Co-overexpression of Escherichia coli RNA Polymerase Subunits Allows Isolation and Analysis of Mutant Enzymes Lacking Lineage-specific Sequence Insertions*

Irina ArtsimovitchDagger , Vladimir SvetlovDagger §, Katsuhiko S. Murakami, and Robert Landick||**

From the Dagger  Department of Microbiology, Ohio State University, Columbus, Ohio 43210, the  Laboratory of Molecular Biophysics, Rockefeller University, New York, New York 10021, and the || Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received for publication, November 3, 2002, and in revised form, December 23, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The study of mutant enzymes can reveal important details about the fundamental mechanism and regulation of RNA polymerase, the central enzyme of gene expression. However, such studies are complicated by the multisubunit structure of RNA polymerase and by its indispensability for cell growth. Previously, mutant RNA polymerases have been produced by in vitro assembly from isolated subunits or by in vivo assembly upon overexpression of a single mutant subunit. Both approaches can fail if the mutant subunit is toxic or incorrectly folded. Here we describe an alternative strategy, co-overexpression and in vivo assembly of RNA polymerase subunits, and apply this method to characterize the role of sequence insertions present in the Escherichia coli enzyme. We find that co-overexpression of its subunits allows assembly of an RNA polymerase lacking a 188-amino acid insertion in the beta ' subunit. Based on experiments with this and other mutant E. coli enzymes with precisely excised sequence insertions, we report that the beta ' sequence insertion and, to a lesser extent, an N-terminal beta  sequence insertion confer characteristic stability to the open initiation complex, frequency of abortive initiation, and pausing during transcript elongation relative to RNA polymerases, such as that from Bacillus subtilis, that lack the sequence insertions.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The amino acid sequences of the two largest subunits of cellular RNA polymerases (RNAPs),1 called beta ' and beta  in bacteria, are remarkably conserved among multisubunit RNAPs from eubacteria to the human homologues, RNAPI, RNAPII, and RNAPIII (1, 2). Recently determined crystal structures of yeast and bacterial RNAPs (3, 4) reveal the structural congruity responsible for this conservation, which extends far beyond the catalytically important residues (5-7). Representing the bulk of the enzyme, beta ' and beta  (and their yeast homologues, Rpb1 and Rpb2) form two halves of a crab claw-shaped molecule, in which the secondary structures of the homologous subunits are nearly identical within 25 Å of the active site (located at the internal junction between the claws). The divergence among RNAP's large subunits generally increases toward the exposed surface of the molecule and frequently is manifest as insertions of up to several hundred amino acids that are characteristic of different evolutionary lineages (3). This pattern of surface sequence variation in RNAPs, which in eukaryotes includes additional subunits bound at the enzyme's periphery, led to the idea that the conserved catalytic core of RNAP is adapted to various environments and cellular milieus by the addition of surface modules that interact with different regulatory factors (4, 7, 8).

In bacteria, the most studied examples of lineage-specific sequence insertions (SIs)2 occur in proteobacteria. In the proteobacteria, three easily recognizable insertions protrude from the surface of the enzyme (Figs. 1 and 6), two in beta  (SI1 between the conserved regions B and C and SI2 between regions G and H) and one in beta ' (SI3 between regions G and H) (9, 10). These insertions are absent in Gram-positive bacteria and in Deinococci, from which the bacterial RNAP structures have been determined; the Deinococci contain a different SI between conserved regions A and B of beta ' (3, 11). Both beta SI1 and beta SI2 tolerate insertions and partial deletions without affecting cell viability or in vitro RNAP activities (9, 12, 13). In contrast, partial deletions in beta 'SI3 are more deleterious; they reduce cell viability and confer defects in transcript cleavage and elongation, whereas the complete removal of beta 'SI3 inhibits assembly of the mutant beta ' into core RNAP (14).


