allosteric activation of bacterial swi2/snf2 (switch/sucrose non

Allosteric Activation of Bacterial Swi2/Snf2 (Switch/SucroseNon-fermentable) Protein RapA by RNA PolymeraseBIOCHEMICAL AND STRUCTURAL STUDIES*

Received for publication, October 13, 2014, and in revised form, July 22, 2015 Published, JBC Papers in Press, August 13, 2015, DOI 10.1074/jbc.M114.618801

Smita Kakar‡1, Xianyang Fang§1, Lucyna Lubkowska¶1, Yan Ning Zhou¶, Gary X. Shaw‡, Yun-Xing Wang§,Ding Jun Jin¶2, Mikhail Kashlev¶3, and Xinhua Ji‡4

From the ‡Macromolecular Crystallography Laboratory, §Structural Biophysics Laboratory, and ¶Gene Regulation andChromosome Biology Laboratory, NCI, National Institutes of Health, Frederick, Maryland 21702

Background: During transcription, RapA recycles RNA polymerase (RNAP) using its ATPase activity.Results: The binding mode and binding site of RapA on RNAP are characterized.Conclusion: The ATPase activity of RapA is inhibited by its N-terminal domain but stimulated by RNAP in an allostericfashion.Significance: Conformational changes of RapA and its interaction with RNAP are essential for RNAP recycling.

Members of the Swi2/Snf2 (switch/sucrose non-fermentable)family depend on their ATPase activity to mobilize nucleic acid-protein complexes for gene expression. In bacteria, RapA is anRNA polymerase (RNAP)-associated Swi2/Snf2 protein thatmediates RNAP recycling during transcription. It is known thatthe ATPase activity of RapA is stimulated by its interaction withRNAP. It is not known, however, how the RapA-RNAP interac-tion activates the enzyme. Previously, we determined the crystalstructure of RapA. The structure revealed the dynamic nature ofits N-terminal domain (Ntd), which prompted us to elucidatethe solution structure and activity of both the full-length proteinand its Ntd-truncated mutant (RapA�N). Here, we report theATPase activity of RapA and RapA�N in the absence or pres-ence of RNAP and the solution structures of RapA and RapA�Neither ligand-free or in complex with RNAP. Determined bysmall-angle x-ray scattering, the solution structures reveal a newconformation of RapA, define the binding mode and bindingsite of RapA on RNAP, and show that the binding sites of RapAand �70 on the surface of RNAP largely overlap. We concludethat the ATPase activity of RapA is inhibited by its Ntd but stim-ulated by RNAP in an allosteric fashion and that the conforma-tional changes of RapA and its interaction with RNAP are essen-tial for RNAP recycling. These and previous findings outline thefunctional cycle of RapA, which increases our understanding ofthe mechanism and regulation of Swi2/Snf2 proteins in generaland of RapA in particular. The new structural information alsoleads to a hypothetical model of RapA in complex with RNAPimmobilized during transcription.

The Swi2/Snf2 (switch/sucrose non-fermentable)5proteinsmediate mobilization of nucleic acid-protein complexes duringreplication, transcription, DNA repair, recombination, andRNA polymerase (RNAP) recycling (1, 2). As the only Swi2/Snf2 protein in bacteria, RapA is an RNAP-associated proteinwith ATPase activity (3, 4). After one or few rounds of tran-scription in vitro, RNAP becomes sequestered or immobilizedin an undefined posttranscription/posttermination complex(PTC), and RapA releases RNAP from the PTC for transcrip-tion reinitiation (5–7). As a Swi2/Snf2 protein, RapA uses itsATPase activity to remodel the PTC (8). However, the mecha-nism of RapA action and, particularly, how its ATPase activity isregulated is largely unknown.

In bacteria, initiation of transcription requires a family of �factors; in Escherichia coli the major sigma factor is �70 thatbinds to the core enzyme (Core) to form the holo enzyme(Holo) of RNAP (9). As abundant as �70 in the cell, RapA isconsistently co-purified with RNAP (3, 7). Using an in vitrocompetition assay, we have previously shown that RapA bindsto the Core but is readily displaceable by �70 (7). Therefore, it isplausible that RapA and �70 either share a common binding siteor have partially overlapping binding sites on RNAP.

The crystal structure of RapA (Protein Data Bank (PDB) ID3DMQ) reveals that the protein contains seven domains: anN-terminal domain (Ntd), two RecA-like domains 1A and 2A,two Swi2/Snf2-specific domains 1B and 2B, a Spacer domain,and a C-terminal domain. Domains 1A, 2A, 1B, and 2B form theSwi2/Snf2-specific ATPase module of the protein. There aretwo molecules in the asymmetric unit. Of the two independentmolecules, the relative positioning of the Ntd with respect to

* This work was supported, in whole or in part, by the Intramural ResearchProgram of the National Institutes of Health, NCI, Center for CancerResearch. The authors declare that they have no conflicts of interest withthe contents of this article.

1 These authors contributed equally to this work.2 To whom correspondence may be addressed. Tel.: 301-846-7684; E-mail:

[email protected] To whom correspondence may be addressed. Tel.: 301-846-1798; E-mail:

[email protected] To whom correspondence may be addressed. Tel.: 301-846-5035; E-mail:

[email protected].

5 The abbreviations used are: Swi2/Snf2, switch/sucrose non-fermentable;Core, the core enzyme of RNA polymerase; Ec, E. coli; Holo, the holoenzyme of RNA polymerase; Ntd, N-terminal domain; PDDF, pair distancedistribution function; PTC, posttranscription/posttermination complex;RapA�N, Ntd-truncated RapA mutant; RNAP, RNA polymerase; SAXS,small-angle x-ray scattering; TtTEC, T. thermophilus ternary elongationcomplex; TIC, transcription initiation complex; Tt, T. thermophilus; Rg,radius of gyration; TtTIC, T. thermophilus transcription initiation complex;DSS, disuccinimidyl suberate; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 39, pp. 23656 –23669, September 25, 2015

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the rest of RapA is different, indicating the dynamic nature ofthe Ntd association with the rest of the protein (7).

In this study we engineered an Ntd-truncated RapA(RapA�N), tested its Core binding and ATPase activity, andcompared the results to those of the full-length protein. Usingsmall-angle x-ray scattering (SAXS), we have also determinedthe solution structures of RapA and RapA�N both standingalone and in complex with the Core. These results provide newinsights into the regulation of the ATPase activity of RapA,shedding light on the mechanism of RapA-facilitated RNAPrecycling during transcription.

Experimental Procedures

Cloning, Protein Expression, and Purification—The cDNAclone encoding E. coli (Ec) RapA�N (rapA�N), containing His6and covering amino acid residues 108 –962 of RapA, was madeby a PCR reaction using the forward primer with BamH1 site(rapA-108 F 5�-TCACGGATCCATGTTCAGCAAACCGCA-GGACCGTCTG) and reverse primer with HindIII site (rapA-31R 5�-GCAAAGCTTACTGATGCGTTACAA). The frag-ment was then cloned into the expression vector pQE-80L(Qiagen, Valencia, CA). The sequence of the rapA�N clone wasvalidated by DNA sequencing.

