Binding of the Transcription Effector ppGpp to Escherichia coli RNA Polymerase Is Allosteric, Modular, and Occurs Near the N Terminus of the beta '-Subunit*

Innokenti I. ToulokhonovDagger, Irina Shulgina, and V. James Hernandez§

From the Department of Microbiology, Center of Microbial Pathogenesis, State University of New York at Buffalo School of Medicine, Buffalo, New York 14214

Received for publication, August 8, 2000, and in revised form, October 13, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Among the prokaryotae, the nucleotide ppGpp is a second messenger of physiological stress and starvation. The target of ppGpp is RNA polymerase, where it putatively binds and alters the enzyme's activity. Previous data had implicated the beta -subunit of Escherichia coli RNA polymerase as containing a single ppGpp binding site. In this study, a photocross-linkable derivative of ppGpp, 6-thioguanosine-3',5'-(bis)pyrophosphate (6-thio-ppGpp), was used to localize the ppGpp binding site. In in vitro transcription assays, 6-thio-ppGpp inhibited transcription from the argT promoter identically to bona fide ppGpp. The thio group of 6-thio-ppGpp is directly photoactivatable and is thus a zero-length cross-linker. Cross-linking of RNA polymerase was directed primarily to the beta '-subunit and could be competed efficiently by native ppGpp but not by GTP or GDP. Cyanogen bromide digestion analysis of the cross-linked beta '-subunit was consistent with an extreme N-terminal cross-link. To assess allosteric consequences of ppGpp binding to RNA polymerase, high level trypsin resistance in the presence and absence of ppGpp was monitored. Trypsin digestion of RNA polymerase bound to ppGpp leads to protection of an N-terminal fragment of the beta '-subunit and a C-terminal fragment of the beta -subunit. We propose that the N terminus of beta ' together with the C terminus of beta  constitute a modular ppGpp binding site.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Within the bacterial domain of the kingdom prokaryotae exists a general and ubiquitous response to nutritional and environmental stress, the stringent response (1). This general stress response is mediated by high level accumulation of the transcription effector guanosine-3',5'-(bis)pyrophosphate (ppGpp). A major effect of elevated ppGpp levels is an immediate and severe reduction of stable rRNA and tRNA gene transcription (2). The cessation of stable RNA syntheses halts the major energy consuming activities of the cell, transcription and translation. This period of metabolic inactivity allows the cell to utilize its remaining energy reserves to adapt to stressful growth conditions through induction of specific "stress genes" (3). Once adaptation is near completion, ppGpp levels decrease and growth resumes. Failure to reduce ppGpp levels results in a severe reduction of cell viability (4).

Several lines of evidence suggest that ppGpp exerts its effects by directly binding to RNA polymerase (RNAP).1 Certain rifampicin-resistant mutants of the beta -subunit of RNAP display increased intracellular sensitivity to ppGpp (5, 6). Spontaneously occurring mutants that confer survival under artificial and prolonged exposure to toxic levels of ppGpp were mapped to the rpoB gene, encoding the beta -subunit of RNAP (7). Mutant strains devoid of ppGpp, ppGpp0 strains, are incapable of surviving nutritional deprivation; however, specific mutants of the sigma 70-, beta -, or beta '-subunits of RNAP restores normal survivability to ppGpp0 strains (8). Fluorescence quenching studies of RNAP in the presence of increasing concentrations of a fluorescently labeled ppGpp analogue (1-aminonapthalene-5-sulfonate-ppGpp) is consistent with binding of ppGpp to a single binding site on RNAP (9). Cross-linking analyses by Chatterji et al. (10) using a radioactive photocross-linkable derivative of ppGpp, 8-azidoguanine-3',5'-(bis)pyrophosphate (8-azido-ppGpp), demonstrated predominant cross-linking of ppGpp to the beta -subunit. In the same study (10), it was also observed that both N- and C-terminal partial trypsin digestion fragments of the beta -subunit were cross-linked by 8-azido-ppGpp, suggesting a modular ppGpp binding site analogous to that of the nucleotide binding site at the catalytic center of RNAP (11). Despite extensive studies on RNAP-ppGpp interactions, a precise localization of the ppGpp binding site on RNAP is lacking.

