Inhibition of Mycobacterium tuberculosis RNA Polymerase by Bindingof a Gre Factor Homolog to the Secondary Channel

Arnab China,a Sonakshi Mishra,a Priyanka Tare,a and Valakunja Nagarajaa,b

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India,a and Jawaharlal Nehru Centre for Advanced Scientific Research,Bangalore, Indiab

Because of its essential nature, each step of transcription, viz., initiation, elongation, and termination, is subjected to elab-orate regulation. A number of transcription factors modulate the rates of transcription at these different steps, and severalinhibitors shut down the process. Many modulators, including small molecules and proteinaceous inhibitors, bind theRNA polymerase (RNAP) secondary channel to control transcription. We describe here the first small protein inhibitor oftranscription in Mycobacterium tuberculosis. Rv3788 is a homolog of the Gre factors that binds near the secondary channelof RNAP to inhibit transcription. The factor also affected the action of guanosine pentaphosphate (pppGpp) on transcrip-tion and abrogated Gre action, indicating its function in the modulation of the catalytic center of RNAP. Although it has aGre factor-like domain organization with the conserved acidic residues in the N terminus and retains interaction withRNAP, the factor did not show any transcript cleavage stimulatory activity. Unlike Rv3788, another Gre homolog from My-cobacterium smegmatis, MSMEG_6292 did not exhibit transcription-inhibitory activities, hinting at the importance of theformer in influencing the lifestyle of M. tuberculosis.

The transcription process of RNA polymerase (RNAP) is con-trolled by many regulators at different steps (6, 11, 28). These

regulators include both general and operon-specific factors whichdetermine the rate and extent of transcription (2, 3, 28). The func-tions of these regulators range from the activation of transcriptionto the repression of the process under different physiological con-ditions. Apart from transcription factors, different small mole-cules and antibiotics also target the RNAP to affect transcription.Being the integral component of the essential process, RNAP is apreferred target of a number of antibiotics. The mechanism ofaction and the binding site for these inhibitors in the multisubunitholoenzyme is distinct. The antibiotics that inhibit RNAP preventthe extension of the nascent RNA beyond the third nucleotide(rifampin and sorangicin) (7, 8, 31), prevent open complex for-mation (myxopyronin) (20), or perturb mobile elements in theactive center (streptolydigin) (35). The antibacterial peptide mic-rocin J25 inhibits transcription by binding within the RNAP sec-ondary channel and inhibiting nucleoside triphosphate (NTP)uptake (21).

A number of structurally similar proteins have been identifiedfrom different bacteria which interact with RNAP through thesecondary channel. The Gre proteins are the first members of thisgroup of factors to assist RNAP in maintaining transcription ac-curacy by stimulating the cleavage of aberrant 3= ends of the RNAto resume RNA synthesis (4, 5, 9, 17). Gfh1, DksA, and TraR arestructural homologs of Gre factors but do not function like Gre;instead they inhibit the transcription process. Gfh1, which is pres-ent in Thermus sp. (13, 14, 16), inhibits both transcription initia-tion and elongation (16, 33), while DksA of Escherichia coli inhib-its some of the rRNA promoters in conjunction with guanosinepentaphosphate (pppGpp) and activates transcription from sev-eral amino acid biosynthesis operons (19, 25, 26). TraR, found inconjugative plasmids, mimics the combined function of pppGppand DksA in both the inhibition and activation of transcription(1). The secondary channel of RNAP is utilized by this group ofstructurally similar proteins for directly accessing the catalytic

center of the enzyme (22, 27, 30, 33) and to influence the mobileelements, the bridge helix, and the trigger loop in the RNAP activecenter (23, 29, 36). However, not all of the proteins that interactwith RNAP at the secondary channel are assigned a specific func-tion. For example, Rnk from E. coli has a shorter N terminus,which is insufficient for it to reach the RNAP active center, henceit probably does not influence transcription (15). Other than theGre factors and the proteins mentioned above, no other secondarychannel binding proteins have been characterized to date in any ofthe bacterial genomes.

