aa11-95: zipper+Zn-finger only) + α-P7

λCI + α-P7

β-G

alac

tosi

dase

ac�

vity

(Mill

er u

nits

)

IPTG (μM)

B

R60

70 region 4

Xo MKDLLNLFNQQRQTLDFDAIKIALASPDLIRSWSYGEVKKPETINYRTFKPERDGLFCAAIFGPIKDYECLCGKYKRMKHRGVVCEKCGTEVTLAKVRRE***** * * :************:*****:************************ ****:************:*****:***** *** ****

Ec MKDLLKFLKAQTKTEEFDAIKIALASPDMIRSWSFGEVKKPETINYRTFKPERDGLFCARIFGPVKDYECLCGKYKRLKHRGVICEKCGVEVTQTKVRRE

MMKDMKDMKDMKMKDMKDMKDMKDMKDMKDMKMKDMKDMMKDMKMKDMKDK LLLLLLLLLLLLLLLLLLLLLNNNNNNNNNNNNNNNNNNNLFNLFNLFNLFNLFNLFNLFNNFNLFNLFNLFNFNNLFNLFNNFNNQQQQQQQQQQQQQQQQ***************************************************** *************************************MMKDMKDMKMKDMKDMKDMKDMKDMKDMKDMMKDMKDMKDMKDMKDMKDK LLLLLLLLLLLLLLLLLLLLKKKKKKKKKKKKKKKKKKFLKFLKFLKFLKFLKFLKKFLKFLKFLKFLKFLKFLKFLKFLKKFLKFLKLKAAAAAAAAAAAAAAAAAAA

KKKKKKKKKKKKKKKKKKIAIAIAIAAAIAIAAAIAAAAAAAALLLLLLLLLASPASASPASPASASPASPAASPASPASPASPASPASPASPASPASPASA DLDDDLDDLDLDLDLDLLDDLDDLDD IIIIIIII*************************************************************************************************************************************:*:*:**:*:*:*:***:**:*:**:** ******************KKKKKKKKKKKKKKKKKKKIAIAIAIAAIAIAIAAAAAAAAIAAAALLLLLLLASPASASPASPASASPASPASPASPASPASPASPASPASPASPASPSPASPAS DMDMDMDMDMDMDMDMDMDMDMDMDMDMDMDMMMIIIIIIII

PPPPPPPPPPPPPPPEEEEEEEEEEEEEETINTINTINTININNINTTINTINTININNNTINTINTINNNYRYRYRYRYYRYYRYRYRYRRYRYRYRYRYRRTTTTTTTTTTTFKFKFKFKFKKFKFKFKKKFKFKFKFKFKKK******************************************************************************************** ***************************************************************************************PPPPPPPPPPPPPPPPEEEEEEEEEEEEEEEEETINTINTINTININNINTTINTINTINTINTINNTINTINTINT YRYRYRYRYYRYRYRYRYRYRYRYRYRYRYRYRRRTTTTTTTTTTTTTFKFKFKFKFKKFKFKKFKFKFKFKFKKKKFK

IFGIFGIFGIFGGGFGGGIFIFGFGGGFGIFGFGPIPIPIPPIPIPIPIPIPPPP KKKKKKKKKKKKKKKKKKDYEDYEDYEDYDYEDYEDYEDYEDYEDYDYEYDYEYEDYEYEDYEECLLCCCCCCCLCCCCCCCCCC**************************************************************************::::::::::************************************************************************* *****************IFGIFGIFGIFGGFGFGGGIFGGIFGFGGFGGFGFFGPVPVPVPVVPVPVPVPVVPVPVPVPVVVPVKKKKKKKKKKKKKKKKKKDYEDYEDYEDYEDYEDYEDYEDYEDYEDDYEDYEDYEDYEDYEYDYEDYEYECCCCLCCCLCLCCCCLCCCCCC

HRHRHRRRRHRRRRRHRRRRRRGVGVGVGVGVGVGVGVGVGVGVGVGVGVVGVGVGG VVVVVVVVVVVVVVVVVCEKCEKCEKCEKCEKCEKCEKCEKCEKCEKCEKCEKCEKCEKEKCEKCEKCEKCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCCGCGTTTTTTTTTTTTT****************************************************:::::::::::::******************************************************************************************HRHRRRRRHRHRRRRRRRHRRGVGVGVGVGVGVGVGVGVGVGVGVGVGVGVGVGVGVGVIIIIIIICEKCEKCEKCEKCEKCEKCEKCEKCEKCEKCEKCEKCEKCEKCEKCEKCEKEKCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGVVVVVVVVVVVVVVVVVV

NTD(1-10) Zipper (38-59) Zn-finger (63-95)1 100

β’βωσ

αIIαI P7

C

0

100

200

300

400

500

600

0 20 40 60 80 100

λCI-β flap + α-P7

λCI + α-P7

λCI-β flap L901A + α-P7

λCI-β flap I905A + α-P7λCI-β flap Δ 900-909 + α-P7

Gal

acto

sida

se a

ctiv

ity(M

iller

uni

ts)

IPTG (µM)

β

ββ

σ

β

Figure 2. P7 interacts with the b subunit flap domain of the RNAp. (A) BTH interaction assay used to detect protein–protein interaction betweendifferent fragments of the Xo b’ subunit (as indicated and see text for details) and P7. The diagram depicts how the interaction between differentfragments of the Xo b’ subunit, fused to the bacteriophage � CI protein (�CI), and P7, fused to the a-NTD (a-P7), activates transcription of the

6 Nucleic Acids Research, 2014

(continued)

at Imperial C

ollege London on February 12, 2014

http://nar.oxfordjournals.org/D

ownloaded from

by P7 in the same way as Xo Es70 (14). We thereforeused the BTH interaction assay to determine if P7interacts with the b flap and/or s70 region 4 domainsusing the Ec b flap domain aa residues 831–1057 and Ecs70 domain 4 residues 528–613 fused to bacteriophage �CI protein. The results show that P7 indeed interacts withEc b flap domain (Figure 2C), while there is little or nointeraction of a-P7 with �CI fused to Ec s70 region 4(Supplementary Figure S2A). The specificity of theinteraction between P7 and the b flap domain isdemonstrated by its abrogation when aa residuescorresponding to the b flap domain tip helix (aa residues900–909) are deleted or alanine substitutions in the b flapdomain tip helix are introduced (L901A and I905A)(Figure 2C). Because the removal of carboxyl-terminusaa residues 67–74 of P7 markedly increases the ability ofP7 to interact with the b flap domain in the context ofthe BTH assay (Figure 2D) [even though the relativeprotein levels of wild-type P7 and P7�67–74 are compar-able under conditions of the BTH assay (SupplementaryFigure S2B)], the folding back of the carboxyl-terminal‘tail’ of P7 on binding to the b’ NTD could unveil a bflap interaction surface, which comprises aa residuesV11, L34 and R60 (Figure 2D and see later). Insummary, the results to a large extent confirm thestructural model of the Ec RNAp-P7 complex andsuggest that the binding of P7 to the b’ NTD is necessarybefore the interaction between P7 and the b flap domaintip helix of the RNAp can take place.

P7-b flap interaction is important for transcriptioninhibition

To analyse the b flap-P7 interaction in more detail weperformed NMR chemical shift mapping with a syntheticpeptide spanning the b flap tip helix of the Xo b flapdomain (Xo aa residues (915–945; Figure 3A). While aninteraction between apo P7 and the Xo b flap tip peptidecould not be observed (Supplementary Figure S3A), theXo b flap tip peptide-dependent backbone amide chemicalshift changes in P7 are detected when fully saturated withthe b’ NTD (Figure 3A). This observation is consistentwith the view that the binding of P7 to the b’ NTD facili-tates the interaction between P7 and the b flap domain tiphelix of the RNAp (see above). Several of the perturbedresidues (S56, L58, K59, R60, N61, L71 and R73) map tothe surface on P7 that is unveiled on the binding to the b’NTD (Figure 3A) and four of these residues (L58, K59,R60 and N61) are proximal to the b flap domain tip helixin our structural model (Figure 2B). Consistent with thisobservation, mutant P7 harbouring a charge reversal sub-stitution at aa residue R60 (R60E) reduces the ability ofP7 to interact with the b flap domain, but not with the b’NTD in the BTH interaction assay (Figure 3B). The inter-action with the b flap domain is important for the abilityof P7 to function as a transcription inhibitor becausemutant P7 harbouring the R60E substitution displays areduced ability to inhibit the transcription initiation byP7SEs70 from the lacUV5 promoter, a well-characterized�35/�10 class of bacterial promoter (Figure 3C).