An allosteric mechanism of ppGpp action on RNAP is generally invoked as mediating its transcriptional effects, although this has not been extensively studied. In this context, allostery refers to the inducement of functionally relevant conformational changes of RNAP as a result of ppGpp binding at a location other than the catalytic site. Consistent with this notion, ribonucleoside triphosphates do not compete with ppGpp for RNAP binding (12). In addition, circular dichroism studies have revealed a small but significant change in total alpha -helical content of RNAP following addition of ppGpp (13). To date, this single previous study (13) represents the only physical evidence of induced conformational change of RNAP by ppGpp. Clearly, further studies characterizing the nature of ppGpp allostery are warranted.

The aim of the present investigation is 2-fold: 1) to substantiate and refine the location of the ppGpp binding site on E. coli RNAP; and 2), to begin to elucidate the nature of ppGpp-induced conformational changes of RNAP. Toward the first of these goals, we synthesized a new photocross-linkable derivative of ppGpp, 6-thioguanosine-3',5'-(bis)pyrophosphate (6-thio-ppGpp). The 6-thio-ppGpp derivative is a zero-length photocross-linking reagent, because the thiol group is directly photoactivatable and has a van der Waals radius similar to the oxygen for which it is substituted (14). Photoaffinity labeling of RNAP with 6-thio-ppGpp indicates that the N terminus of the largest subunit of RNAP, beta ', is the predominant location of cross-linking. To begin to investigate the conformational consequences of ppGpp-RNAP interactions, we assessed alterations in trypsin sensitivity of RNAP in the presence and absence of ppGpp. Three major ppGpp-dependent trypsin-resistant fragments of RNAP were observed. Identification of these trypsin-resistant fragments and their superimposition onto the three-dimensional structure of RNAP indicates an overlap with and close proximity to the 6-thio-ppGpp cross-linking site. These results have ramifications for both definitive identification of the ppGpp binding site on RNAP as well as for the allosteric consequences of ppGpp-RNAP interaction.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Enzymes and Chemicals-- -Escherichia coli RNA polymerase was purified from E. coli K12 strain MG1655 using the method of Burgess and Jendrisak (15) with the modifications of Lowe et al. (16). Final sigma 70 holoenzyme separation from core enzyme was accomplished by heparin-Sepharose chromatography (17). Crude RelA-ribosome preparations were prepared according to Cashel (18). Chemicals and solvents were reagent grade, used without further purification, and purchased from Sigma-Aldrich. All radioactive nucleotides were obtained from ICN Pharmaceuticals Inc.

Synthesis of ppGpp and Derivatives-- Synthesis conditions for ppGpp were as described (18). For synthesis of radioactive ppGpp, 0.5 mCi of [gamma -32P]ATP (4500 Ci/mmol) were diluted with 1 mM cold ATP and 0.5 mM GDP or 6-thioguanosine-5'-triphosphate (6-thio-GTP) in a 0.1 ml reaction volume mixed with 60 A260 units of crude RelA-ribosomes at room temperature for 12 h. Final purification of ppGpp was on a QAE-Sephadex A-25 column as described (19). Nucleotides were typically eluted with a 0-0.5 M linear gradient of triethylammonium bicarbonate, pH 7.5. Elution products were visualized by autoradiography after separation by polyethyleneimine-cellulose thin layer chromatography (20). It was noted that 6-thioguanosine derivatives chromatographed with lower mobility on polyethyleneimine-cellulose plates than the corresponding guanosine containing compounds. The ppGpp-containing fractions were pooled and concentrated through removal of the triethylammonium bicarbonate solvent by lyophilization in a vacuum centrifuge at room temperature. The product was resuspended in 50 µl of water followed by precipitation with 2% NaI in acetone, the pellet was washed three times with 1 ml of acetone and a final wash with 1 ml of ether. The final product was resuspended in 50 µl of water. 6-Thio-ppGpp exhibited an ultraviolet light spectrum typical for ppGpp with a characteristic maximum at 253 nm and a shoulder peak between 265 and 280 nm at pH 7.0 (19).

Synthesis of 6-Thioguanosine-5'-triphosphate-- 6-mercaptoguanosine was converted into the corresponding triphosphate as described by Sergiev et al. (14).