The EcRapA�N protein was expressed and purified using thesame protocol as described for the full-length protein (7). Ther-mus thermophilus (Tt) Core was obtained from the T. thermo-philus HB8 strain (ATCC 27634; Manassas, VA). The cells weregrown at 65 °C as described (10). Cells were lysed by sonicationand spun down at 12,000 rpm for 45 min. This was followed bypolyethyleneimine P and ammonium sulfate precipitation, andthe TtCore was purified as described (11).

Gel-shift Assay—4 �g of TtCore was incubated with EcRapAand EcRapA�N in molar ratios 1:0.5, 1:1, 1:2, and 1:3 in a 20-�lreaction mixture. The reaction buffer consisted of 20 mM Tris,pH 8.0, 200 mM NaCl, 5 mM MgCl2, and 5% glycerol. The reac-tion mixtures were incubated and shaken for 1.5 h at roomtemperature; 6% DNA retardation gel in 1� Tris borate EDTAbuffer (Life Technologies) was run for 75 min at 2 watts fol-lowed by Coomassie staining.

ATPase Assay—The ATPase assay was performed to deter-mine the amount of [�-32P]ADP released from [�-32P] ATP asdescribed (4). The reaction mixture was prepared in 10� tran-scription buffer consisting of 40 mM Tris-HCl, pH 7.8, 40 mM

KCl, 5 mM MgCl2, and 3 mM �-mercaptoethanol. The sampleswere prepared by mixing 1 �l of 10� transcription buffer, 1 �lof 500 mM NaCl, 1 �l of proteins (0.5 mg/ml), and 1 �l of 1 mM

ATP containing 0.2– 0.5 �Ci of [�-32P]ATP, and the final vol-ume was made to 10 �l with water. The samples were incubatedat 37 °C for 60 min. 1.5 �l of each sample was applied on apoly(ethyleneimine)-cellulose plate (Sigma), and thin-layerchromatography was used to separate the ADP from ATP. Theplate was dried for 15 min, and the sample spots were firstwashed with water and then with methanol. The TLC plate wasdried for 30 min and developed in 1 M formic acid, 1 M LiClsolution in water. After developing, the plate was dried for 20min and exposed for 10 – 60 min to a phosphorus screen. Thescreen was scanned using Typhoon 8600 PhosphorImager (GE

Healthcare). The experiment was performed in triplicate underthe same reaction conditions.

RNAP Recycling Assay—In vitro recycling of T. thermophilusRNAP (TtRNAP) was tested with EcRapA on a circular DNAtemplate carrying E. coli Tac promoter, pTac (5). The assaywas performed as described (5) except that the final reactionmixture contained 40 mM Tris-HCl, pH 7.9, 0.05 mM EDTA,5 mM MgCl2, 3 mM DTT, 200 mM NaCl, 0.2 �g/ml acetylatedbovine serum albumin, 1� SUPERase*In RNase Inhibitor (LifeTechnologies), and nucleotide triphosphates mix (1/0.2/0.2/0.2mM ATP/CTP/GTP/UTP) with 0.1– 0.2 mCi/ml [32P]UTP(PerkinElmer Life Sciences). For a single-round transcriptionreaction, the DNA competitor heparin (50 �g/ml) and nucleo-tide triphosphates were added at the time of initiation. Thereaction samples contained TtHolo alone, TtHolo in the pres-ence of heparin, TtHolo plus RapA, and TtHolo plus RapA inthe presence of heparin. For each sample, two mixtures (1.3 �gTtHolo, 10 �g plasmid DNA (pDJ631) with or without 10.4 �gEcRapA in 90 �l of reaction mix (A)) and (nucleotide triphos-phates in the presence or absence of heparin in 30 �l of reactionmix (B)), were pre-incubated at 37 °C for 5 min to form the openpromoter complex (8). The in vitro transcription was initiatedby mixing of A and B followed by collecting 10-�l aliquots after5, 15, 30, 60, 120, 180, 240, and 480 min. The reaction wasstopped by adding 12 �l of stop solution (10 M urea, 250 mM

EDTA, pH 8.0, and 0.05% xylene cyanol). Five microliters ofeach sample was analyzed on 8% (19:1) acrylamide sequencinggels (in 7 M urea, 1� Tris borate EDTA (TBE)) with 1� TBErunning buffer. The gels were scanned using Typhoon 8600PhosphorImager. The assay was performed in triplicate, andthe results were plotted in the form of a bar graph (see below).

Structural Analyses by Small-angle X-ray Scattering—TheEcRapA and EcRapA�N samples for SAXS data collection wereprepared by dialyzing 300 �g of each purified protein in 20 mM

Tris, pH 7.9, 200 mM NaCl, 5 mM MgCl2, and 2 mM �-mercap-toethanol. The samples were concentrated to 100 �l at the finalconcentration of 3 mg/ml. The EcRapA-TtCore complex wasprepared by incubating 240 �g of TtCore with 60 �g of EcRapAfor 1 h at 4 °C in 1 ml of buffer containing 20 mM Tris, pH 7.9,200 mM NaCl, 5 mM MgCl2, and 2 mM �-mercaptoethanol.After incubation, the complex was transferred to a 4-ml Ami-con centrifugal filter with a membrane cut-off of 100 kDa (Mil-lipore, Taunton, MA), washed with the buffer, and concen-trated to a total volume of 200 �l. The complex was washed 5times to exactly match the sample buffer with the referencebuffer and concentrated down to 100 �l. The final concentra-tion of the EcRapA-TtCore complex used for SAXS data collec-tion was 3 mg/ml. The EcRapA�N-TtCore complex was pre-pared using the same protocol as described above.

SAXS measurements were carried out at room temperatureat beamline 12ID-B of the Advanced Photon Source, ArgonneNational Laboratory. The wavelength (�) of x-ray radiation was0.8856 Å. The scattered x-ray photons were recorded with aPILATUS 1M detector (Dectris) for SAXS and a PILATUS 100kdetector (Dectris) for WAXS. The setups were adjusted toachieve scattering q values of 0.006 � q � 2.8Å�1, where q �(4�/�)sin�, and 2� is the scattering angle. Thirty two-dimen-sional images were recorded for each buffer and sample solu-

Allosteric Activation of RapA by RNA Polymerase

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tion using a flow cell, with the exposure time of 1–2 s per imageto minimize radiation damage and obtain good signal-to-noiseratio. The absence of systematic signal changes in sequentiallycollected x-ray scattering images confirmed that there was noradiation damage. The two-dimensional images were reducedto one-dimensional scattering profiles using the Matlab soft-ware package on site. The scattering profile of a sample solutewas calculated by subtracting the buffer contribution from thesample-buffer profile using the program PRIMUS (12) follow-ing standard procedures (13). Concentration series measure-ments (4- and 2-fold dilution and stock solution) for the samesample were carried out to remove the scattering contributiondue to interparticle interactions and to extrapolate the data toinfinite dilution. The forward scattering intensity I(0) andthe radius of gyration (Rg) were calculated from the data ofinfinite dilution at low q values in the range of qRg � 1.3, usingthe Guinier approximation: ln I(q) � ln (I(0)) � Rg