In the genus Mycobacterium, which includes several patho-genic species, open reading frames (ORFs) which showed similar-ity to the Gre factor were identified. The most similar in the ge-nomes of M. tuberculosis and M. smegmatis are Rv3788 (9) andMSMEG_6292, respectively. Homology modeling of the ORFs in-dicate that their domain organizations are similar to that of theGre factor and hence are likely to interact with RNAP in a similarfashion. However, these proteins do not have transcript cleavagestimulatory activity. Instead, Rv3788 of M. tuberculosis, but notMSMEG_6292 of M. smegmatis, inhibits transcription by bindingnear the RNAP secondary channel and possibly perturbing thecatalytic center.

MATERIALS AND METHODSSequence alignment and homology modeling. Multiple-sequence align-ments were carried out by using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2). The figures were generated using the GenDoc multiple-

Received 8 September 2011 Accepted 7 December 2011

Published ahead of print 22 December 2011

Address correspondence to V. Nagaraja, vraj@mcbl.iisc.ernet.in.

Supplemental material for this article may be found at http://jb.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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sequence alignment editor (http://www.psc.edu/biomed/genedoc).Homology models of Rv3788 (using the Thermus thermophilus Gfh1[TthGfh1] crystal structure as the template) and MSMEG_6292 (using theEscherichia coli GreA structure as the template) were generated using thecomparative protein structure modeling program Modeler (version 9.3).

Expression and purification of the proteins. The Rv3788 gene fromM. tuberculosis was cloned in pET20b vector with a C-terminal His tagbetween the NdeI and HindIII sites (9). E. coli BL21 cells with pET20b-Rv3788 were grown to an optical density at 600 nm (OD600) of 0.6 andinduced with 0.3 mM isopropyl-�-D-thiogalactopyranoside (IPTG). Thecells were lysed by sonication and centrifuged at 100,000 � g for 2 h. Thesupernatants were subjected to 45 to 60% ammonium sulfate precipita-tion and resuspended in 3 ml of TGE buffer (10 mM Tris-HCl, pH 8.0, 5%glycerol, 0.1 mM EDTA) and purified through DEAE-Sephacel chroma-tography by elution with a linear NaCl gradient of 50 to 400 mM. His-tagged Rv3788 was purified from E. coli BL21 cells transformed withpET20b-rv3788his using nickel-nitrilotriacetic acid (Ni-NTA) affinitychromatography. V36W, S126E, and D45A/D48A His-tagged mutants ofthe protein (see Table S1 in the supplemental material) were generated bythe megaprimer inverse PCR method and purified by Ni-NTA chroma-tography. MSMEG_6292 was PCR amplified from M. smegmatis mc2155genomic DNA using the primers listed in Table S1 in the supplementalmaterial and cloned into pET20b (at NdeI and HindIII sites) with aC-terminal His tag and purified by following the same method as that forHis-Rv3788. M. tuberculosis and M. smegmatis Gre (MtbGre and MsGre,respectively) proteins were purified by following the methods describedearlier (9). M. tuberculosis and M. smegmatis RNAP (MtbRNAP andMsRNAP, respectively), which were used for in vitro transcription assaysand protein-protein interaction studies, were purified by following themethod described earlier (9, 10).

Ni-NTA pulldown assays. MtbRNAP without any tag was used forinteraction analysis with His-tagged Rv3788 or MSMEG_6292. Five-�galiquots of both proteins were incubated at room temperature for 15 minin 50 �l of reaction buffer (50 mM Tris-HCl [pH 8.0], 100 mM potassiumglutamate, 5% glycerol, and 20 mM imidazole). Twenty �l of Ni-NTApreequilibrated in reaction buffer then was added to the protein mixtureand incubated for 30 min in a rotary mixer. The supernatant was sepa-

rated and the pellet was washed thrice with 400 �l of the reaction buffer.The pellet was resuspended in 50 �l of the buffer followed by the additionof 6� SDS gel loading dye. The samples were incubated at 95°C for 5 minand centrifuged briefly, and the supernatant fractions were subjected to11% SDS-PAGE. The gels were silver stained. The interaction of MsGreand MSMEG_6292 with the MsRNAP also was carried out by following asimilar procedure.

In vitro transcription assays. (i) Stalled TEC preparation. Ternaryelongation complexes (TECs) were generated on a 5= biotinylated T7A1promoter DNA template using M. tuberculosis RNAP by following themethod described earlier (9, 18).