-

200

400

600

800

- 20 40 60 80 100

Gal

acto

sida

se a

ctiv

ity(M

iller

uni

ts)

IPTG (µM)

λCI-β flap + α -P7Δ67-74

λCI-β flap + α-P7

λCI-β flap + α

D β

β

Figure 2. ContinuedlacZ gene. Because the expression of the fusion proteins are under the control of IPTG-inducible promoters, and the cells used for the b-galactosidaseassays were grown in the presence of increasing concentrations of IPTG, in the graph the b-galactosidase activity (in Miller units) is expressed as afunction of IPTG concentration. The alignment of the aa sequence corresponding to residues 1–100 of the b’ subunit of Ec and Xo RNAp is shownfor comparison with the b’ NTD, zipper and Zn-finger domains boxed. In the alignment, ‘*’ and ‘:’ indicate identical and similar aa residues,respectively. (B) Surface representation of the structural model of P7 bound to the Ec Es70. The aI, aII, b, b’, o and s70 subunits coloured asindicated. The boxed region is enlarged and shows the region around the P7-RNAp interface in more detail. The s70 region 4 and the b flap domaintip helix are shown as cartoon representation and circled, and the black arrow points to the b’ NTD; the side-chain of aa residue R60 of P7 is shownas red surface representation and circled. (C) As in (A) but the BTH interaction assay used to detect protein–protein interaction between the Ec bflap domain (and mutants thereof, as indicated) and P7. (D) Left. Surface representation of free and bound forms of P7 showing the conformationalchanges in the C-terminus ‘tail’ of P7. The region in P7 unveiled on binding to the b’ NTD is shown in pink. Right. As in (A), but BTH interactionassay is used to detect protein–protein interaction between the Ec b flap domain and P7 and a truncated P7 mutant lacking the C-terminal ‘tail’(P7�67–74).

Nucleic Acids Research, 2014 7

at Imperial C

ollege London on February 12, 2014

http://nar.oxfordjournals.org/D

ownloaded from

A

-

200

400

600

800

0 20 40 60 80 100

-

2,000

4,000

6,000

8,000

0 20 40 60 80 100

’ NTD

-Gal

acto

sida

se a

ctiv

ity(M

iller

uni

ts)

IPTG (µM)

-Gal

acto

sida

se a

ctiv

ity(M

iller

uni

ts)

IPTG (µM)

λCI-β flap + α-P7

λCI-β flap + α-P7 (R60E)λCI-β flap + α

λCI-β’ NTD + α-P7

λCI-β’ NTD + α-P7 (R60E)

λCI-β’ NTD + α

B

C

Xo KVTPKGESQLTPEEKLLRAIFGEKASDVKDS

Ec KVTPKGETQLTPEEKLLRAIFGEKASDVKDS

915 945

886 916

*******:***********************

P7 +

P7 + flap tip helix peptide (1:1:1)

P7

Lane 1 2 3 4 5

ApApUpU

100 <10 12 60 93%A

P7

P7(R60E)+ +

+ +

P7SE 70:P7 1:4 1:2 1:4 1:2

β

ββ β

β β

ββ

σ

Figure 3. P7-b flap interaction is important for transcription inhibition. (A) 1H-15N-HSQC NMR spectra of either 15N,13C-labelled P7 (left) or thep7- b’ NTD complex (right) in the presence and absence of the b flap domain tip helix peptide based on Xo b flap domain aa sequence. The aaresidues significantly affected in P7 are labelled. The alignment of the aa sequence corresponding to the b flap domain tip helix from the Ec and Xo bsubunit is shown for comparison. In the alignment, ‘asterisk’ and ‘colon’ indicate identical and similar aa residues, respectively. (B) BTH interactionassay used to detect protein–protein interaction between the b’ NTD or Ec b flap domain and P7 and P7 (R60E) mutant. The diagram depicts howthe interaction between the b’ NTD or Ec b flap domain, fused to the bacteriophage � CI protein (�CI), and P7 and P7 (R60E), fused to the a-NTD

8 Nucleic Acids Research, 2014

(continued)