2q2/3. Theseparameters were also estimated from the scattering profile witha broader q range of 0.006 – 0.30 Å�1 using the indirect Fouriertransform method implemented in the program GNOM (14)along with the pair distance distribution function (PDDF, orP(r)) and the maximum dimension of the protein, Dmax. Theparameter Dmax (the upper end of distance r) was chosen so thatthe resulting PDDF has a short, near zero-value tail to avoidunderestimation of the molecular dimension and consequentdistortion in low resolution structural reconstruction. ThePorod volume of solutes (Vporod) and the volume-of-correlation(Vc) were calculated using the program PRIMUS (12) and Scat-ter (15), respectively. The molecular weights of solutes werecalculated on a relative scale using the Rg/Vc power law (15), theSAXS MoW method (16), and AUTOPOROD (17), indepen-dent of protein concentration and with minimal user bias. Thetheoretical scattering intensity of the atomic structure modelwas calculated and fitted to the experimental scattering inten-sity using CRYSOL (18), and the model-data agreement param-eter, 2

free, was calculated using Scatter (15).Low resolution ab initio shape envelopes were derived using

DAMMIN (19), which generates models represented by anensemble of densely packed beads, using scattering profileswithin the q range of 0.006 – 0.30 Å�1. A total of 32 indepen-dent runs were performed. The resulting models were averagedby DAMAVER (20), superimposed by SUPCOMB (21) based onthe normalized spatial discrepancy criteria, and filtered usingDAMFILT to generate the final model.

The all-atom rigid body modeling of RapA in solution wasperformed using the Xplor-NIH package (22, 23). The startingstructure was prepared from the crystal structure of RapA (7).The Ntd (residues from 1–102) and the other six domains (res-idues from 121–962) of the molecule were defined as two rigidbodies, and the relative orientation of these two rigid bodies wasoptimized, resulting in models that best fit into the experimen-tal scattering profile.

The flexibility of EcRapA was analyzed using the EnsembleOptimization Method (EOM) (24), which allows for coexis-tence of different conformations of the protein in solution. Apool of 10,000 random structures of EcRapA was generatedwith the same definition of rigid body domains as above. Scat-tering curves generated from these structures were filtered

using a genetic algorithm against the SAXS scattering curves toselect an ensemble of conformers consistent with the experi-mental data. Rigid body modeling for the EcRapA-TtCore andEcRapA�N-TtCore complexes was first conducted using theprogram CORAL (25), which is applicable to SAXS-based rigidbody modeling of multisubunit complexes, one or several ofwhose components lack some fragments (e.g. terminal portionsor interdomain linkers are missing). The CORAL programcombines rigid body and ab initio modeling; the missing termi-nal portions or interdomain linkers are treated as random poly-peptide chains. The starting coordinates of RapA�N and RapAwere taken from the SAXS solution structures (this work), andthe starting coordinates of TtCore were taken from the crystalstructure of T. thermophilus transcription initiation complex(TtTIC) (26). A simulated annealing protocol was employed inCORAL to find the optimal positions and orientations ofRapA�N (or RapA) and TtCore and the approximate confor-mations of the missing portions of polypeptide chain(s) inTtCore, against the experimental scattering profiles.

To account for the polydispersity resulting from the associa-tion/dissociation equilibrium of the complexes, rigid body model-ing for the EcRapA-TtCore and EcRapA�N-TtCore complexeswas performed using the recently developed program SASREFMX(27). The algorithm implemented in SASREFMX performs rigidbody modeling of multisubunit complexes against the scatter-ing profiles from polydisperse samples that contain dissocia-tion products. The optimized parameters are the positions, ori-entations, and volume fractions of the intact complexes anddissociation products. The starting coordinates and scatteringprofiles used in SASREFMX were the same as those used inCORAL modeling.

Cross-linking Analysis—The cross-linking analysis was doneusing disuccinimidyl suberate (DSS) as the cross-linking agent(Sigma). 26 �g of TtCore was incubated with 8.4 �g of EcRapAand 6.25 �g of EcRapA�N, respectively, in 45 �l of transcrip-tion buffer containing 40 mM KCl. The proteins were incubatedfor 1 h at room temperature to generate complexes. Five sam-ples, 9 �l each of EcRapA, EcRapA�N, TtCore, EcRapA-TtCore, and EcRapA�N-TtCore were mixed with 1 �l of dif-ferent concentrations of freshly prepared solution of DSS indimethylformamide. These were incubated at 30 °C for 30 min.The final concentrations of DSS used were 0.025, 0.05, 0.1, and0.25 mM. We optimized the DSS concentration by titratingincreasing amounts of DSS to the protein samples followed bySDS-PAGE analysis. The highest concentration of DSS usedwas enough to lead to the disappearance of �� subunit ofTtCore and its complete cross-linking to higher molecularweight bands. The lowest DSS concentration was enough topartially cross-link the proteins and visualize all the subunits ofTtCore and EcRapA by SDS-PAGE at the same time. The cross-linking was quenched by the addition of 1 M ammonium bicar-bonate to a final concentration of 100 mM and incubation for 30min. The samples were fixed with 2� SDS loading buffer, boiledfor 10 min, and loaded to a 4 –12% Bis-Tris SDS gel. The gelswere run in MOPS SDS running buffer for 1 h and 20 min at 20watts and stained with Coomassie Blue.

In-gel Digestion and Mass Spectrometry—Coomassie Blue-stained gel samples were destained and digested with trypsin




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(Promega, Madison, WI) overnight at 37 °C. Extracted peptideswere lyophilized and reconstituted in 0.1% trifluoroacetic acidbefore being analyzed by nanoflow reversed-phase LC-MS/MS.The nanoflow reversed-phase LC-MS/MS was performed usinga Thermo Scientific EASY-nLC 1000 nanoflow LC system cou-pled online to a Velos Pro dual-pressure linear ion trap massspectrometer (ThermoElectron, San Jose, CA). The LC-MS/MS data were searched using Proteome Discoverer 1.4.Dynamic modification of methionine oxidation was included inthe database search. Only tryptic peptides with up to twomissed cleavage sites meeting a specific SEQUEST scoring cri-teria (delta correlation (�Cn) 0.1 and charge state-dependentcross-correlation (Xcorr) 1.9 for [MH]1, 2.2 for[M2H]2, and 3.5 for [M3H]3] were considered as legit-imate identifications. The normalized spectral abundance fac-tor quantitative analysis function of Scaffold (Version 4.4.5,Proteome Software Inc., Portland, OR) was used to compare theabundance of proteins identified in the gel samples. For each ofthe EcRapA-TtCore and EcRapA�N-TtCore complexes, twosamples were chosen. To remove any bias in the quantitativedetection of peptides resulting from the difference in sequencelengths of �� and �, control samples for the two complexes werealso analyzed. Bands of both EcRapA-TtCore and EcRapA�N-TtCore complexes were cut from non-denaturing gels to get theabsolute number of �� and � in a 1:1 complex that was notcross-linked. The results are normalized to the mass spectrom-etry (MS) data of the control samples.