(ii) Single-round transcription assays. Fifteen nM T7A1 promotercontaining template DNA and 50 nM RNAP were incubated in STB (50mM Tris-HCl, pH 7.5, 5 mM magnesium acetate, 100 �M dithiothreitol[DTT], 5% glycerol, 50 �g ml�1 bovine serum albumin [BSA], and 50mM KCl) at 37°C for 15 min. Reactions were initiated by the addition of100 �M NTP mix, 1 �Ci of [�-32P]UTP, 50 �g ml�1 heparin and incu-bated further at 37°C for 15 min. In transcription inhibition reactionswith Rv3788, the protein was preincubated with the template andMtbRNAP for 15 min at 37°C prior to the addition of the NTPs. Subse-quently, the reactions were stopped with 2� gel loading buffer (0.025%[wt/vol] bromophenol blue, 0.025% [wt/vol] xylene cyanol FF, 0.08%amaranth, 5 mM EDTA, 0.025% SDS, and 8 M urea) and analyzed by 10%urea-PAGE. For carrying out transcription inhibition assays at differentmycobacterial promoters, 50 nM different promoters containing DNA(gel purified) and 100 nM MtbRNAP were used. Transcription inhibitionreactions were carried out as described above with 2 �M Rv3788.

Electrophoretic mobility shift assay (EMSA). (i) Closed complex. M.tuberculosis PrrnPCL1 promoter labeled at the 5= end with [�-32P]ATP wasincubated with MtbRNAP in the presence of increasing concentrations ofRv3788 on ice for 15 min in STB before being loaded onto a 4% native-PAGE run at 4°C.

(ii) Open complex. To form competitor-resistant open complexes(RPO), MtbRNAP, promoter, and increasing concentrations of Rv3788were incubated for 15 min at 37°C, followed by the addition of 50 �g ml�1

heparin. The DNA-protein complexes were resolved by native PAGE runat room temperature and quantified using Multi Gauge software (version

FIG 1 Sequence comparison of Rv3788 and MSMEG_6292 to Gre factor and its homologs. (A) Multiple-sequence alignment of Rv3788 and MSMEG_6292 withE. coli GreA (Ec GreA), GreB (Ec GreB), Rnk (Ec Rnk); M. tuberculosis Gre (Mtb Gre); and T. thermophilus Gfh1 (Tth Gfh1) were carried out using Clustal W. (B)The table indicates the percentages of identity and similarity (shaded) of the proteins to each other.

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2.3; Fujifilm). The rate of RPO decay was determined by initially incubat-ing 100 nM RNAP and 1 nM promoter DNA in STB at 37°C for 30 min toform RPO, which subsequently was challenged with 50 �g ml�1 heparinand 2 �M Rv3788 protein. The aliquots were taken out at different timepoints between 1 and 32 min and subjected to 4% native PAGE run atroom temperature.

(iii) Ternary elongation complex. RPO were formed with MtbRNAPand the PrrnPCL1 promoter, followed by the addition of 100 �M NTP mixto form TECs. Two �M protein was preincubated with RPO prior to theaddition of NTPs in the reaction mixture containing Rv3788. For inhibi-tion assays with pppGpp, RNAP (100 nM) was incubated with pppGpp(100 �M) in transcription buffer for 15 min, followed by the addition of50 �g ml�1 heparin, and resolved by 4% native PAGE. pppGpp was pre-pared by using M. tuberculosis Rel protein with GTP and ATP as sub-strates.

Western blotting. Polyclonal antibodies raised in rabbits againstRv3788 were used for the detection of protein in M. tuberculosis H37Racells grown for 6, 8, 12, and 20 days. Antibody against M. tuberculosis �A

was used as a control.

RESULTS AND DISCUSSIONRv3788 and MSMEG_6292 have domain organizations similarto those of RNAP secondary channel binding proteins. The ho-mology model of Rv3788 was generated using T. thermophilusGfh1 as the template (9). The model showed an overall sequenceand structural similarity with the Gre factors (see Fig. S1A in thesupplemental material), similar molecular weights, and the pres-ence of the signature motifs of secondary channel binding pro-teins (see Fig. S1A and B). A coiled-coil domain with acidic resi-dues in the loop between the two helices in the N terminus and aglobular domain at the C terminus found in the protein are typicalfeatures of Gre factors (see Fig. S1A). The Rv3788 homologueswere found only in slow-growing mycobacteria and were absentfrom fast-growing mycobacteria and other sequenced bacterialgenomes (see Table S2 in the supplemental material). The proteinwhich has been annotated as Rnk in the M. tuberculosis genomeshows 21% identity and 33% similarity to MtbGre (Fig. 1A and B).