at Imperial C

ollege London on February 12, 2014

http://nar.oxfordjournals.org/D

ownloaded from

P7 displaces p70 from the RNAp

The results so far establish that P7 can simultaneouslyinteract with the b’ NTD and the b flap domain tip helixof the RNAp. Based on an observation that RNAppurified from Xo cells infected by Xp10 phage is largelydevoid of the s70 (compared with RNAp isolated fromuninfected cells) (32) and the functionally obligatory rolethe b flap domain plays during Es70 formation (5), wehypothesized that P7 could inhibit transcription bydestabilizing the interaction between s70 and RNAp.Initially, we probed directly whether P7 causes s70 dis-placement using an electrophoretic mobility shift assay(EMSA) with s70-32P. As shown in Figure 4A, theaddition of P7-sensitive core RNAp (P7SE) to s70-32Presults in a slower migrating radioactive complex that rep-resents the RNAp holoenzyme (P7SEs70-32P) (comparelanes 1 and 2). The addition of P7 to P7SEs70-32P doesnot result in any detectable changes in the mobility ofthe P7SEs70-32P or any obvious decrease in the intensityof the radioactive signal originating from the P7SEs70-32P(in fact an intensification of the radioactive signal inthe P7SEs70-32P complex was observed) and both theP7SEs70-32P and P7SEs70-32P:P7 complexes run as adoublet on the native gel. Thus, the results suggest thatP7 does not displace the s70 from the RNAp (Figure 4A,lane 2 and 3). The addition of lacUV5 promoter probe toP7SEs70-32P results in a new complex that migrates slowerthan the P7SEs70-32P complexes (Figure 4A, lane 4). Thenew complex represents the RPo because it is resistant tothe polyanion heparin [heparin-resistance is a hallmarkfeature of the RPo formed on the lacUV5 promoter(Figure 4A, inset)]. However, when the lacUV5promoter probe is added to the P7SEs70-32P:P7 complex,the radioactivity originating from the band correspondingto the RPo decreases by �50%, thus indicating the dis-placement of s70-32P from the RNAp in the presence of P7(Figure 4A, compare lane 4 and 5). Additional experi-ments with 32P-P7 confirm that P7 displaces s70 fromP7SEs70:P7 complex on interaction with the lacUV5promoter probe: As expected, 32P-P7 and s70 can bindto the RNAp at the same time (Figure 4B, lanes 1–3),but the addition of the lacUV5 promoter probe to theP7SEs70:32P-P7 results in a complex that migrates at theidentical position on the native gel as the P7SE:32P-P7complex (Figure 4B, compare lanes 2–4). Further experi-ments with 32P-lacUV5 promoter probe corroborate theview that P7 causes the displacement of s70 from theRNAp on interaction with DNA: Results shown inFigure 4C reveal that the presence of P7 results in afaster-migrating (relative to the RPo) and heparin-sensi-tive complex that is likely composed of P7, core RNAp

subunits and lacUV5 (Figure 4C, lanes 6–9; Figure 4B,lane 4; Figure 4A, lane 5). To independently verify thatP7 displaces s70 from the RNAp, which indeed results inthe formation of a non-specific and thus transcriptionallyinactive complex between RNAp core and DNA, weanalysed promoter complexes formed on the s70-depend-ent T5 N25 promoter by potassium permanganate(KMnO4) probing. KMnO4 is a single-stranded thymine-reactive DNA oxidizing agent that is widely used to detects70-dependent local DNA melting around and down-stream of the consensus �10 element on promoters bythe RNAp that is indicative of RPo formation. WhereasKMnO4-mediated DNA cleavage is seen on the unpairedthymine at positions �12 to �3 on the non-template and�4 on the template strands by P7SEs70, as expected, noDNA cleavage at these positions on either the non-template on template strands of the T5 N25 promoter isdetected in reactions containing P7, which is indicative ofa non-specific and thus transcriptionally inactive complexbetween the promoter and core RNAp (SupplementaryFigure S4).Next, we repeated the EMSA experiments with 32P-

lacUV5 at �4�C to determine if P7 causes s70 dissociationon initial engagement of RNAp with the promoter (i.e.during RPc formation) or during the RPo formation.Recall that the majority of promoter complexes formedon the lacUV5 promoter at �4�C correspond to RPc(33). As shown in Figure 4D, the complex formed in thepresence of P7 and lacUV5 promoter (lane 5) has an iden-tical mobility as the P7SE:32P-lacUV5:P7 complex (lane 3)and migrates faster than the P7SEs70:32P-lacUV5:P7 (RPc)complex (lane 4). This result suggests that P7-induced dis-placement of s70 from the RNAp occurs at the earlystages of engagement of promoter DNA by the RNApholoenzyme.Collectively, the results indicate that P7 simultaneously

interacts with the b’ and b subunits of the bacterial RNApand inhibits transcription by s70 displacement whenRNAp attempts to interact with the promoter. P7 firstdocks onto the accessible b’ NTD and positions itselfproximal to the b flap domain. A new interactionsurface is unveiled on P7 that interfaces with the b flapdomain tip helix, thereby (i) interfering with s70 region 4/bflap interaction and (ii) weakening the interaction betweens70 and core RNAp. The latter effect apparently results inthe dissociation of s70 when RNAp undergoes additionalconformational changes while engaging promoter DNA(see below). Results from the following observationssupport this scenario: Under identical experimental par-ameters of the BTH assay, the binding of s70 region 4 tothe b flap domain is �4 - to 5-fold weaker than the

Figure 3. Continued(a-P7), activates transcription of the lacZ gene. Because the expression of the fusion proteins (�CI-b’ NTD and flap �CI-b flap and a-P7) are underthe control of IPTG-inducible promoters, and the cells used for the b-galactosidase assays were grown in the presence of increasing concentrations ofIPTG, in the graphs the b-galactosidase activities (in Miller units) are expressed as a function of IPTG concentration. (C) Autoradiograph of 20%(w/v) denaturing urea gel showing the synthesis of ApApUpU transcript (underlined nucleotides are a32P labeled) from the lacUV5 promoter byP7SEs70 in the absence (lane 1) and presence of P7 (lanes 2 and 3) and P7 (R60E) (lanes 4 and 5). The percentage of ApApUpU transcriptsynthesized (%A) in the reactions with P7 with respect to reactions without P7 is normalized to unincorporated [a-32P]-UTP (not shown) andgiven at the bottom of the gel for each reaction. All values for %A obtained from at least three independent experiments fell within 5% of the valueshown.