Structural Illustration—All of the structural illustrationswere generated using the PyMOL Molecular Graphics System,Version 1.3 Schrodinger, LLC.

Results

EcRapA Forms a 1:1 Complex with TtCore—As shown in lane1 of Fig. 1A, TtCore by itself migrates as a diffused band in anon-denaturing gel, indicating a mixture of various oligomericspecies (28). However, the EcRapA-TtCore complex migratesas a sharp band, indicating its homogenous state; furthermore,the titration of EcRapA in increasing amounts suggests reason-ably tight binding of EcRapA to the TtCore (Fig. 1A, lanes 2–5).Using the same gel-shift assay, we observed that EcRapA�Nalso forms a 1:1 complex with TtCore (Fig. 1B).

The Ntd of EcRapA contains two subdomains, each folded asa highly bent antiparallel �-sheet (7). The functional roles ofNtd homologs in other proteins suggest that the Ntd of RapAinteracts with both nucleic acids and protein. In RapA, the Ntdcontacts domains 1A, Spacer, and C-terminal domain, amongwhich the 1A domain is part of the ATPase module. As afore-mentioned, RNAP recycling requires the ATPase activity ofRapA, which is stimulated upon its binding to RNAP (5, 6).Contacting the ATPase module in a dynamic fashion, the Ntdmay play a role regulating the ATPase activity of the protein.We then compared the ATPase activities of RapA and RapA�Nin the absence or presence of the Core RNAP.

Ntd Inhibits the ATPase Activity of RapA—The ATPase activ-ity of RapA in the presence of Core is approximately four timesthat in the absence of Core (4). Strikingly, RapA�N exhibits anATPase activity approximately five times that of RapA in theabsence of Core, and this activity is not significantly stimulated

by the Core (Fig. 1, C and D). These observations suggest thatthe ATPase activity of RapA is regulated by the Ntd, the struc-tural basis for which may be the relative positioning of Ntd withrespect to the rest of the molecule as our structural data indi-cated. In the crystal structure, the Ntd is in close contact withthe 1A domain (7), which may limit the internal motion of theATPase module during ATP hydrolysis and thereby inhibit itsactivity. The dynamic nature of Ntd and the stimulated ATPaseactivity of RapA�N (Fig. 1D) suggest that the observed stimu-

FIGURE 1. Formation of the RapA-Core complex and ATPase activity. Aand B, binding of EcRapA and EcRapA�N to TtCore by EMSA using 6%acrylamide gel. Lane 1 shows TtCore. Lanes 2–5 show EcRapA-TtCore orEcRapA�N-TtCore complex with increasing amounts of EcRapA or EcRapA�Nas labeled. C, ATPase activity was measured for EcRapA, EcRapA�N, and theircomplexes with their cognate EcCore by an in vitro ATPase assay (see “Exper-imental Procedures”). The scan shows the separation of ADP from ATP on aTLC poly(ethyleneimine)-cellulose plate. Lane 1 shows [�-32P]ATP, lanes 2– 6show results of incubation of [�-32P]ATP with RNAP (lane 2), EcRapA�N (lane3), EcRapA�N-EcCore (lane 4), EcRapA (lane 5), and EcRapA-EcCore (lane 6). D,percent conversion of [�-32P]ATP to [�-32P]ADP quantified for the six samplesof Fig. 1C. The graph shows data from three independent experiments.

FIGURE 2. Activation of TtRNAP recycling by EcRapA. The percentage oftotal radioactively labeled RNA is shown for time points from 5 min up to 8 h.The black arrow indicates the approximate time required for completion of asingle-round transcription. The results for four samples from three experi-ments were normalized over the total radioactively labeled RNA of the con-trol sample, TtHolo, at the 8-h time point, which was assumed a value of 50%.RNA degradation became apparent when the reactions were longer than 8 h.




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FIGURE 3. Structural analysis of EcRapA and EcRapA�N by SAXS. A, top, Overlay of experimental scattering profiles with back-calculated scattering profilesfor EcRapA and EcRapA�N. The inset shows the Guinier analysis (30) for the experimental scattering curves of EcRapA and EcRapA�N with qmax � Rg � 1.3.Bottom, residual differences in the scattering curves between the back-calculated and experimental ones. B, overlay of the model (blue) with experimental(green) PDDFs for EcRapA and overlay of the model (cyan) with experimental (magenta) PDDFs for EcRapA�N. The PDDFs were not normalized to theirmolecular masses. The PDDFs for the models were calculated using Xplor-NIH (22, 23); 100 models were calculated, out of which an ensemble of 20 with thelowest 2 values was averaged to obtain the representative curve. C, shown in two views, the EcRapA�N structure (in cyan, residues 108 –962 from the crystalstructure of RapA; PDB ID 3DMQ) was docked into the SAXS envelope (in magenta). D, the crystal structure of EcRapA (in cyan with Ntd highlighted in red; PDBID 3DMQ) was docked into the SAXS envelope (in green). E, the best-fit rigid-body model of EcRapA (in cyan with Ntd highlighted in red; PDB ID 3DMQ) usingXplor-NIH was docked into the SAXS envelope (in green).




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lation of ATPase activity by the Core is mediated by Ntd posi-tioning. We speculated that the Ntd could move even furtheraway from the ATPase core and utilized the SAXS method foran answer.

EcRapA Facilitates the Recycling of TtRNAP in Vitro—Next,we tested if EcRapA was active in the recycling of heterologousTtRNAP in vitro on the plasmid DNA template carrying E. coliTac promoter (pTac), which was previously used for EcRNAPrecycling assay (5). Because EcRapA was not functional at42 °C temperature (data not shown), we performed thisexperiment at 37 °C where TtRNAP (the optimal temperature55– 65 °C) transcribed DNA at a 1000-fold slower rate com-pared with EcRNAP (29). We confirmed that TtRNAP effi-ciently initiated transcription at pTac under these conditions.Adding heparin prevented re-initiation at pTac, which revealedthat a single round transcription required �4 h to complete inthe presence or absence of RapA. Thus, the efficiency of initia-tion and elongation by TtRNAP were not significantly affectedby EcRapA under the single-round conditions. In the multir-ound assay lacking heparin, the RNA synthesis continuedbeyond 4 h in the presence, but not in the absence of RapA (Fig.2). We concluded that RapA was active in recycling TtRNAP ina trend similar to its effect on the cognate EcRNAP on the sametemplate (7).