FIG 2 Interaction of RNAP with Rv3788 and MSMEG_6292. (A) Pulldown assays were carried out using His-tagged Rv3788 and MtbRNAP (without any tag).The scheme of the assay is described in the left panel. Proteins were incubated together at room temperature for 15 min, followed by the addition of Ni-NTAbeads. Both pellet (pel) and supernatant (sup) fractions were loaded on an 11% SDS-PAGE. Lane 4 represents the pulldown of MtbRNAP in the pellet fractionalong with Rv3788. (B) Pulldown assays also were carried out using His-tagged MSMEG_6292 and M. smegmatis RNAP without any tag. Lanes 4 and 6 representthe pellet fraction consisting of precipitated MsGre (lane 4) or MSMEG_6292 (lane 6) with MsRNAP. Gre homologs in mycobacteria lack transcript cleavagestimulatory activity. Rv3788 (C) and MSMEG_6292 (D) do not possess transcript cleavage stimulatory activity. Stalled elongation complexes of M. tuberculosisRNAP formed on the T7A1 promoter based template were incubated with the proteins, and the reaction products were resolved by 22% urea PAGE. Lane 1, onlystalled elongation complex; lanes 2 and 3, stalled elongation complexes with 1 and 5 �M MtbGre (Fig. 2A) or MsGre (Fig. 2B), respectively; lanes 4 and 5, stalledcomplexes with 1 and 5 �M Rv3788 (A) or MSMEG_6292 (B), respectively.

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Rnk is a secondary channel binding protein that was identifiedearlier in Gram-negative proteobacteria (15, 32). However, M.tuberculosis Rv3788 does not share much similarity with Rnk (only12% identity and 25% similarity) (Fig. 1B), as demonstrated bythe experimental results described below. The M. smegmatis Grefactor homolog MSMEG_6292 also has the structural features of asecondary channel binding protein (see Fig. S1A) and shareshigher sequence similarity to the Gre factors than Rv3788 (21%identity to E. coli GreA, 39% identity to both M. smegmatis and M.tuberculosis Gre factors, and only 16% identity to Rv3788) (Fig.1B). The homologs of MSMEG_6292 could be found in the ge-nomes of all fast-growing isolates of mycobacteria sequenced todate except Mycobacterium abscessus. It is not found in the M.

tuberculosis genome (see Table S2 in the supplemental material)and the other slow-growing species.

The proteins interact with RNAP but lack Gre like activity.Gre factors and Gfh1 bind RNAP at the entry to the secondarychannel (24, 34, 37). The sequence similarity and resemblance inthe overall domain organization of Rv3788 and MSMEG_6292with these proteins suggest that they also interact with RNAP in asimilar manner. The direct interaction of Rv3788 with MtbRNAPwas tested by a pulldown assay as described in Materials andMethods (Fig. 2A). His-tagged Rv3788 bound MtbRNAP (lane 4of Fig. 2A), while RNAP alone did not bind to the Ni-NTA matrix(lane 2 of Fig. 2A), confirming the direct physical interaction be-tween the two proteins. MSMEG_6292 also interacted with the M.

FIG 3 Rv3788 inhibits transcription. (A) In vitro single-round transcription assays were carried out using T7A1 promoter-containing template in the presence of 0.5,1, 2, and 5 �M Rv3788. The bar diagram represents the quantification of the runoff transcripts. Transcription in the absence of any factor was considered 100%. (B) Invitro transcription assays were carried out in the absence and the presence of 2 �M Rv3788 using templates having different promoters from M. smegmatis (Ms) and M.tuberculosis (Mtb). (C) Effect of MSMEG_6292 on in vitro transcription. Two different concentrations of the protein (2 and 5 �M) were incubated with MsRNAP, anda runoff transcription assay was carried out on T7A1 promoter-containing template. Transcription inhibition by 2 �M Rv3788 was used as a positive control (lane 2).

FIG 4 Mechanism of transcription inhibition. EMSAs were carried out using the end-labeled M. tuberculosis PrrnPCL1 promoter and MtbRNAP. The effects of theincreasing concentrations of Rv3788 on closed complex (RPC) formation (A), open complex (RPO) formation (B), and rate of decay of the open complexes (C)were determined by resolving the promoter-RNAP complexes in 4% native PAGE.