Nucleic Acids Research, 2014 9

at Imperial C

ollege London on February 12, 2014

http://nar.oxfordjournals.org/D

ownloaded from

A

EP7

P7SE + + + +

70

+ +++

Lane 1 2 3 4 5

70-32P

P7SEσ70-32P /P7SEσ70-32P:P7

xF 1.7 5.0

C D

Lane 1 2 3 4 5 6 7 8 9

32P-lacUV5

P7

Heparin+ + + +

+ +

P7SEσ70WTEσ70

RPo

P7SE:P7:32P-lacUV5

+ +

%CH 100 46 100 58

%CP7 100 90 100 35

%CP7+H 100 40 <1

Lane 1 2 3 4

32P-P7

P7SE:32P-P7

P7SEσ70:32P-P7

lacUV5

70

P7SE

+

+ + +++

%C n/a 47 43 45

100

lacUV5P7

P7SE

RPo

70-32P

Lane 1 2 3 4 5 6

+

P7SEσ70-32P /P7SEσ70-32P:P7

++ +

+ + + +

+

%C n/a 88 93 66 34 <1

B

σ

σ

σ

σ

P7σ70

+ +++

P7SE:32P-lacUV5

P7SEσ70:32P-lacUV5 (RPc)

P7SE 32P l UV5 P7

P7SE

Lane 1 2 3 4 5

32P-lacUV5

P7SE:32P-lacUV5:P7

%C / 19 20 30 26%C n/a 19 20 30 26

Figure 4. P7 displaces s70 from the RNAp. (A) Autoradiograph of a 4.5% (w/v) native polyacrylamide gel showing results from experimentsconducted with s70-32P to determine if P7 causes the displacement of s70 from the RNAp. The %C indicates the radioactivity in the differentcomplexes as a percentage of free s70-32P in lane 1 (please note that the sum of total radioactivity in all lanes except lane 1 is equal because theEs70-32P poorly enters the gel matrix under our experimental conditions). The inset shows an autoradiograph of a 4.5% (w/v) native polyacrylamidegel demonstrating that the slower migrating complex that forms when promoter DNA is added to the P7SEs70-32P is resistant to heparin and thusrepresents the RPo. The percentage P7SEs70-32P-lacUV5 complexes (i.e. RPo) remaining following challenge with heparin compared with reactionswith no heparin added is given at the bottom of the gel. (B) As in (A), but experiments were conducted with 32P-P7. The %C indicates theradioactivity in the different complexes as a percentage of free 32P-P7 in lane 1. (C) As in (A), but experiments were conducted with 32P-lacUV5promoter DNA and wild-type RNAp was used as a negative control in lanes 2–5. The %CH indicates the relative percentages of WTEs70:32P-lacUV5and P7SEs70:32P-lacUV5 complexes (i.e. RPo) remaining following challenge with heparin compared with reactions with no heparin added (lanes 1and 6, respectively). The %CP7 indicates the relative percentages of promoter complexes formed in the presence of P7 compared with reactions withno P7 added (lanes 1 and 6, respectively). The %CP7+H indicates the relative percentages of heparin-resistant promoter complexes formed in thepresence of P7 compared with reactions with no P7 or heparin added (lanes 1 and 6, respectively). (D) As in (C), but experiments were conducted at4�C and only with the P7-sensitive RNAp (see text for details). The %C indicates the radioactivity in the different complexes as a percentage offree 32P-lacUV5 in lane 1. (E) As in (A), but P7SEs70-32P and P7SEs70-32P:P7 complexes were challenged with x5-fold molar excess of non-radioactive ‘cold’ s70 in lanes 3 and 4. The fold decrease (%F) in the intensity of the radioactive signal originating from s70-32P in P7SEs70-32Pand P7SEs70-32P:P7 complexes in the presence of ‘cold’ s70 relative to complexes formed in its absence (lanes 1 and 2) are given at the bottom of thegel. In (A–E) the components present in each lane are indicated on the top of each gel and the identity of the different protein–protein and protein–DNA complexes are indicated. In (A–E) representative results from at least three independent experiments are shown and the percentages/folddifferences calculated represent averages obtained from three independent experiments and fell within 3–5% of the percentage shown.