Solution Structures of EcRapA and EcRapA�N by SAXS—Toelucidate the structure of EcRapA by the SAXS method,EcRapA and EcRapA�N were exposed at multiple concentra-tions, and the experimental data were normalized and pro-cessed as described (13). The experimental scattering profiles,with the scattering intensity I(q) plotted versus momentumtransfer q for each sample, along with PDDF are shown in Fig. 3,A and B. The Guinier region of the scattering profiles (Fig. 3A,inset) is linear, indicating that the two samples are monodis-perse and homogeneous in solution (30). The overall structuralparameters are summarized in Table 1, among which themolecular weights estimated from the SAXS data are consistentwith those predicted, indicating that both proteins are mono-meric in solution. The experimental scattering profiles for thetwo samples were fitted to scattering profiles back-calculatedfrom the crystal structure for full-length EcRapA and the crystalstructure with the Ntd removed for EcRapA�N. The back-calculated scattering profile for EcRapA�N fits well to theexperimental data ( � 1.64, 2

free � 4.3), indicating thatEcRapA�N under the SAXS conditions has a conformationvery similar to that in the crystal structure. In contrast, theback-calculated scattering profile for EcRapA fits poorly tothe experimental data ( � 5.14, 2

free � 36.8). Furthermore,the predicted Rg value based on the crystal structure ofEcRapA, is 35.5 Å, which is much smaller than that derivedfrom the SAXS data (40.8 Å). Therefore, EcRapA must adopta conformation significantly different from that observed inthe crystal lattice.

To gain more specific information on the solution structuresof EcRapA and EcRapA�N, ab initio shape envelopes were builtfor each sample using the program DAMMIN (19). The crystalstructure of EcRapA with the Ntd removed can be well fittedinto the SAXS envelope of EcRapA�N (Fig. 3C), whereas

fitting the crystal structure in its full length to the SAXSenvelope of EcRapA indicates that the Ntd of EcRapA is posi-tioned differently (Fig. 3D). Indeed, SAXS-based rigid bodyrefinement of EcRapA using Xplor-NIH (22, 23), duringwhich the relative positions of the Ntd and the other sixdomains of the EcRapA molecule were refined and opti-mized as rigid bodies against the SAXS data, resulted in anaverage conformation of RapA (Fig. 3E), which is much moreextended than that of the crystal structure and fits well to thescattering data ( � 0.52, 2

free � 0.91).SAXS experiments have been used as a powerful technique to

assess macromolecular flexibility. The visual features in thedimensionless Kratky plots of EcRapA and EcRapA�N, whichprovide a direct means to qualitatively assess the degree of flex-ibility of the scattering solute on a common scale, show a morebell-shaped curve for EcRapA�N but a significant enrichmentin the curve at higher scattering angles and a decrease of peakheight at qRg � 1.73 for EcRapA (Fig. 4A). This indicates thatEcRapA�N is more compact in solution than EcRapA, whichhas increased flexibility. This is further supported by the Porod-Debye plots (31) of EcRapA and EcRapA�N, which show a pla-teau for EcRapA�N (Fig. 4B). The loss of plateau for EcRapAsuggests its increased flexibility. To further assess the flexibilityof EcRapA in solution, we employed the Ensemble Optimiza-tion Method (EOM) (24). In this approach, the Ntd and all theother six domains of EcRapA were defined as two rigid bodies,which were used as inputs to generate a pool of 10,000 con-formers. A genetic algorithm selection process was performedto generate ensembles containing multiple conformers that

TABLE 1Data collection and structural parameters derived from SAXSexperiments

a The MWpred was calculated from the primary sequences of components.b The MWsaxs1 was calculated using the Rg/Ve power law developed by Rambo et

al. (15).c The MWsaxs2 was calculated from Porod volume using AUTOPOROD (27).d The MWsaxs3 was calculated using the web portal “SAXS MoW” (16).




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best fit the experimental scattering curve. A plot of the fitting between the experimental data and that calculated from theselected ensemble versus ensemble size (Ne) shows that an opti-mized ensemble with two conformers versus one single con-former significantly improves the fitting but that furtherenlarging the ensemble size does not, indicating that a minimalensemble size of 2 is sufficient to describe EcRapA in solution(Fig. 4C). The frequency distribution of conformers for opti-mized ensemble of Ne � 2 as a function of Rg shows that thescattering curve is best described as the coexistence of two con-formers with Rg value of 38.4 and 41.4, respectively (Fig. 4D).Interestingly, this is in broad agreement with the Rg values forEcRapA�N and the EcRapA in its extended conformationderived above (see Table 1).

We wondered whether EcRapA adopts the open or closedconformation to bind to RNAP. To characterize the EcRapA-TtCore complex in solution, we first tested the applicability ofthe SAXS method to the RNAP system by determining the

SAXS solution structure of TtHolo. The crystal structure ofTtTIC available at the time of our SAXS experiment containedan incomplete �� subunit (32). Therefore, this experiment alsoprovided an opportunity to test whether the SAXS method isable to reveal the missing structure.

Applicability of SAXS to the RNAP System: Proof of Prin-ciple—The experimental scattering profile and the PDDF forTtHolo are shown in Fig. 5, A and B. The Guinier region of thescattering profile (Fig. 5A, inset) is linear, indicating thatTtHolo is monodisperse and homogeneous (30). As shown inTable 1, the molecular weight estimated from the SAXS data isconsistent with that predicted, indicating that the TtHolo ismonomeric. The ab initio shape reconstruction for TtHolo byDAMMIN reveals a unique bowling pin shape (Fig. 5C). Fittingthe incomplete TtHolo structure with the incomplete �� sub-unit (32) into the bowling-pin-shaped SAXS envelope revealedelectron density for the missing portion of the �� subunit (Fig.5C). Accordingly, we built the TtHolo with a complete �� sub-

FIGURE 4. Flexibility analysis of EcRapA and EcRapA�N by SAXS. A, the dimensionless Kratky plots for EcRapA and EcRapA�N. B, the Porod-Debye plots (31)for EcRapA and EcRapA�N. C, overlay of experimental scattering profile with that back-calculated for optimized ensemble with two conformers for EcRapA. Theinset is the plot of fitting against the ensemble size. D, distribution of the optimized ensemble with two conformers and the initial pool of 10000 randomconformers (gray area) as a function of Rg.




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unit, and our structure was soon validated by the recent crystalstructure of TtTIC that contains a complete �� subunit (26).Indeed, the head of the envelope corresponds to the missingportion of the �� subunit (Fig. 5D). As shown in Fig. 5A, theexperimental scattering profile of TtHolo can be well fitted intothe back-calculated curve from the crystal structure of TtHolo( � 0.68, 2

free � 0.51). This experiment demonstrates theapplicability of SAXS to the RNAP system. Next, we used theSAXS method to determine the solution structures of EcRapA-TtCore and EcRapA�N-TtCore.