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smegmatis RNAP in a similar fashion (Fig. 2B). From these data itis evident that the C-terminal domains (CTD) of both proteins aresimilar to Gre factors, and the differences (if any) in the propertieslie in the N-terminal half of the protein.

The presence of conserved acidic residues in the tip of the Nterminus coiled-coil domain and the similarity in overall domainorganization of Gre factors led us to examine whether these ho-mologs have Gre-like activity. Transcript cleavage assays were car-ried out with MtbRNAP using T7A1 20-nucleotide-long stalledelongation complexes. There was no detectable transcript cleavageinducing activity with Rv3788 (Fig. 2C) or MSMEG_6292 (Fig. 2D)in the standard assay conditions. The examination of multiple-sequence alignments of Rv3788 with E. coli and M. tuberculosisGre factors showed that several residues important for Gre activityare absent from Rv3788 (Fig. 1A). These include Asn47, Tyr50 (ofMtbGre), and several other basic residues in the N terminus (Fig.1A). MSMEG_6292 also lacked several conserved residues in theN-terminal coiled-coil region flanking the acidic patch (see Fig. S2in the supplemental material), resulting in a shorter N-terminaldomain (NTD) compared to those of the Gre factors and Rv3788,which may be insufficient for it to reach the RNAP active center(see Fig. S1A).

Rv3788 is an inhibitor of transcription. Although the Gre fac-tor homologs did not exhibit any transcript cleavage stimulatoryactivity, their interaction with RNAP (Fig. 2A and B) is suggestiveof their role in the regulation of transcription. In vitro transcrip-tion analysis showed that Rv3788 inhibited the runoff transcrip-tion from the T7A1 promoter (Fig. 3A). That the inhibition oftranscription is an intrinsic property of Rv3788 and not due to anycopurifying RNase was verified by incubating the transcriptionproduct with Rv3788 (see Fig. S3 in the supplemental material).

The transcription inhibition also was not promoter specific, sinceRv3788 efficiently inhibited transcription from different promot-ers from M. smegmatis (PrrnPCL1, PrrnB, and Pgyr) and M. tubercu-losis (PrrnPCL1, PgyrP1, PgyrR, and PmetU) with comparable efficiency(Fig. 3B). In contrast, the M. smegmatis protein MSMEG_6292 didnot show any transcription inhibitory activity in similar experi-ments from T7A1 promoter-containing template (Fig. 3C), indi-cating the functional difference between the two Gre factor ho-mologs from the two species of the same genus. The transcriptioninhibition activity of Rv3788 seems to be restricted to mycobacte-rial transcription machinery, as it did not inhibit E. coli RNAP (seeFig. S4A in the supplemental material). The lack of inhibitoryactivity was due possibly to its inability to interact with the E. coliRNAP (see Fig. S4B), indicating the requirement for species-specific protein-protein interactions for secondary channel-mediated regulation.

Rv3788 inhibits transcription during ternary complex for-mation. We analyzed the mechanism of transcription inhibitionby Rv3788 by carrying out EMSA on the M. tuberculosis PrrnPCL1

promoter. MtbRNAP forms promoter-specific complexes on bothPrrnPCl1 and T7A1 promoters (see Fig. S5 in the supplemental ma-terial). Closed complex (Fig. 4A) or open complex (Fig. 4B) for-mation was not inhibited by Rv3788. Unlike the action of DksA,which affects the open complex stability of the E. coli RNAP (25,27, 30), the stability of RPO was not altered by the factor (Fig. 4C),indicating that in this case the inhibition of transcription is at astep beyond open complex formation. A reduction in the ternaryelongation complex with NTPs in the presence of Rv3788 sug-gested that the protein inhibits that particular step (Fig. 5A).When NTPs were added to RPO prior to the addition of the pro-tein, inhibitory activity was not observed (Fig. 5A, lane 5). Fur-