10 Nucleic Acids Research, 2014

at Imperial C

ollege London on February 12, 2014

http://nar.oxfordjournals.org/D

ownloaded from

interaction between P7 and the b flap domain (5), and theinteraction between P7 and b flap is important for themode of transcription inhibition by P7 because mutantP7 harbouring the R60E mutation displays significantlycompromised (by �50%) ability to interact with the bflap and inhibit transcription initiation by P7SEs70

(Figure 3B and C). However, the interface between s70

and RNAp is extensive in which the interaction betweens70 region 2 and the b’ coiled-coiled motif represents themajor interaction interface (34), whereas the interfacebetween s70 region 4 and the b flap is relatively weak.Therefore, it is a formal possibility that P7 binding tothe RNAp adversely affects the affinity of s70 to theRNAp. In support of this view, s70-32P dissociates muchmore readily from the RNAp on challenging the Es70-32Pwith ‘cold’ unlabeled s70 in the presence of P7 than in itsabsence (Figure 4E), thus indicating that P7 contributes toweaken the interaction between s70 and core RNAp,which is evidently amplified in the presence of promoterDNA. In summary, our results provide a detailed molecu-lar mechanism that explains the previous observation thatRNAp isolated from Xp10 infected cells is largely devoidof s70 compared to RNAp isolated from uninfectedcells (32).

CONCLUSION

Many bacteria and phages encode a specialized class ofproteins, called anti-s factors, to modulate transcriptionby controlling the availability and activity of s-factors(35). Anti-s factors interact with their cognate s-factorsto either inhibit their activity or, as in the case of the T4phage encoded protein AsiA, alter the promoter-specificityof the bacterial RNAp (36). Unlike other phage-encodedbacterial transcription initiation inhibitors described todate, which function through direct interactions with s70

(26), our results provide no evidence for an interactionbetween P7 and s70. However, it seems that P7 functionsas a type of anti-s factor by inhibiting transcription initi-ation by s70 displacement on engagement of the RNApwith the promoter DNA.

Because many phage-encoded antiterminators, such as� phage proteins N and Q (37,38) and Thermusthermophilus phage P23-45 Gp39 protein (39), interactwith the b flap domain, it is conceivable that theantitermination function of P7, like its transcription initi-ation inhibition function, also requires interactions withthe b flap domain. In conclusion, this study not onlyunderscores the b flap as a nexus for regulation of thebacterial RNAp by bacterial and phage transcriptionregulators, but also highlights the remarkable variationand efficiency in the design of phage-encoded bacterialRNAp inhibitors and thus serves to inspire novel waysfor developing antibacterial drugs targeting the bacterialRNAp based on strategies used by bacteriophages.

SUPPLEMENTARY DATA

Supplementary data are available at NAR Online.

ACKNOWLEDGEMENTS

The authors are grateful to Dr Ernesto Cota for usefuldiscussions and thank Dr Ellen James for help with dataanalysis. The authors also thank Dr Bryce Nickels forplasmids for the BTH experiments.

FUNDING

Biotechnological and Biological Research Council (toS.W. and S.M.) and Wellcome Trust Investigator awards(to S.W. and S.M.). Funding for open access charge:Imperial College (through Wellcome Trust and BBSRCopen access policy).

Conflict of interest statement. None declared.

REFERENCES

1. Nechaev,S. and Severinov,K. (2008) The elusive object of desire—interactions of bacteriophages and their hosts. Curr. Opin.Microbiol., 11, 186–193.

2. Nechaev,S. and Severinov,K. (2003) Bacteriophage-inducedmodifications of host RNA polymerase. Annu. Rev. Microbiol.,57, 301–322.

3. Paget,M.S. and Helmann,J.D. (2003) The sigma70 family ofsigma factors. Genome Biol., 4, 203.

4. Callaci,S., Heyduk,E. and Heyduk,T. (1999) Core RNApolymerase from E. coli induces a major change in the domainarrangement of the sigma 70 subunit. Mol. Cell, 3, 229–238.

5. Kuznedelov,K., Minakhin,L., Niedziela-Majka,A., Dove,S.L.,Rogulja,D., Nickels,B.E., Hochschild,A., Heyduk,T. andSeverinov,K. (2002) A role for interaction of the RNApolymerase flap domain with the sigma subunit in promoterrecognition. Science, 295, 855–857.

6. Saecker,R.M., Record,M.T. Jr and Dehaseth,P.L. (2011)Mechanism of bacterial transcription initiation: RNA polymerase- promoter binding, isomerization to initiation-competent opencomplexes, and initiation of RNA synthesis. J. Mol. Biol., 412,754–771.