NMR Analyses of ppGpp and 6-Thio-ppGpp-- The proton and phosphorus NMR spectra of purified ppGpp and 6-thio-ppGpp were obtained at 500 MHz on a Varian INOVA 500 (Chemistry Instrumentation Center, State University of New York at Buffalo) in 10% D2O pH 4.0, spectra was collected for 27 h. 1H and 31P chemical shifts were compared and found to be nearly identical between ppGpp and 6-thio-ppGpp. Assignments of proton shifts were based on previous published spectra for 6-thio-GTP (14).

In Vitro Transcription-- RNA synthesis was carried out with a mixture of argT and lacUV5 (10 nM each) promoter-bearing linear templates in 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 250 µM GTP, ATP, and CTP, and 20 µM UTP with 15 µCi of [alpha -32P]UTP (800 Ci/mmol) in 20-µl reaction volumes at 37 °C for 10 min. Increasing amounts of GDP, ppGpp, or 6-thio-ppGpp were incubated as indicated with 100 nM RNA polymerase for 15 min at room temperature and then warmed to 37 °C for 2 min prior to initiating the reaction by adding an equal volume of prewarmed template/substrate mix. Reactions were terminated and processed according to Hsu (21). RNA was fractionated on a 7 M urea-6% polyacrylamide gel (19:1 acrylamide:bisacrylamide) in Tris-boric acid-EDTA buffer.

Cross-linking-- All cross-linking experiments were carried out in 25 mM HEPES, pH 7.9, 5 mM MgCl2, 100 mM KCl, 25 µg/ml bovine serum albumin, 5% glycerol. E. coli RNA polymerase (1 µM) was incubated with radioactively labeled 6-thio-ppGpp in a 20-µl reaction volume. Samples were mixed, incubated for 15 min at room temperature, and then transferred to ice and irradiated with 302 nm ultraviolet light (MacroVue UV-20 transilluminator; surface intensity, 9000 µW/cm2) for 20 min at a distance of 4 cm in an open Eppendorf tube covered with a polystyrene filter to remove stray radiation of <290 nm, thus avoiding protein-protein cross-linking. Photoaffinity labeling reactions were terminated by the addition of 5× SDS sample buffer (0.625 M Tris-HCl, pH 6.8, 5% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 0.2% bromphenol blue, 25% glycerol) to a 1× concentration. Samples were heated to 65 °C for 5 min and then resolved by electrophoresis through a SDS-10% polyacrylamide gel (30:0.8 acrylamide:bisacrylamide) to separate all RNA polymerase subunits and through a SDS-4% polyacrylamide gel to resolve beta - and beta '-subunits using Tris-glycine buffer.

Mapping of Cross-links-- Radioactive 6-thio-ppGpp cross-linked beta '-subunit was extracted from a denaturing SDS-4% polyacrylamide gel following electrophoresis. The region of the polyacrylamide gel containing beta '-protein was excised. Protein was eluted by diffusion out of polyacrylamide by overnight shaking of macerated gel slices in 0.03% SDS at 37 °C. Eluted protein was concentrated by freeze-drying and then resuspended in water to a final concentration of 1-2% SDS (22). Partial cyanogen bromide (CNBr) degradation of isolated beta '-protein was performed as described previously (23). Cleavage products were fractionated by electrophoresis on a SDS-7-16% gradient polyacrylamide gels in a Tris-glycine buffer system.

Trypsin Digestion Studies-- RNA polymerase (0.25 µM) was incubated for 15 min at room temperature with 200 µM of either GDP or ppGpp in 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM magnesium acetate, 5% glycerol, 0.1 mM EDTA. After a 2-min warming at 37 °C, samples were mixed with increasing amounts of tosyl-L-phenylalanine chloromethylketone-treated trypsin (0, 0.1, 0.3, 0.6, 1.2, and 2.5 µM) in a 20-µl reaction volume and incubated at 37 °C for an additional 5 min. Trypsin digestions were stopped by the addition of phenylmethylsulfonyl fluoride to a final concentration 10 mM, immediately followed by the addition of 5× SDS-sample buffer to a 1× concentration and incubation at 105 °C for 2 min. Total trypsin fragments were resolved in a SDS-12% polyacylamide gel and visualized by colloidal Coomassie G-250 staining (24). Specific trypsin fragments were identified by Western blot analyses using monoclonal antibodies against N-terminal, middle, and C-terminal epitopes of beta ' (7RC78, 7RC74, and NT73, respectively) and beta  (7RB145, 7RB135, and NT63, respectively) (Ref. 25 and courtesy of Richard Burgess, University of Wisconsin-Madison). Reactive bands were visualized by chemiluminescence using the Renaissance Western blot Chemiluminescence Reagent Plus detection kit (PerkinElmer Life Science Products)