FIG 5 Rv3788 inhibits NTP binding to RNAP, preventing formation of TEC. (A) EMSAs were carried using M. tuberculosis PrrnPCL1 promoter-containing DNAto check the TEC formation between the RNAP promoter and the NTPs. Lane 1, RPO; lane 2, RPO in the presence of 2 �M Rv3788; lane 3, TEC formation in thepresence of NTPs; lane 4, RPO treated with 2 �M Rv3788 prior to the addition of NTPs; lane 5, NTPs were added to the RPO before the addition of Rv3788. Thebar diagrams in the right panel represent the quantification of the complexes. (B) Rv3788 prevents inhibition by pppGpp. The assays were carried out using M.tuberculosis PrrnPCL1 to determine the action of pppGpp in the presence of Rv3788. Lane 1, RPO; lane 2, RPO incubated with pppGpp; lane 3, RPO in the presenceof 2 �M Rv3788; lane 4, RPO in the presence of both pppGpp and Rv3788. The bar diagram in the right panel represents the quantification of % RPO formation.

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ther, Rv3788 also prevented the inhibitory activity of pppGpp onthe M. tuberculosis PrrnPCL1 promoter (Fig. 5B). These results to-gether suggest that the protein prevents the access of the nucleo-tides and the effector molecules to the catalytic center directly orindirectly. Alternatively, Rv3788 could modulate the activity ofthe enzyme by inducing a conformational change in the active site.

Transcription inhibition is mediated by the N-terminal do-main. Despite the conservation of the acidic residues at the tip ofthe predicted coiled-coil domain, instead of inducing cleavage,Rv3788 inhibited transcription. Thus, the property of Rv3788 issimilar to that of TthGfh1 (16). The crystal structure of the Gfh1-RNAP complex revealed that the N-terminal coiled-coil domainenters the RNAP secondary channel and possibly modulates thebridge helix in the RNAP active center. Within the secondarychannel, the Gfh1 NTD fits particularly well with the narrowestregion of the secondary channel (34). Val 36 of Rv3788 probably islocated in the narrowest region of the secondary channel, similarlyto Leu33 of Gfh1 (Fig. 6A). A mutant of Rv3788 with Val36 re-placed by Trp (V36W) significantly reduced transcription inhibi-

tory activity (Fig. 6B). The bulky side chain of Trp probably pre-vents the Rv3788 NTD from penetrating into the secondarychannel to reach the RNAP active center. However, it also is pos-sible that the introduced bulky amino acid distorts the coiled-coildomain. However, this mutation did not abolish the interaction ofthe protein with RNAP (Fig. 6C), indicating that the interactingregion in the C terminus is independent of the N terminus coiled-coil domain. Further, a mutant protein with changes in the acidicresidues at the tip of the coiled-coil domain (D45A/D48A doublemutant) showed significantly reduced inhibitory activity (Fig.6B), indicating that the acidic residues are required for the tran-scription inhibitory activity.

The structure of Gfh1 in complex with RNAP revealed that thehydrophobic region in the CTD is involved in the interaction withthe RNAP �= coiled-coil domain at the edge of the secondarychannel (34). The hydrophobic region of the Gfh1 CTD is con-served in Rv3788. In the TthRNAP, the interaction involvesPhe982 and Leu983 in the �= coiled-coil domain and Ile119,Met117, and Met125 in the Gfh1 CTD (34). The �= coiled-coil

FIG 6 Rv3788 interacts with RNAP through its C-terminal domain and accesses the secondary channel of RNAP by using the N terminus to inhibit transcription.(A) The conserved residue V36 of Rv3788 was mutated to V36W to introduce a bulky group in the N-terminal coiled-coil domain. The S126E mutation wasintroduced in the C-terminal RNAP interaction domain. The acidic residues D45 and D48 in the coiled-coil domain were mutated to D45A/D48A (doublemutant). (B) In vitro transcription inhibition assays with the WT and the mutant proteins using T7A1 promoter-containing template. (C) Pulldown assays werecarried out using His-tagged WT, V36W, and S126E with MtbRNAP as described in Materials and Methods, and the samples were analyzed by 11% SDS-PAGE.Lanes 4, 6, and 8 represent the pellet fractions of the interaction assays with WT, V36W, and S126E proteins, respectively. The loss of RNAP interaction in theS126E mutant is indicated by the absence of RNAP from the pellet fraction (lane 8). S126E showed anomalous mobility in the gel, possibly caused by a structuralchange in the protein due to mutation.