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Fig. 1.   Linear maps of the beta ' and beta  subunits of RNAP. Conserved regions of beta ' (A-H) and beta  (A-I) are shown in pink and blue, respectively, with the structural domains of RNAP indicated by the wider segments of the subunit maps and labeled using the nomenclature of Darst and Kornberg (3, 4). SI indicates the positions of amino acid sequence insertions in E. coli RNAP that are not present in most bacterial species; respective boundaries are shown above each SI. The endpoints of deletions studied here are given at the ends of dotted lines (numbers listed are the last deleted residues). Black triangles marked with letters (a-k) indicate positions of other sequence insertions in various organisms. a (at beta 184), 178-aa insertion in Odontella sinensis chloroplast; b (at beta 391), 25-aa insertion in Xanthomonas campestris; c (at beta 774), 70-aa insertion in Leptospira biflexa; d (at beta 1135), 69-aa insertion in Wolbachia pipientis; e (at beta '144), 290-aa insertion in Thermotoga maritima; f (at beta '164), 115-aa insertion in Thermus aquaticus; g (at beta '204), 115-aa insertion in Aquifex aeolicus; h (at beta '618), 76-aa insertion in Mycoplasma pulmonis; i (at beta '869), 99-aa insertion in T. maritima; j (at beta '1161), 53-aa insertion in A. aeolicus; k (at beta ' 1302), 68-aa insertion in T. maritima. These sequence insertions represent only a subset of a growing number of apparent lineage-specific insertions intercalated between the conserved regions of beta  and beta ' subunits in bacterial RNAPs.

The observations that SIs can tolerate significant structural alterations without the loss of RNAP function led to them being described as dispensable (13-15). SIs in related bacteria exhibit somewhat greater variability than the sequences of full-length beta  and beta ' (e.g. SIs are ~70% conserved between Escherichia coli and Hemophilus influenzae and ~60% conserved between E. coli and Shewanella violacea, whereas the remaining parts of beta  and beta ' are ~85 and ~80% conserved between the two pairs, respectively). Nonetheless, their retention in distinct bacteria with significant sequence conservation suggests that they play some functional role. One role could be the recruitment of cellular regulatory proteins. For instance, beta SI1 appears to be the target of the phage T4 Alc termination factor (15). SI modules could also affect biochemical properties of RNAP more directly; both beta 'SI3 and beta SI1 are proposed to make downstream DNA contacts that explain the greater stability and longer downstream footprints of E. coli RNAP open complexes (relative to, for instance, Bacillus subtilis RNAP) (7, 16).

We became interested in the sequence insertions in proteobacterial RNAP as possible explanations for the inability of B. subtilis RNAP to recognize hairpin-dependent pause signals as well as the decreased open complex longevity and decreased abortive initiation of B. subtilis RNAP (17). To facilitate study of RNAPs with precise SI deletions, we developed a polycistronic co-overexpression system for E. coli RNAP that relies on T7 RNAP-dependent transcription of all three core RNAP subunit genes (rpoA, rpoB, and rpoC) on a single plasmid. To facilitate purification of the recombinant RNAPs, we appended an intein-chitin-binding protein (CBP) module to the beta ' C terminus and a hexahistidine-hemagglutinin tag to the N terminus of beta . This system allowed us to purify and test precise deletions of all three SIs in E. coli RNAP and provides a generally useful method for the study of mutant E. coli RNAP enzymes that avoids the loss of activity and assembly problems that sometimes arise with in vitro reconstitution methods (18, 19).

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Proteins-- Oligonucleotides (listed in Table I) were obtained from Operon Technologies (Alameda, CA); dNTPs were from U.S. Biochemical Corp.; NTPs were from Amersham Biosciences; [alpha -32P]CTP was from PerkinElmer Life Sciences; and other chemicals were from Sigma. Restriction and modification enzymes were obtained from New England Biolabs. Linear DNA templates for in vitro transcription were generated by PCR amplification and purified using reagents (cat. A7170) from Promega (Madison, WI).

Construction of Deletion Mutants-- To delete beta SI1, an rpoB fragment was PCR-amplified from pRL702 with oligonucleotides 343 and 3135. The PCR product was digested with NruI and cloned between two NruI sites of pRL702. To delete beta SI2 and beta 'SI3, site-directed PCR mutagenesis with two fully complementary oligonucleotides flanking the deleted fragment in pIA160 (beta ) or pRL663 (beta ') was performed. Each oligonucleotide (see Table I) annealed on both sides of the deletion, forcing the intervening fragment to loop out. The shortened rpoB fragment located between the unique NcoI and ClaI sites was sequenced, excised, and recloned into NcoI, ClaI-cut pIA160 (pIA319) or NcoI, and ClaI-cut pIA178 (pIA302). The shortened rpoC fragment located between the unique SalI and BspEI sites was sequenced, excised, and recloned into SalI-, BspEI-cut pRL663. To obtain overexpression plasmids encoding beta  Delta SI1 and SI2, the mutant rpoB fragments were transferred to the T7 RNAP-based expression plasmid pIA423 (Fig. 2) on an NcoI to SdaI fragment. To obtain overexpression plasmids encoding beta ' Delta (943-1130), the mutant rpoC fragments were transferred to the same expression plasmid on a BsmI to XhoI fragment.