7. Nickels,B.E., Roberts,C.W., Roberts,J.W. and Hochschild,A.(2006) RNA-mediated destabilization of the sigma(70) region 4/beta flap interaction facilitates engagement of RNA polymeraseby the Q antiterminator. Mol. Cell, 24, 457–468.

8. Twist,K.A., Campbell,E.A., Deighan,P., Nechaev,S., Jain,V.,Geiduschek,E.P., Hochschild,A. and Darst,S.A. (2011) Crystalstructure of the bacteriophage T4 late-transcription coactivatorgp33 with the beta-subunit flap domain of Escherichia coli RNApolymerase. Proc. Natl Acad. Sci. USA, 108, 19961–19966.

9. Osmundson,J., Montero-Diez,C., Westblade,L.F., Hochschild,A.and Darst,S.A. (2012) Promoter-specific transcription inhibition inStaphylococcus aureus by a phage protein. Cell, 151, 1005–1016.

10. Yuan,A.H., Nickels,B.E. and Hochschild,A. (2009) Thebacteriophage T4 AsiA protein contacts the beta-flap domain ofRNA polymerase. Proc. Natl Acad. Sci. USA, 106, 6597–6602.

11. Semenova,E., Djordjevic,M., Shraiman,B. and Severinov,K. (2005)The tale of two RNA polymerases: transcription profiling andgene expression strategy of bacteriophage Xp10. Mol. Microbiol.,55, 764–777.

12. Djordjevic,M., Semenova,E., Shraiman,B. and Severinov,K. (2006)Quantitative analysis of a virulent bacteriophage transcriptionstrategy. Virology, 354, 240–251.

13. Nechaev,S., Yuzenkova,Y., Niedziela-Majka,A., Heyduk,T. andSeverinov,K. (2002) A novel bacteriophage-encoded RNApolymerase binding protein inhibits transcription initiation andabolishes transcription termination by host RNA polymerase.J. Mol. Biol., 320, 11–22.

14. Yuzenkova,Y., Zenkin,N. and Severinov,K. (2008) Mapping ofRNA polymerase residues that interact with bacteriophage Xp10transcription antitermination factor p7. J. Mol. Biol., 375, 29–35.

Nucleic Acids Research, 2014 11

at Imperial C

ollege London on February 12, 2014

http://nar.oxfordjournals.org/D

ownloaded from

15. Ederth,J., Mooney,R.A., Isaksson,L.A. and Landick,R. (2006)Functional interplay between the jaw domain of bacterial RNApolymerase and allele-specific residues in the product RNA-binding pocket. J. Mol. Biol., 356, 1163–1179.

16. Nechaev,S. and Severinov,K. (1999) Inhibition of Escherichia coliRNA polymerase by bacteriophage T7 gene 2 protein. J. Mol.Biol., 289, 815–826.

17. Sattler,M., Schleucher,J. and Griesinger,C. (1999) Heteronuclearmultidimensional NMR experiments for the structuredetermination of proteins in solution employing pulse fieldgradients. Prog. NMR Spectrosc., 34, 93–158.

18. Marchant,J., Sawmynaden,K., Saouros,S., Simpson,P. andMatthews,S. (2008) Complete resonance assignment of the firstand second apple domains of MIC4 from Toxoplasma gondii,using a new NMRView-based assignment aid. Biomol. NMRAssign., 2, 119–121.

19. Pavlova,O., Lavysh,D., Klimuk,E., Djordjevic,M., Ravcheev,D.A.,Gelfand,M.S., Severinov,K. and Akulenko,N. (2012) Temporalregulation of gene expression of the Escherichia coli bacteriophagephiEco32. J. Mol. Biol., 416, 389–399.

20. Bogdanova,E., Djordjevic,M., Papapanagiotou,I., Heyduk,T.,Kneale,G. and Severinov,K. (2008) Transcription regulation ofthe type II restriction-modification system AhdI. Nucleic AcidsRes., 36, 1429–1442.

21. Battiste,J.L. and Wagner,G. (2000) Utilization of site-directedspin labeling and high-resolution heteronuclear nuclear magneticresonance for global fold determination of large proteins withlimited nuclear overhauser effect data. Biochemistry, 39,5355–5365.

22. Tzeng,S.R., Pai,M.T. and Kalodimos,C.G. (2012) NMR studiesof large protein systems. Methods Mol. Biol., 831, 133–140.

23. Murakami,K.S. (2013) X-ray crystal structure of Escherichia coliRNA polymerase sigma70 holoenzyme. J. Biol. Chem., 288,9126–9134.