Imaging and Quantification-- Radioactive samples were visualized by autoradiography and, when necessary, imaged on a Bio-Rad Molecular Imager and quantified using the Molecular Analyst f software (Bio-Rad Laboratories Inc.).

Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry (MALDI-TOF MS)-- Protein fragments were excised from Coomassie-stained gels and analyzed at Borealis Biosciences Inc., Toronto Canada by MALDI-TOF MS.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Synthesis and Structure of 6-Thio-ppGpp-- The nucleotide derivative 6-thioguanosine-5'-triphosphate (6-thio-GTP) was synthesized from 6-mercaptoguanosine and used as a substrate in the enzymatic reaction depicted in Reaction 1, to yield 6-thioguanosine-3',5'-(bis)pyrophosphate (6-thio-ppGpp; Fig. 1). The reaction occurs in two steps, using crude RelA-ribosomes (see "Experimental Procedures").



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Fig. 1.   Structure of 6-thioguanosine-3',5'-(bis)pyrophosphate. The chemical structure of 6-thio-ppGpp is depicted with the thio group in the mercapto and thio conformation, as indicated. NMR chemical shift in parts per million (ppm) of the total applied magnetic field are indicated for phosphorus (31P) and proton (1H) spectra. The putative assignments of proton shift are indicated for either a base (H8) or ribose (e.g. H1') proton followed by the number of protons per signal, with apparent peak splitting occurring for H1'. The proton and phosphorus NMR were collected at 500 MHz on a Varian INOVA 500 in 10% D2O, pH 4.0.


<UP> 6-thio-GTP + ATP → 6-thio-pppGpp + AMP</UP> (Step 1)

<UP>6-thio-pppGpp → 6-thio-ppGpp+P<SUB>i</SUB></UP> (Step 2)

<UP><SC>Reaction</SC> 1</UP>
The reactivity of 6-thio-GTP was comparable with GTP in the RelA-mediated reaction (data not shown). The conversion of pppGpp to ppGpp is mediated by the enzyme guanosine pentaphosphatase, which is present in crude RelA-ribosome preparations (18). Highly purified 6-thio-ppGpp was separated and obtained as described by Cashel (18).

The structure of purified 6-thio-ppGpp was verified by nuclear magnetic resonance (NMR). Both phosphorus (31P) and proton (1H) spectra of bona fide ppGpp and 6-thio-ppGpp were comparable and indicative of very similar structures (Fig. 1). Unlike the proton NMR spectra reported for 6-thio-GTP (14), 6-thio-ppGpp shows a strong chemical shift specific for a mercapto group proton (Fig. 1). Thus, in solution at pH 4.0, most of 6-thio-ppGpp bears a mercapto group at position 6 of the guanine moiety rather than a thio group as depicted in Fig. 1. The predominance of the mercapto over the thio form of 6-thio-ppGpp may simply be because of the acidic pH under which NMR was performed. This is unclear, however, because the absolute pKa of the thio group of 6-thio-ppGpp is unknown, and sufficient material was not obtained to be able to determine this experimentally. Because the photoreactive conformation of the 6-thioguanosine moiety is the thio form, cross-linking to RNA polymerase is only possible if the bound form of 6-thio-ppGpp is in the thio form and not the mercapto form, as appears to be the case (Fig. 3).