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Fig. 2.   Purification of RNAP via the chitin protein binding tag. A, overexpression plasmid pIA423 contains the rpoA-rpoB-rpoC gene cassette flanked by a single T7 gene 10 promoter and T7 terminator. The rpoC gene is fused in-frame to sequences encoding the CBP/intein module. B, purification scheme with times indicated. C, purification of beta 'Delta SI3 mutant RNAP. 4-12% NuPAGE SDS-PAGE gel was loaded with (lanes 1 and 2) protein molecular weight standards (Bio-Rad) with sizes indicated on the left; lane 3, cleared lysate; lane 4, flow-through fraction; lane 5, bead fraction prepared by boiling the beads in SDS; lane 6, eluate from chitin column; lane 7, eluate from heparin column; lane 8, MRE600 holoenzyme prepared by the conventional method (27).

Design of the Polycistronic Overexpression Plasmid-- The original plasmid for co-overexpression of E. coli RNAP subunit genes, pIA423 (Fig. 2; GenBankTM accession number AF533984), contains a polycistronic operon rpoA-rpoB-rpoC*, flanked by a single T7 promoter and terminator sequences derived from pET21 (Novagen, Madison, WI); each ORF is preceded by a separate ribosome-binding site (Fig. 2). Introduction of a XhoI site at the 3'-end of the rpoC ORF resulted in the addition of two amino acids (LE) to the C terminus of the beta ' subunit, whereas a new EagI site in rpoB ORF is silent. In the absence of induction, expression is repressed by the product of the lacIq gene (carried on the same plasmid) through lacO positioned upstream of rpoA. To facilitate purification of the recombinant RNAP, the rpoC* fusion contains a CBP-intein module from pTYB3 plasmid (New England Biolabs) fused in-frame to the 3'-end of the rpoC ORF. Derivatives of pIA423 containing hexahistidine and a hemagglutinin epitope tag at the N terminus of beta  were prepared by ligation of NcoI-SdaI fragments from pIA160 and pIA178 (rpoBSF531) into the corresponding sites of pIA423.

The co-overexpression plasmid pIA423 was created in the following steps (see also Table I). pET21alpha was constructed to express rpoA under the control of an IPTG-inducible T7 RNAP promoter. The HindIII site in the pET21alpha rpoA gene was eliminated without altering the encoded amino acid sequence by site-directed PCR mutagenesis with primers 3683 and 3684 to yield pIA287. pIA287 was converted to pIA299 first by introducing a new sequence between the BamHI and NcoI sites downstream of rpoA. The resulting plasmid was then modified by insertion between its NcoI and HindIII sites of an NcoI to SbfI fragment from pRW408 carrying most of rpoB, followed by an SbfI to BsmI fragment from pNF1346, carrying the remainder of rpoB, the rpoBC intergenic region, and the first half of rpoC, followed by a BsmI to HindIII fragment from pRL663, carrying the remainder of rpoC, a C-terminal XhoI site, and a His6 tag. pIA423 was then created by insertion between XhoI sites of pIA299 of a XhoI-SalI-digested PCR product from pTYB3 (amplified with primers 3741 and 3742; Table I).