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domain loop region is highly conserved in MtbRNAP (9), suggest-ing the requirement of this conserved set of residues for its inter-action with MtbGre and Rv3788. Ser123 and Ser126 of Rv3788 aretwo well-conserved residues found among the Gre factors andGfh1. These conserved serine residues have been shown to be im-portant for interactions with RNAP in the case of E. coli GreB (37),TthGfh1 (34), and M. tuberculosis Gre (9). The residues are locatedclose to the conserved glutamate residue in the RNAP �= subunit(34) and may hydrogen bond with it. Indeed, a mutation in theS126 residue of Rv3788 (S126E) (Fig. 6A) disrupted the interac-tion between the protein and MtbRNAP and also abolished itstranscription inhibitory activity (Fig. 6B and C).

To test whether MtbGre and Rv3788 both compete for thesame binding site in RNAP, interaction assays in the presence ofincreasing concentrations of MtbGre were carried out. MtbGre(no tag) could prevent Rv3788 (His tagged) from binding withRNAP, as indicated by the decrease in RNAP in the pellet fractionsin the Ni-NTA pulldown assays (Fig. 7A). Similarly, when thetranscription assays were carried out in the presence of Rv3788and increasing concentrations of MtbGre, transcription inhibi-tion was partially rescued by MtbGre (Fig. 7B). MSMEG_6292also competed with MsGre for binding with RNAP. Increasingconcentrations of MSMEG_6292 could compete with the Gre fac-tor and inhibit the transcript cleavage activity, probably by pre-venting Gre factor accessibility to the secondary channel of theRNAP (Fig. 7C).

Comparison of Rv3788 to Thermus species Gfh1. Secondarychannel binding proteins have emerged as one set of regulators ofRNAP activity in different bacteria. Among them, Gfh1, DksA,and TraR are the only transcription factors that have been identi-fied to date as inhibiting transcription (16, 30). Rv3788 shares themechanism of inhibition with Gfh1, i.e., by preventing NTP bind-ing. The inhibitory activity of Gfh1 was more robust at acidic pH,possibly due to the conformational toggle of the N-terminal do-main at low pH (16). The conserved residue Gly86 in Gfh1 waspredicted to be responsible for the conformational switch re-quired for transcription inhibition (14), and residue Gly88 is alsoconserved in Rv3788. The inhibition assay carried out at differentpHs showed that the transcription inhibitory activity of Rv3788was greater at pH 6.0 than at pH 8.0 (Fig. 8A), indicating possiblemechanistic similarity between Rv3788 and Gfh1. Similarly toGfh1, Rv3788 also inhibited both runoff and abortive transcrip-tions (Fig. 8B). However, unlike Gfh1, it did not inhibit the intrin-sic cleavage activity of RNAP (Fig. 8C). A few differences in theprimary sequence of the N-terminal coiled-coil domains of thetwo proteins may account for these differences in activity. Gfh1has four Asp residues in the loop region of the coiled-coil domain(Fig. 1), whereas the M. tuberculosis protein has only two acidicAsp residues at the same region. The Asp residues of Gfh1 wereshown to interact with Mg(II) in the RNAP active center, render-ing it unavailable to bind substrate NTPs and thus leading to tran-scription inhibition (16). A recent study, however, indicates thatN-terminal acidic residues are not solely responsible for transcrip-tion inhibition by Gfh1 (34). The binding of the protein to thesecondary channel and locking the RNAP in an alternative ratchetconformation is also considered to be important for transcriptioninhibition in conjunction with the acidic tip (34). Notably, in thecase of Rv3788, mutations of the acidic residues in the N-terminalcoiled-coil loop region significantly reduced transcription inhib-itory activities but did not abolish the inhibition completely (Fig.

6B). Thus, it appears that both the Asp residues and the binding ofthe coiled-coil domain to the secondary channel, which possiblyalters RNAP conformation, contribute collectively to the mecha-nism of inhibition. The major target of the secondary channelbinding regulators is predicted to be the trigger loop of the RNAP�= subunit, which undergoes substrate-induced refolding duringthe nucleotide addition cycle. The trigger loop has emerged asboth a central regulatory element and as a key determinant for theprocessivity and fidelity of the transcription (29, 36). The highsensitivity of the structure of the loop to subtle alterations inneighboring domains and the modulation of its folding to triggerhelix by many transcription factors appears to be a major mode of