Transcription Activity of 6-Thio-ppGpp-- To test the activity of 6-thio-ppGpp compared with ppGpp, their ability to inhibit transcription of the tRNA promoter, PargT, was assayed. The activity of the argT promoter is negatively affected by increased ppGpp levels in vivo (26). Here, we demonstrate for the first time a specific inhibition of PargT in vitro by ppGpp under standard ionic and transcription conditions. At low ionic conditions, <50 mM KCl, the inhibitory effects of ppGpp were diminished (data not shown). A 4-6-fold inhibition of RNA synthesis from PargT was observed at the highest concentration of ppGpp tested (Fig. 2). As an internal control, transcription from a PlacUV5 template was monitored in an equimolar mixed template reaction. The transcription from PlacUV5 was also inhibited between 20-35%, as previously noted (27), but ppGpp preferentially inhibited transcription from PargT. The inhibition of transcription of PargT by 6-thio-ppGpp paralleled that of ppGpp.



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Fig. 2.   Inhibition of in vitro transcription by ppGpp and 6-thio-ppGpp. Multiple-round in vitro transcription of an equimolar mixture of linear templates bearing the argT and lacUV5 promoters was performed in the presence of increasing concentrations of GDP, ppGpp, or 6-thio-ppGpp, as indicated. The argT promoter produced a terminated transcript of 230 nucleotides, and the lacUV5 promoter produced a pair of run-off transcripts of ~250 nucleotides in length. The percentage of radioactivity in each transcript was normalized to untreated samples and plotted against GDP, ppGpp, or 6-thio-ppGpp concentrations. PargT transcription is represented by open symbols and PlacUV5 transcription by closed symbols.

Cross-linking of 6-Thio-ppGpp to RNA Polymerase-- Increasing amounts of radioactive 6-thio-ppGpp were mixed with 1 µM RNA polymerase and subjected to photoactivating ultraviolet light irradiation. Electrophoretic analysis of radioactively labeled RNAP on denaturing polyacrylamide gels revealed the presence of cross-linker on all RNAP subunits. At limiting concentrations of cross-linker, however, labeling appeared predominant for beta - and/or beta '-subunits (Fig. 3A). Further cross-linking experiments were all performed at an equimolar concentration of RNAP and 6-thio-ppGpp (1 µM ea). In competition experiments, cross-linking of beta '- and beta -subunits was dramatically reduced in the presence of a 200-fold excess of cold genuine ppGpp but not GTP (Fig. 3B). Electrophoresis of photoaffinity-labeled RNAP on low percentage denaturing 4% polyacrylamide gels to resolve beta '- and beta -subunits showed that the majority (~90%) of label was associated with the beta ' subunit (Fig. 3C). Competition with cold ppGpp had little effect on residual beta -subunit cross-linking, but it essentially eliminated cross-linking of the beta '-subunits (Fig. 3D). No competition with 6-thio-ppGpp binding was observed in experiments with either GTP or GDP (Fig. 3D). In addition, we observed no effect on cross-linking in the presence of nonspecific or promoter containing DNA, RNA, or rifampicin (data not shown).



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Fig. 3.   Cross-linking of 6-thio-ppGpp to RNAP. All cross-linking reactions were performed with 1 µM RNAP and radioactively labeled 6-thio-ppGpp. A, increasing concentrations of 6-thio-ppGpp were incubated with RNAP, as indicated, followed by photocross-linking and fractionation on a denaturing 10% polyacrylamide gel. B, 1 µM 6-thio-ppGpp was competed with 200 µM GTP or ppGpp prior to photocross-linking and fractionation as described in A. C, six duplicate cross-linking reactions with 1 µM 6-thio-ppGpp and RNAP were performed followed by fractionation on a denaturing 4% polyacylamide gel to resolve beta - and beta '-subunits. D, 1 µM 6-thio-ppGpp was competed with 200 µM GTP, GDP, or ppGpp prior to photocross-linking and fractionation as described for panel C.

Mapping of 6-Thio-ppGpp Cross-linking on the beta '-Subunit of RNAP-- The labeled beta '-subunit shown in Fig. 3C was excised and extracted from the 4% polyacrylamide gel and subjected to partial CNBr digestion. Following digestion, the fragments were resolved on a gradient polyacylamide gel and visualized by phosphorimaging. The actual partial CNBr digest is shown in Fig. 4 in comparison with idealized computer-generated profiles of a single-hit partial CNBr digest of beta '. Idealized digestions were generated assuming either extreme C- or N-terminal labeling of beta ', respectively. The profile of the experimental CNBr digestion fragments aligns well with the idealized CNBr N-terminally labeled fragment ladder, particularly the three very specific bands of 102, 130, and 152 amino acid residues (Fig. 4). The pattern and intensity of bands is comparable with what has been obtained previously for N-terminally labeled beta ' (28). These results are consistent with 6-thio-ppGpp cross-linking between amino acid residues 29 and 102 of the beta '-subunit of RNAP, because no label was found in association with the extreme N-terminal 29 amino acid residue fragment.