RNAP Purification-- Plasmids encoding variants of E. coli RNAP (wild type and mutants) were transformed into BL21 lambda DE3 (20). A single colony was inoculated into 500 ml to 2 liters of LB + 100 µg of ampicillin/ml at 37 °C and grown until apparent A600 reached 0.3-0.5, at which point protein production was induced by the addition of IPTG to 1 mM. Cells were grown for 3 h at 37 °C, rapidly chilled on ice, collected by centrifugation for 15 min at 5000 × g and 4 °C, and resuspended in 50 ml of column buffer (20 mM Tris, pH 7.9, 5% glycerol, 500 mM NaCl, 1 mM EDTA). Protease inhibitor mixture (Sigma catalog no. P8465) was then added as recommended by the manufacturer, and the cells were disrupted by sonication. The resulting lysate was cleared by centrifugation for 20 min at 27,000 × g and 4 °C and then filtered through the 0.4-µm syringe filter (Nalgene). Chitin beads (5 ml; New England Biolabs) were equilibrated with 10 volumes of column buffer in a 20-ml disposable column (Bio-Rad Econo-Pac), and cleared lysate was passed through the column by gravity flow, followed by 20 volumes of column buffer. To induce intein cleavage, the column was washed with 3 bed volumes of column buffer containing 50 mM DTT (to exchange buffer) and then incubated at 4-8 °C for 8-16 h (overnight). To elute the protein, column buffer (~4 ml) was added, and 0.2-ml fractions were then collected and tested for protein by Bradford assay and SDS-PAGE (4-12% NuPAGE gels; Invitrogen). Fractions containing RNAP were pooled, concentrated using Centriplus 100 or Ultrafree concentrators (Millipore Corp.) to 1-5 ml (depending on total RNAP recovered), and then dialyzed against loading buffer for heparin affinity or anion exchange chromatography. Chromatography was carried out using HiTrap columns and an Akta Prime low pressure chromatography system (Amersham Biosciences). For heparin affinity separation, samples were loaded onto the HiTrap Heparin HP column in 50 mM sodium phosphate (pH 6.9), 0.1 mM DTT buffer. For quaternary amine chromatography (Hi-Trap Q Sepharose Fast Flow), protein was loaded in 50 mM Tris-HCl (pH 8.0), 5% glycerol, 0.1 mM Na-EDTA, 0.1 mM DTT. Columns were washed with 10 column volumes of the loading buffer and eluted with a gradient (0-1.5 M) of NaCl in loading buffer over 20 column volumes. Elution peaks were identified by monitoring A280, and their contents were further characterized using SDS-PAGE. Fractions containing RNAP were pooled and dialyzed against storage buffer (10 mM Tris, pH 7.9, 50% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 0.1 M NaCl) for 12-14 h at 4 °C. Enzymes containing deletions in the beta  subunit were additionally purified by adsorption to Ni2+-nitrilotriacetic acid beads (Qiagen), followed by washing and imidazole elution prior to dialysis against storage buffer. Typical yields from 2-liter cultures were 0.5-2 mg of purified RNAP, depending on properties of the particular enzyme. For the wild type RNAP, versions containing or lacking hexahistidine tags on beta  were purified and found to behave indistinguishably in the assays used here. B. subtilis core RNAP (21) and sigma 70 (22) were purified as described. Wild type and mutant RNAP holoenzyme (core beta 'beta alpha 2 plus sigma 70) was prepared by incubating a 5-fold molar excess of sigma 70 with core enzyme for 30 min at 30 °C.

Open Complex Longevity Assays-- Linear DNA template (40 nM) carrying the T7A1 promoter (pIA171) (23) was incubated with 50 nM RNAP holoenzyme (with sigma 70) for 15 min at 37 °C in 50 µl of transcription buffer (20 mM Tris·HCl, 20 mM NaCl, 10 mM MgCl2, 14 mM 2-mercaptoethanol, 0.1 mM EDTA, pH 7.9). Heparin was added to 14 µg/ml to sequester any free RNAP (at time 0); reaction aliquots were withdrawn at various times and combined with nucleotide substrates (ApU to 150 µM; ATP, GTP, and CTP to 2.5 µM; and 5 µCi of [alpha -32P]CTP). Following a 10-min incubation at 37 °C, reactions were quenched by the addition of an equal volume of STOP buffer (10 M urea, 20 mM EDTA, 45 mM Tris borate, pH 8.3). For the zero time point, an aliquot was taken prior to heparin addition and incubated with substrates.

Abortive Initiation Assays-- Reactions were assembled on ice in 50 µl of transcription buffer with ApU at 150 µM, ATP and CTP at 20 µM, and 10 µCi of [alpha -32P]CTP (3000 Ci/mmol). Linear pRL550 (24) DNA template was at 40 nM, and RNAP holoenzyme containing sigma 70 was at 50 nM. Transcription was initiated by shifting samples to 37 °C. Samples (5 µl) were removed at times indicated in the legend to Fig. 3B and after a final 5-min incubation with 500 µM each NTP (chase) and quenched by the addition of STOP buffer.