Solution Structures of EcRapA-TtCore and EcRapA�N-TtCore by SAXS—The experimental scattering profiles and thePDDFs for the two complexes are shown in Fig. 6, A and B.The molecular weights for the two complexes estimated fromthe SAXS data are consistent with predicted ones for 1:1 com-plexes (Table 1). The three-dimensional shapes generated withDAMMIN (19) for the two complexes are shown in Fig. 6, C andD. Whereas the envelope of EcRapA-TtCore shares a similaroverall shape with that of TtHolo, the envelope of EcRapA�N-

TtCore is slightly different from those of the other twocomplexes (Fig. 6, C, D, and E). Superposition of the enve-lope of EcRapA-TtCore with that of EcRapA�N-TtCorereveals the electron density for the Ntd in the EcRapA-TtCore complex (Fig. 6E), which is in agreement with theopen conformation of RapA where the Ntd is disengagedfrom the rest of RapA. We further performed SAXS-basedrigid body refinement for the two complexes using CORAL(25), However, the resulting structures could not fit well tothe experimental data, as indicated by the values betweenexperimental and back-calculated scattering curves of 1.8and 2.2 for EcRapA-TtCore and EcRapA�N-TtCore, respec-tively (Fig. 6A).

Both complexes were measured at a stoichiometry of 1:1.1,and so there was a small amount of free EcRapA or EcRapA�Nin the sample. Therefore, the effects of dissociation products(EcRapA or EcRapA�N) on the scattering profiles and rigidbody refinement should be taken into account. Recently, theSASREFMX method (27) was developed to reconstruct three-

FIGURE 5. Structural analysis of TtHolo by SAXS. A, top, overlay of experimental scattering profile with back-calculated scattering profile (PDB ID 4G7H) forTtHolo. The inset shows the Guinier analysis for the experimental scattering curves of TtHolo with qmax � Rg � 1.3. Bottom, residual differences in theexperimental and back-calculated scattering curves. B, overlay of the PDDFs for the TtHolo model (cyan) with the experimental data (green). The PDDF for themodel was calculated using Xplor-NIH (22, 23); 100 models were calculated, out of which an ensemble of twenty with lowest the 2 values was averaged toobtain the representative curve. C, fitting the crystal structure of TtHolo with incomplete �� subunit (PDB ID 1IW7) into the SAXS envelope of TtHolo. The headof the bowling-pin-shaped envelope (boxed) was attributed to the missing portion of the �� subunit. D, the crystal structure of TtTIC (in blue with �70 highlightedin red; PDB ID 4G7H) is superimposed with the SAXS envelope (green mesh).




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dimensional structures for transient multisubunit complexesfrom SAXS. This method not only provides low resolutionthree-dimensional structures but also estimates the volumefractions of both the complex and dissociation products. Wethus used SASREFMX to probe the association behavior and

conformations of the complexes. The resulting SAXS struc-tures fit to the experimental data significantly better, with values of 0.74 and 0.88 for EcRapA-TtCore and EcRapA�N-TtCore, respectively. The final structures for the two com-plexes are shown in Fig. 6, C–E.




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The Binding Sites of RapA and �70 on RNAP Largely Over-lap—As depicted in Fig. 7A, superposition of the SAXS struc-ture of EcRapA-TtCore with the crystal structure of TtTIC (26)demonstrates that the binding sites of RapA and �70 on theCore largely overlap. The surface charge potential distributionfor the contacting surfaces of RapA and Core in the solutionstructure of EcRapA-TtCore exhibits high complementarity.As shown in Fig. 7B, the RNAP-contacting RapA surfaceappears to be a positively charged island in the middle of anegatively charged sea, whereas the RapA-contacting RNAPsurface looks like a negatively charged island in the middle of apositively charged ocean. Recently, the E. coli Holo structurebecame available (33). The superposition of EcRapA with theEc�70 subunit embedded in the E. coli Holo structure also indi-cates the complementarity of surface charge potential distribu-tion for the EcRapA-EcCore complex (not shown). The solu-tion structure of EcRapA-TtCore suggests 77 amino acid sidechains in EcRapA may interact with RNAP electrostatically.Among the 77 residues, 21 are conserved in TtRapA.

Fig. 7C shows that RapA mainly contacts the �� and � sub-units of TtCore. The structure also suggests that 54 and 21 sidechains from the �� and � subunit, respectively, may form elec-trostatic interactions with RapA. All of the 75 residues are con-served in EcCore. To validate the general contacting areas ofRapA on RNAP, we performed a cross-linking assay (Fig. 7D).EcRapA, EcRapA�N, and TtCore were used as controls. In thecase of EcRapA-TtCore and EcRapA�N-TtCore complexes, weobserved that increasing the amount of DSS leads to gradualcross-linking of �� more than �. Upon the addition of RapA, the�� subunit cross-links to RapA to a higher degree than �, sug-gesting that RapA interacts more with �� than �. We analyzedthe products of cross-linking assay by in-gel digestion and MSto characterize the binding interface. Two bands, 1 and 2 (Fig.7D), were cut from lane a of EcRapA-TtCore, digested withtrypsin, and total unique peptide counts were determined byMS. A band with a 1:1 complex of EcRapA-TtCore on a 6%DNA retardation gel (lane 3, Fig. 1A) was analyzed as a control(see “Experimental Procedures”). MS analysis shows the pres-ence of ��, �, and RapA in both the control and test samples. Acomparison of the spectral count of peptides in the control andtest bands shows �1.28 times more �� than � peptides forEcRapA-TtCore. Comparable results (�1.35 times) wereobtained for EcRapA�N-TtCore using the test bands (1� and 2�in Fig. 7D) with the control band (lane 3 in Fig. 1B). This dem-onstrates that RapA has a more extensive binding interface with�� than �, as suggested by our SAXS structures of the com-plexes (Fig. 6). In addition, we show here that the deletion of

Ntd does not change the general binding area of RapA on RNAP(Fig. 7D).

Discussion

Accurate Structural Information from SAXS—Despite theresolution limit of SAXS, we have applied rigorous metrics andanalyzed data in comparison to crystal structure information,which provides accurate structural analysis in solution (34, 35).Comparison of the ab initio shape envelopes of RapA�N andRapA revealed electron density for the Ntd (Fig. 3, C and D),which was confirmed by rigid body modeling against the scat-tering profiles (Fig. 3E). Similarly, superposition of the enve-lopes of RapA�N-Core and RapA-Core revealed the electrondensity for the Ntd in the full-length RapA complex (Fig. 6E),which was also confirmed by rigid-body modeling against theexperimental data (Fig. 6, C–E). The PDDFs of SAXS solutionstructures of EcRapA, EcRapA�N (Fig. 3B), TtHolo (Fig. 5B),EcRapA-TtCore, and EcRapA�N-TtCore (Fig. 6B) are also ingood agreement with those calculated from experimental dataas judged on the basis of previous studies (36, 37). Furthermore,we show here that SAXS can be used to complete large, multi-subunit structures with missing components. When we deter-mined the SAXS solution structure of TtHolo, the availablecrystal structure of TtTIC contained an incomplete �� subunit(32). Fitting the incomplete TtHolo taken from the TtTICstructure into the ab initio bowling-pin-shaped SAXS enveloperevealed electron density for the missing portion of the �� sub-unit (Fig. 5C). We completed the �� subunit accordingly, andour SAXS solution structure of TtHolo was soon validated bythe recent crystal structure of TtTIC (26), which contains acomplete �� subunit (Fig. 5D).