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Fig. 4.   Partial cyanogen bromide digestion of cross-linked beta '-subunit. Partial cyanogen bromide digestion of the 6-thio-ppGpp cross-linked beta '-subunit isolated from the gel of Fig. 3C was carried out as described under "Experimental Procedures" under single-hit partial digestion conditions. Digestion products were fractionated on a denaturing 7-16% polyacrylamide gradient gel and visualized by phosphorimaging. N-terminal (N) and C-terminal (C) theoretical ladders of single-hit cyanogen bromide digestion patterns are shown adjacent to the beta ' digestion.

ppGpp-dependent Trypsin-resistant Fragments of RNAP-- To begin to explore the allosteric consequences of ppGpp binding to RNAP, resistance to trypsin proteolysis was used to probe for ppGpp stabilized domains of RNAP. Limiting proteolysis has been used as a means of defining subtle alterations in the conformation of RNAP upon promoter binding (29) as well as for the definition of major RNAP structural domains (30). In contrast, we employed in this study an excess of trypsin to assay for protection of small highly structured domains of RNAP formed upon ppGpp binding. A range of trypsin concentration between 0 and 2.5 µM was used to digest a solution of 0.25 µM RNAP preincubated with 250 µM GDP or ppGpp. Trypsin fragments were resolved on denaturing polyacylamide gels. Following Coomassie staining, three prominent ppGpp-dependent trypsin-resistant fragments were revealed with an apparent molecular mass of ~100, 40, and 28 kDa, respectively (Fig. 5A, indicated by asterisks). The same fragments were also obtained in the presence of GDP; however, they had a much lower resistance to trypsin in comparison with ppGpp (Fig. 5A). The binding of ppGpp does not, therefore, appear to induce formation of unique conformers of RNAP. Instead, it seems to stabilize the existing domain structure and/or sterically hinder trypsin accessibility. In control experiments, bovine serum albumin trypsin sensitivity was identical in the presence or absence of either GDP or ppGpp (data not shown). Attempts to cleave RNAP cross-linked with radioactive 6-thio-ppGpp did not give a clear or consistent pattern of digestion; we conjecture that this failure was the result of technical problems, e.g. an interference with trypsin activity by the presence of nonspecific UV-induced cross-links. Perhaps in future experiments, the exact identification of the conformational change of RNAP induced following 6-thio-ppGpp cross-linking may be achieved using methods other than trypsin digestion to probe for changes in protein conformation.



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Fig. 5.   Trypsin-resistant fragments of RNAP in the presence of ppGpp. Increasing concentrations of trypsin, as indicated, were incubated with 250 nM RNAP for 5 min and fractionated on denaturing 10% polyacylamide gels. A, Coomassie-stained gel of total protein. Known proteins and the position of migration of relative molecular weight markers are indicated. The major trypsin-resistant fragments are indicated by asterisks. B, Western blot of the same total protein samples as shown in A probed with monoclonal antibody 7RC78, which recognizes an epitope between amino acid residues 115 and 236 of the beta '-subunit. C, Western blot of the same total protein samples as shown in A probed with monoclonal antibody NT63, which recognizes an epitope between amino acid residues 922 and 1090 of the beta  -subunit.