24. Yang,Y., Berry,A.A., Lee,W.C., Garnett,J.A., Marchant,J.,Levine,J.A., Simpson,P.J., Fogel,S.A., Varney,K.M.,Matthews,S.J. et al. (2011) Complete 1H, 13C and 15N NMRassignments for donor-strand complemented AafA, the majorpilin of aggregative adherence fimbriae (AAF/II) fromenteroaggregative E. coli. Biomol. NMR Assign., 5, 1–5.

25. Sawmynaden,K., Saouros,S., Friedrich,N., Marchant,J.,Simpson,P., Bleijlevens,B., Blackman,M.J., Soldati-Favre,D. andMatthews,S. (2008) Structural insights into microneme proteinassembly reveal a new mode of EGF domain recognition. EMBORep., 9, 1149–1155.

26. James,E., Liu,M., Sheppard,C., Mekler,V., Camara,B., Liu,B.,Simpson,P., Cota,E., Severinov,K., Matthews,S. et al. (2012)Structural and mechanistic basis for the inhibition of Escherichiacoli RNA polymerase by T7 Gp2. Mol. Cell, 47, 755–766.

27. Camara,B., Liu,M., Reynolds,J., Shadrin,A., Liu,B., Kwok,K.,Simpson,P., Weinzierl,R., Severinov,K., Cota,E. et al. (2010) T7

phage protein Gp2 inhibits the Escherichia coli RNA polymeraseby antagonizing stable DNA strand separation near thetranscription start site. Proc. Natl Acad. Sci. USA, 107,2247–2252.

28. Thibodeau,S.A., Fang,R. and Joung,J.K. (2004) High-throughputbeta-galactosidase assay for bacterial cell-based reporter systems.Biotechniques, 36, 410–415.

29. Dove,S.L. and Hochschild,A. (2004) A bacterial two-hybridsystem based on transcription activation. Methods Mol. Biol., 261,231–246.

30. Young,B.A., Gruber,T.M. and Gross,C.A. (2004) Minimalmachinery of RNA polymerase holoenzyme sufficient forpromoter melting. Science, 303, 1382–1384.

31. Yuzenkova,Y., Tadigotla,V.R., Severinov,K. and Zenkin,N.(2011) A new basal promoter element recognized by RNApolymerase core enzyme. EMBO J., 30, 3766–3775.

32. Lin,S.H., Liu,J.S., Yang,B.C. and Kuo,T.T. (1998) Disassociationof sigma subunit from RNA polymerase of Xanthomonas oryzaepv. oryzae by phage Xp10 infection. FEMS Microbiol. Lett., 162,9–15.

33. Buc,H. and McClure,W.R. (1985) Kinetics of open complexformation between Escherichia coli RNA polymerase and thelacUV5 promoter. Evidence for a sequential mechanism involvingthree steps. Biochemistry, 24, 2712–2723.

34. Schleucher,J., Schwendinger,M., Sattler,M., Schmidt,P.,Schedletzky,O., Glaser,S.J., Sorensen,O.W. and Griesinger,C.(1994) A general enhancement scheme in heteronuclearmultidimensional NMR employing pulsed field gradients.J. Biomol. NMR, 4, 301–306.

35. Campbell,E.A., Westblade,L.F. and Darst,S.A. (2008) Regulationof bacterial RNA polymerase sigma factor activity: a structuralperspective. Curr. Opin. Microbiol., 11, 121–127.

36. Hinton,D.M., Pande,S., Wais,N., Johnson,X.B., Vuthoori,M.,Makela,A. and Hook-Barnard,I. (2005) Transcriptional takeoverby sigma appropriation: remodelling of the sigma70 subunit ofEscherichia coli RNA polymerase by the bacteriophage T4activator MotA and co-activator AsiA. Microbiology, 151,1729–1740.

37. Santangelo,T.J. and Artsimovitch,I. (2011) Termination andantitermination: RNA polymerase runs a stop sign. Nat. Rev.Microbiol., 9, 319–329.

38. Deighan,P., Diez,C.M., Leibman,M., Hochschild,A. andNickels,B.E. (2008) The bacteriophage lambda Q antiterminatorprotein contacts the beta-flap domain of RNA polymerase. Proc.Natl Acad. Sci. USA, 105, 15305–15310.

39. Berdygulova,Z., Esyunina,D., Miropolskaya,N.,Mukhamedyarov,D., Kuznedelov,K., Nickels,B.E., Severinov,K.,Kulbachinskiy,A. and Minakhin,L. (2012) A novel phage-encodedtranscription antiterminator acts by suppressing bacterial RNApolymerase pausing. Nucleic Acids Res., 40, 4052–4063.

12 Nucleic Acids Research, 2014

at Imperial C

ollege London on February 12, 2014

http://nar.oxfordjournals.org/D

ownloaded from