The Ntd of RapA Regulates the ATPase Activity of RapA viaConformational Transformation—In the crystal structure ofRapA, the Ntd packs against the 1A domain of the ATPasemodule (Fig. 8A). We show here that the Ntd can indeed movefarther away from the ATPase module, leading to the open con-formation of EcRapA (Fig. 8B), which is dramatically differentfrom the closed conformation in the crystal structure. We alsoshow that removing the Ntd stimulates ATPase activity, whichis not further stimulated in the presence of RNAP (Fig. 1, C andD). These data suggest that eliminating the physical contactbetween the Ntd and the ATPase module stimulates theATPase activity of RapA. Hence, stabilizing the open confor-mation of RapA could also lead to the stimulated ATPase activ-ity similar to the Ntd truncation.

The Ntd is tethered to the 1A domain in the ATPase moduleby Linker1, which is �40 residues in length (7). The flexibility of

FIGURE 6. Structural analysis of EcRapA-TtCore and EcRapA�N-TtCore by SAXS. A, top, overlay of experimental scattering profiles with that back-calcu-lated for the rigid-body models from CORAL and SASREFMX for EcRapA-TtCore and EcRapA�N-TtCore. The starting models for rigid-body modeling usingCORAL are the same as that using SASREFMX. The inset shows Guinier analysis (30) for the experimental scattering curves of EcRapA-TtCore and EcRapA�N-TtCore with qmax � Rg � 1.3. Bottom, residual differences in the scattering curves between the rigid-body models and experimental ones. B, overlay of themodel (magenta) and experimental (orange) PDDFs for EcRapA-TtCore; also shown is the overlay of model (red) and experimental (blue) PDDFs for EcRapA�N-TtCore. The PDDFs for the models were calculated using Xplor-NIH (22, 23); 100 models were calculated, out of which an ensemble of 20 with the lowest 2

values was averaged to obtain the representative curve. C, the rigid-body docking model for the EcRapA-TtCore complex (EcRapA, in cyan with Ntd highlightedin red; this work; TtCore, in gray, PDB ID 4G7H) is superimposed with the SAXS envelope (orange mesh). D, the rigid-body docking model for the EcRapA�N-TtCore complex (EcRapA�N, in cyan; this work; TtCore, in gray, PDB ID 4G7H) is superimposed with the SAXS envelope (in light blue). E, superimposition of theenvelopes of EcRapA-TtCore and EcRapA�N-TtCore. The major difference highlighted with a box indicates the location of Ntd in the EcRapA-TtCore complex(shown as a schematic in gray for TtCore (PDB ID 4G7H)) and in cyan for EcRapA with Ntd in red; this work). Panels C and D show the common binding area of�70 and RapA on the Core, whereas D and E define the binding site of Ntd.




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Linker1, especially that of its N-terminal end, enables RapA totransform between the closed and open forms (Fig. 8, A and B).These two forms of RapA suggest that the protein exists insolution as an equilibrium between two major conformations.It appears that the closed form is suitable for forming the crystallattice, and the consumption of the closed form shifts the equi-librium to sustain crystal growth. In the closed conformation,however, the Ntd appears to inhibit the ATPase activity. Only

when the physical impact of Ntd on the ATPase module is com-pletely removed, either upon the truncation of Ntd or upon thebinding of RapA to the Core RNAP, the stimulated ATPaseactivity of RapA is observed (Fig. 1, C and D). Our SAXS struc-ture of RapA-Core shows that RapA binds to the Core in theopen conformation (Fig. 8C), and the overall shape of the RapA-Core complex resembles that of the �70-Core complex (Fig.8D). Hence, stabilizing the open form of RapA is equivalent to

FIGURE 7. The largely overlapped binding sites of RapA and �70 on the Core enzyme of RNAP. A, superposition of the SAXS structure of EcRapA-TtCore(this work) with the crystal structure of TtTIC (PDB ID 4G7H). The EcRapA (in cyan with Ntd highlighted in red) and Tt�70 (in yellow) are shown as ribbon diagrams(�-helices as cylinders, �-strands as arrows, and loops as tubes). The TtCore is shown as a molecular surface in white. B, the observed EcRapA-TtCore interface.Note that the view is different from that in panel A, optimized to expose the main contacting area of the interface. On the left, charge potential distribution(positive in blue, negative in red) of the EcRapA surface that contacts the TtCore. On the right, charge potential distribution of the TtCore surface that contactsthe EcRapA. Ellipses indicate major contacting areas of the RapA-Core interface. C, surface view of TtCore showing the �� and � subunits in yellow and cyan,respectively. The view is the same as that in panel B. The rest of the molecule is shown in light gray. D, cross-linking experiment; DSS was added to EcRapA,EcRapA�N, TtCore, EcRapA-TtCore, and EcRapA�N-TtCore to a final concentration of 0.025 (a), 0.05 (b), 0.1 (c), and 0.25 mM (d). The reaction mixtures wereseparated on a 4 –12% Bis-Tris SDS-gel using MOPS SDS running buffer. The cross-linked product bands that were analyzed by in-gel digestion and massspectrometry are indicated with numbers 1, 2, 1�, and 2�. The mobility of ��, �, EcRapA, EcRapA�N, �, and cross-linked products is indicated with arrows on theright. The gels were run for 80 min to achieve better separation of the higher molecular weight cross-linked products. The � subunit (�10 kDa) ran out of thegel under these conditions. Ref1 is EcRapA-TtCore, and Ref 2 is EcRapA�N-TtCore in the absence of DSS.




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the removal of the Ntd, each resulting in the stimulated ATPaseactivity of the enzyme, which also explains why the ATPaseactivity of RapA�N is not stimulated upon its binding to theCore (Fig. 1D). Together, these structural and functional datademonstrate that the Ntd of RapA inhibits the ATPase activityof RapA via its physical contact with the ATPase module andthat RNAP stimulates the activity in an allosteric fashion.

The Functional Cycle of RapA—Our new structural and bio-chemical data together with previous findings provide criticalinsights into the functional cycle of RapA. As abundant as thehousekeeping, promoter-specific transcription factor �70 in thecell, RapA exists as the equilibrium between the open andclosed conformations (Fig. 8, A and B), exhibiting inhibitedATPase activity. In addition to these two major conformations,there must be many minor ones either clustered around the twoor as short-lived intermediates between them. In fact, theclosed form of RapA represents two slightly different confor-mations (7). RapA binds to the available Core RNAP in its openform to form the RapA-Core complex (Fig. 8, B to C), but �70

readily replaces RapA in RapA-Core, leading to the formationof the �70-Core, i.e. the Holo complex (Fig. 8, C to B) as we havepreviously shown in vitro using a competition assay (7). Usingan in vitro transcription assay, we have also demonstrated thatRapA binds to and remodels the PTC, mediating the recyclingof RNAP (7). To connect these two processes, structural infor-mation for the RapA-Core complex is critical. Our SAXS solu-tion structures show that the binding sites of RapA and �70

largely overlap (Fig. 7A), providing the structure basis for thefact that RapA binds to the Core only and not to the Holoenzyme (7). Our SAXS structures also show that RapA binds tothe Core in its open form, which stimulates the ATPase activityfor remodeling the PTC because the process depends on ATPhydrolysis (8).