The identity of the tryptic fragments stabilized in the presence of ppGpp was determined by Western blot analyses. The trypsinized samples shown in Fig. 5A were processed for Western analyses and probed with a battery of monoclonal antibodies (mAb) against various epitopes of the beta - and beta '-subunits (see "Experimental Procedures"). The beta '-subunit-specific mAb, 7RC78, recognizes an epitope located between amino acid residues 115 and 236. Western blots of trypsin fragments with 7RC78 reveals that the 100- and 28-kDa ppGpp-protected fragments contain the N terminus of the beta '-subunit (Fig. 5B). The beta -subunit-specific mAb, NT63, recognizes an epitope between amino acid residues 922 and 1099 and is the most C-terminal epitope recognized by available RNAP mAbs (Burgess and colleagues (15, 16, 25, 29, 37)). Western blot analysis with NT63 is consistent with the 40kDa fragment containing the C terminus of beta  (Fig. 5C). Given the fact, however, that the epitope of NT63 is a considerable distance from the actual C terminus of the beta -subunit, it was difficult to make a definitive C-terminal assignment to this fragment. For this reason, MALDI-TOF MS analysis was performed on the 40-kDa trypsin stable fragment. MALDI-TOF MS analysis (see "Experimental Procedures") indicated that the 40-kDa beta  fragment spans amino acid residues 958-1328 ± 10. Thus, the C-terminal assignment of the 40-kDa beta -subunit band was confirmed. Assignment of the approximate end points of the 28-kDa fragment by MALDI-TOF MS was not successful because of contamination by identical size fragments of the alpha -subunit. Fortunately, the close proximity of the beta '-specific mAb, 7RC78, allows confident N-terminal assignment to the trypsin stable 28-kDa fragment.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The global stress regulator, ppGpp, a ligand and effector of RNAP, has been studied since its discovery by Cashel and Gallant (31) over 30 years ago, yet little is known about how it binds to and modifies RNAP. Here, we report the synthesis of a novel photocross-linkable derivative of ppGpp, 6-thioguanosine-3',5'-(bis)pyrophosphate. This cross-linkable ppGpp derivative has allowed us to obtain the first indication that ppGpp binds near the N terminus of the large subunit of RNAP, beta '. This compound has the distinct advantage over preceding cross-linkable ppGpp derivatives of carrying a zero-length cross-linking group. The chemical structure of 6-thio-ppGpp in solution, confirmed by NMR spectroscopy, reveals a predominance of the mercapto group (analogous to an enol group in ppGpp) rather than the thio group (analogous to a keto group in ppGpp) at the sixth position of the guanine moiety at pH 4.0 (Fig. 1). At this same pH level, a mercapto group was not noted or reported for the precursor, 6-thio-GTP (14). Thus, the presence of the 3'-pyrophosphate on ppGpp may change the local chemistry of the thio group on the guanine ring. No enol proton was detectable in bona fide ppGpp, indicating that this property is unique for the thiolated ppGpp. It is not clear which is the preferred form of 6-thio-ppGpp at pH 7.9, at which cross-linking and transcription analyses were performed. Equilibrium competition experiments and relative affinity measurements of 6-thio-ppGpp compared with ppGpp could not be performed because of the nonequilibrium conditions necessary for efficient 6-thio-ppGpp cross-linking (see "Experimental Procedures"). If 6-thio-ppGpp at pH 7.9 is predominantly in the mercapto conformation, however, it does not appear to affect dramatically its efficacy in inhibiting transcription (Fig. 2). Fundamental chemistry dictates that the thio and not the mercapto conformation of the 6-thioguanosine moiety is subject to photoactivation. With this in mind, the observed cross-linking of RNAP by 6-thio-ppGpp (Fig. 3) is consistent with a preferred binding to RNAP of the thio form of 6-thio-ppGpp. It is likely, therefore, that in natural ppGpp the keto group at position 6 of the guanine moiety is important for the binding of ppGpp to RNAP.

Predominant cross-linking of radioactively labeled 6-thio-ppGpp occurs within the first 102 amino acid residues of the N terminus of the beta '-subunit of RNAP (Figs. 3 and 4). This location overlaps with the conserved region A of beta ' (beta 'A). In the recently solved crystal structure model of Thermus aquaticus RNAP, the extreme N terminus of the beta '-subunit, including beta 'A, is disordered and lacks electron density (32). The crystal data together with our finding that ppGpp binds to and induces trypsin resistance to the N-terminal portion of beta ' (Fig. 5B) leads us to propose that ppGpp binding induces a higher order structure of this region.