FIG 7 Rv3788, MSMEG_6292, and the Gre factor share the same binding siteon MtbRNAP. (A) Ni-NTA pulldown assays with MtbRNAP and Rv3788 werecarried out in the presence of non-His-tagged MtbGre protein (5 and 10 �g) asdescribed in Materials and Methods. (B) Inhibition of in vitro transcriptionfrom the T7A1 promoter by a fixed concentration of Rv3788 (1 �M) and in thepresence of increasing concentrations (0.25 to 5 �M) of MtbGre (lanes 3 to 7).Lane 1, in vitro transcription from T7A1 promoter-containing template; lane2, transcription in the presence of only Rv3788; lane 8, only MtbGre. (C)MSMEG_6292 competes with MsGre. The T7A1-TECs were preincubatedwith 1 to 5 �M MSMEG_6292 followed by the addition of 1 �M MsGre. OnlyMsGre was added in lane 2, and in lane 6 only 5 �M MSMEG_6292 was added.

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regulation of transcription. From the present studies, we suggestthat it also could be the target of Rv3788 in the RNAP activecenter.

Apart from Gfh1 and Rv3788, peptide inhibitor microcinJ25 also was shown to inhibit transcription by binding to theRNAP secondary channel and occluding NTPs (21). Other in-hibitors of bacterial RNAP described so far employ differentmechanisms to perturb transcription (12). RNAP is evolution-arily conserved across different species, and a common mech-anism of enzyme inhibition by proteins from diverse species(Gfh1 and Rv3788) indicates the importance of regulationthrough secondary channels. These results also suggest the co-evolution of these proteins along with the RNAP to utilize thesecondary channel. Despite similar mechanisms of action,these structurally similar proteins exhibit some differences intheir properties, suggesting the evolutionary optimization oftheir function in different species to cope with the physiologi-cal needs to survive in a particular environment. A point infavor of this argument is that the Gre factor paralogs of the twospecies of mycobacteria are different. While one shows tran-scription inhibition, the other could not modulate the RNAP

active center, possibly due to the reduced length of the N ter-minus. These results also are suggestive of specific require-ments for transcriptional downregulation in M. tuberculosisunder certain physiological conditions. The analysis of the M.tuberculosis cell lysates indicated that Rv3788 is present in allgrowth phases (see Fig. S6 in the supplemental material). How-ever, the expression level was lower in the early exponentialphase, increased in the mid-exponential phase, and remainedconstant thereafter.

In conclusion, Rv3788 inhibits transcription, possibly by inter-acting with RNAP to modulate activities at the active center. Thepresence of several secondary channel-binding proteins in the ge-nome could result in a competition between them to access theactive center through the channel. The inhibition of transcriptionby proteins from diverse species of bacteria by utilizing the RNAPsecondary channel appears to be a general theme of transcriptioncontrol. The identification of secondary channel-specific tran-scription inhibitors like Rv3788 in mycobacteria and the elucida-tion of the structural features of their interaction with RNAPcould form the basis for the development of small-molecule in-hibitors which block the channel and, as a result, inhibit RNAP.

FIG 8 Properties of Rv3788. (A) The inhibition of transcription from T7A1 promoter template at two different pHs, 6.0 and 8.0. The quantification of thetranscription inhibition is represented in the graph. Transcription in the absence of Rv3788 is represented as 100%. (B) Inhibition of abortive transcription fromM. smegmatis PrrnPCL1 promoter by Rv3788. (C) Intrinsic cleavage activity of MtbRNAP in the absence (lanes 1 to 3) and in the presence (lanes 4 to 6) of 2 �MRv3788 in a �20 T7A1-TEC.

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ACKNOWLEDGMENTS

We thank Sergei Borukhov of the University of Medicine and Dentistry ofNew Jersey for valuable suggestions, Anirban Mitra for Table S2 in thesupplemental material, and Dipankar Chatterji of the Molecular Biophys-ics Unit, IISc, for the M. tuberculosis Rel protein. We thank the phosphor-imager facility of IISc, supported by the Department of Biotechnology,Government of India.

A.C. was a Senior Research Fellow from the Council of Scientific andIndustrial Research, Government of India, P.T. is a Senior Research Fel-low from the University Grants Commission, Government of India, andV.N. is a J. C. Bose Fellow of the Department of Science and Technology.

The work was supported by a Center for Excellence in TuberculosisResearch grant from the Department of Biotechnology, Government ofIndia.

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