Our new data contribute to the completion of the transcrip-tion cycle proposed previously (2, 38). Briefly, RapA binds to thePTC, forming the RapA-PTC assembly; RapA remodels thePTC in an ATP-dependent manner, leading to the release ofthe Core from the PTC in the form of the RapA-Core complex.

Then, �70 displaces RapA and binds to the Core, resulting inthe formation of the �70-Core, i.e. the Holo complex, readyfor transcription reinitiation (Fig. 8). Additional structuraland biochemical data are needed to fully describe the func-tional cycle of RapA and complete the transcription cycle,among which high resolution structures of the PTC andRapA-PTC complexes are essential for the identification ofnucleic acid components and characterization of protein-nucleic acid interactions.

A Hypothetical Model for the RapA-PTC Complex—Previ-ously, we modeled a RapA-dsRNA complex showing how RapAmay bind to and translocate on dsDNA (7). The up-to-datedata, including the structural information we have gained inthis study, allows us to extend the RapA-dsRNA model toinclude the PTC. The crystal structures of TtTIC (26) andTtTEC (39) exhibit consistent non-coding and coding DNAstrands. However, the non-coding strand is longer in the TtTICstructure, and the coding strand is longer in the TtTEC struc-ture. We modeled a hypothetical RapA-PTC complex by firstincluding the non-coding DNA strand from the TtTIC and thecoding DNA strand from the TtTEC to the SAXS structure ofRapA-Core, resulting in a model of RapA-Core-DNA (Fig. 9).Intriguingly, superposition of the hypothetical RapA-dsDNA(7) and RapA-Core-DNA models indicates that the non-codingDNA strand joins a strand of the dsDNA meaningfully in termsof both strand polarity and structural arrangement, suggestingthat RapA binds to the upstream dsDNA. All of the availablestructural data suggest that RapA translocates along theupstream dsDNA (Ref. 40 and this work). The gap between theupstream dsDNA and the coding DNA strand is �6 – 8 nucle-otides in length. Although no structural information is availablefor this gap, the DNA components of the RapA-PTC assemblymay include the upstream dsDNA, the coding and non-codingstrands in the transcription bubble, and the downstreamdsDNA. This DNA architecture mimics the bent-and-openDNA model recently proposed for the T7 RNAP (41), and thepolarity of the upstream dsDNA is consistent with the unified

FIGURE 8. The functional cycle of RapA. A, the closed form of EcRapA (PDB ID 3DMQ) shows that the Ntd is packed against the ATPase module of RapA. B, theopen form of EcRapA by SAXS shows that the Ntd is disengaged from the ATPase module. C, the SAXS structure of EcRapA-TtCore shows that the open formof EcRapA is consistent with its binding mode to the TtCore. Proteins are shown as transparent molecular surfaces except that Linker1 is shown as a ribbondiagram and colored in white except that the Ntd, 1A, 2A, 2B, and Linker1 of EcRapA are colored in red, cyan, yellow, orange, and black, respectively. D, crystalstructure of TtHolo from the transcription initiation complex (TtTIC; PDB ID 4G7H) is illustrated as a molecular surface in white with �70 highlighted in red. Thepromoter DNA is omitted from the structure. It also represents the SAXS solution structure of �70-TtCore (TtHolo, Fig. 5D).




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mechanism for the Swi2/Snf2 enzymes and DEXX box helicases(42).

Our RapA-PTC model suggests that RapA facilitates a for-ward hypertranslocation of RNAP (Fig. 9). Weakening the tran-scription complex via forward hypertranslocation is a generalmechanism for complex dissociation (43, 44). Another RNAP-associated transcription factor that promotes forward hyper-translocation of RNAP is the Mfd, an ATPase-dependent DNAtranslocase (45, 46). It binds to the upstream DNA and pro-motes the forward hypertranslocation of stalled RNAP, leadingto the removal of RNAP from the DNA template (47).

RapA is the only Swi2/Snf2 protein in bacteria (3, 4). Toestablish the molecular mechanism of RapA action, high reso-lution structures of the PTC and the RapA-PTC complex areessential.

Author Contributions—D. J. J., X. J., M. K., and Y.-X. W. conceivedthe project and supervised the experiments. S. K. and G. X. S. pre-pared the RapA proteins and built the hypothetical PTC and RapA-PTC model complexes. X. F., S. K., and Y.-X. W. determined thesolution structures of RapA proteins and RapA-RNAP complexesusing the SAXS method. L. L. and S. K. purified the RNAP proteins,prepared the RapA-RNAP complexes, and performed the cross-link-ing experiment and gel-shift, ATPase activity, and in vitro transcrip-tion assays. Y. N. Z. cloned Ntd-truncated mutant of RapA andtested protein expression and solubility. X. J., S. K., X. F., L. L., D. J. J.,and M. K. wrote the manuscript with inputs from all authors. Allauthors analyzed the results and approved the final version of themanuscript.

Acknowledgments—We thank Drs. Xiaobing Zuo and Randall Win-ans for assistance with the SAXS experiments, Dr. Charles D. Schwi-eters for helpful discussions of Xplor-NIH, and Dr. Genbin Shi forcritical reading of the manuscript. SAXS was collected at theAdvanced Photon Source (APS), Argonne National Laboratory. Use ofthe APS was supported by the United States Department of Energy,Office of Science, Office of Basic Energy Sciences under ContractDE-AC02-06CH11357 and under Partner User Proposal PUP 22978.In-gel digestion and mass spectrometry were performed at the ProteinChemistry Laboratory, Frederick National Laboratory for CancerResearch.

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FIGURE 9. Hypothetical model of the RapA-PTC complex. The model iscomposed by using the SAXS structure of EcRapA-TtCore (this work, Fig. 8C),the dsDNA from the model of RapA-dsDNA (7), the non-coding DNA strandfrom the crystal structure of T. thermophilus transcription initiation complex(TtTIC, PDB ID 4G7H, Fig. 8D), and the coding DNA strand from the crystalstructure of TtTEC (PDB ID 2PPB). Proteins are illustrated and colored as in Fig.8. The nucleic acids are represented by tube-and-sticks, showing their polarityand colored in red for the coding or green for non-coding DNA strand. Thedashed line represents a portion of the DNA coding strand for which structuralinformation is not available.




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Yun-Xing Wang, Ding Jun Jin, Mikhail Kashlev and Xinhua JiSmita Kakar, Xianyang Fang, Lucyna Lubkowska, Yan Ning Zhou, Gary X. Shaw,

STUDIESProtein RapA by RNA Polymerase: BIOCHEMICAL AND STRUCTURAL

Allosteric Activation of Bacterial Swi2/Snf2 (Switch/Sucrose Non-fermentable)

doi: 10.1074/jbc.M114.618801 originally published online August 13, 20152015, 290:23656-23669.J. Biol. Chem.

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