The N terminus of beta ' is implicated as playing a crucial role in many aspects of the transcription process. Region beta 'A contains a zinc finger, which is essential for stable DNA association of the elongating transcription complex (33) and has been cross-linked to the double-stranded DNA at the lagging end of the transcription bubble (34). Additionally, within the transcription complex the extreme N terminus of the beta '-subunit cross-links along the entire length of the "extruded" nascent RNA (34, 35). This close proximity of the extruded RNA to the N terminus of beta ' is "replaced" in a paused transcription complex by an alternate proximity of the RNA to the so-called "flap" structure of the beta -subunit (36). This repositioning or "switching" of the path of RNA within the complex is thought to be part of the mechanism of transcriptional pausing (36). Finally, a primary determinant of sigma 70 binding is found between amino acid residues 260 and 309 of the beta '-subunit (37). These multifaceted functions associated with the N terminus of beta ' will be considered below in light of the putative binding of ppGpp near this region and the known effects of ppGpp.

The binding of ppGpp to RNAP has been proposed to destabilize specifically the open complex of rRNA promoters (27, 38). Thus, ppGpp binding and restructuring of the region of RNAP near the N terminus could disrupt the function of the beta 'A zinc finger and/or sigma 70 interaction and could lead to collapse of the open complex. Another well documented effect of ppGpp is that it decreases transcription elongation rates, primarily by increasing the pause times at naturally occurring pause sites (12, 39-41). Because the path of nascent RNA, extruded across the N terminus of beta ' in elongating RNAP, is altered to putatively bring about transcriptional pausing (36), ppGpp binding might directly influence this process. Thus, the binding of ppGpp to this particular region of RNAP may be linked mechanistically to many of the observed effects of ppGpp-RNAP interactions.

Previous data (see the Introduction) have indicated that ppGpp binding is localized to the beta -subunit of RNAP. However, in this study we have determined that the N terminus of the beta '-subunit is in close proximity to bound ppGpp. Although our results seemingly conflict with these previous observations, upon closer examination they actually corroborate earlier data concerning ppGpp-RNAP binding. We have synthesized and used for the first time a zero-length cross-linking ppGpp derivative, 6-thio-ppGpp. Preceding studies (10) used an azido group cross-linker, which replaces the proton at position 8 of the guanine moiety of ppGpp with an azido group (-N3). The thio group at position 6 of the guanine moiety of 6-thio-ppGpp is 8-10 Å distant from the azido group of 8-azido-ppGpp. Given the considerable separation of these two cross-linking groups, the thio group of 6-thio-ppGpp predictably would be in a different location and thus provide unique cross-links compared with 8-azido-ppGpp. Our trypsin resistance studies (Fig. 5) are congruent with the formation of a highly structured N-terminal portion of the beta '-subunit and C-terminal portion of the beta -subunit of RNAP as a consequence of ppGpp binding. These results are consistent with the fact that the N and C termini of beta ' and beta , respectively, are spatially close in the three-dimensional model of prokaryotic RNAP and constitute an intertwined interface of the two subunits (32). These observations lead us to propose that the binding of ppGpp is allosteric and that the site of binding is modular and located close to the intersubunit interface comprising the N- and C-terminal portions of the beta '- and beta -subunits, respectively.


    ACKNOWLEDGEMENTS

We thank and gratefully acknowledge Dr. Mike Cashel for providing high purity ppGpp for NMR and in vitro transcription studies as well as for offering continuing encouragement. We thank Richard Burgess and Nancy Thompson for providing RNA polymerase monoclonal antibodies, Arkady Mustaev for advice on cross-linking, and Sudha Chakrapani for initiating the trypsin-sensitivity studies. We also acknowledge Dr. Dinesh Sukumaran for executing NMR spectroscopy and analysis through the Chemistry Instrumentation Center, State University of New York at Buffalo. Finally, we thank Drs. Maria Laura Avantaggiati, Karen Fien, Gerald Koudelka, and Edward Niles for reading of and offering suggestions regarding this manuscript


    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM57189.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger On leave from the Limnological Institute of the Russian Academy of Sciences, Irkutsk, Russia. Current address: Dept. of Bacteriology, University of Wisconsin, Madison, WI 53706.

§ To whom correspondence should be addressed. Tel.: 716-829-2141; Fax: 716-829-2158; E-mail: vjh@buffalo.edu.

Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007184200


    ABBREVIATIONS

The abbreviations used are: RNAP, RNA polymerase; mAb, monoclonal antibody; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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