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Fig. 3.   Transcription initiation of E. coli, B. subtilis, and Delta SI mutant RNAPs. A, open complex stability in vitro. Plot of open complexes formed at T7 A1 promoter remaining as a function of time after the addition of heparin (see "Experimental Procedures"). B. subtilis (BS), E. coli (EC), and mutant holoenzymes lacking SIs were prepared with E. coli sigma 70 initiation factor. The half-lives of open complexes were estimated by nonlinear regression of the amount of transcript produced as a function of the time elapsed between the heparin addition and NTP addition to preformed open complexes. Relative to the amount of transcript produced by wild type RNAP with no heparin added, the other RNAPs yielded 0.2, 0.6, 0.5, and 1.1 equivalents of transcript in the absence of heparin (B. subtilis, Delta beta SI1, Delta beta SI2, and Delta beta 'SI3, respectively; adjusted to 1.0 for the zero time point). B, electrophoretic separation of [alpha -32P]CTP-labeled RNAs formed on the pRL550 template as a function of time (during 5-, 10-, 20-, 30-, and 60-min incubation with substrates; see "Experimental Procedures"). Positions of the run-off RNA and the predominant abortive product, ApUpC, are indicated. The ratio of ApUpC trimer to the full-length RNA (corrected for the number of C residues) is shown below for each enzyme.

Single Round Pause Assays-- Linear DNA template encoding the his pause signal was generated by PCR amplification of pIA171 (23). Halted A29 elongation complexes were formed during a 15-min incubation of 40 nM DNA template and 50 nM RNAP holoenzyme at 37 °C in 50 µl of transcription buffer in the absence of UTP, with ApU at 150 µM, ATP and GTP at 2.5 µM, and CTP at 1 µM, with 32P derived from [alpha -32P]CTP (3000 Ci/mmol). Transcription was restarted by the addition of 20 µM GTP; 150 µM ATP, UTP, and CTP; and heparin to 100 µg/ml. Samples were removed at the times listed in the figure legends and after a final 5-min incubation with 250 µM each NTP (chase) and were quenched as above.

Sample Analysis-- Samples were heated for 2 min at 90 °C and separated by electrophoresis in denaturing acrylamide (19:1) gels (7 M urea, 0.5× TBE; 8% for pause assays, 15% for abortive initiation and open complex stability assays). RNA products were visualized and quantified using a PhosphorImager and ImageQuant Software (Amersham Biosciences). Pause half-life (the time during which half of the complexes reenter the elongation pathway) was determined by nonlinear regression analysis as described previously (25).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Co-overexpression of RNAP Subunits Allows Study of the Role of Proteobacterial Sequence Insertions-- To allow facile expression, assembly, and purification of poorly assembled or toxic mutant RNAPs, such as beta 'Delta SI3, we designed a vector that expresses alpha , beta , and beta ' polypeptides from a single T7 promoter; the beta ' subunit is fused to the CBP tag (see "Experimental Procedures"). We predicted that expressing the mutant core RNAP from this plasmid would facilitate its assembly in vivo and would allow purification via the CBP tag (Fig. 2). This approach has several advantages. First, only the fully assembled core (alpha 2beta beta ') RNAP is purified because beta ' recruitment is the last step in the assembly pathway (26) and because the expression levels from this vector follow the assembly pathway of RNAP (alpha >beta >beta '; data not shown). Second, intein-mediated cleavage removes the CBP tag to release the assembled RNAP from the matrix; therefore, the purified enzyme does not carry additional protein segments. Third, the entire purification can be completed quickly, is relatively inexpensive, and yields RNAP that is sufficiently pure for in vitro transcription (Fig. 2). Fourth, the plasmid-encoded beta  and beta ' subunits assemble preferentially with each other (and not with the chromosomal subunits; data not shown), allowing combination of substitutions in different subunits and more homogenous population of purified RNAPs. To purify RNAP with altered beta  subunit, we also constructed a version of the co-overexpression plasmid with a hexahistidine tag at the N terminus of beta  (see Table I).


                              
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Table I
Strains, plasmids, DNA oligonucleotides, and transcription templates

Using the co-overexpression plasmid, we were able to obtain active Delta beta 'SI3 RNAP (Fig. 2) as well as RNAPs with precise excisions of beta SI1 or beta SI2. We found that RNAP eluted directly from chitin beads was suitable for in vitro transcription but sometimes exhibited reduced activity relative to RNAP purified by conventional methods (27). We hypothesized that this low activity arose from residual binding of nucleic acids to the RNAPs; the addition of one chromatography step on heparin- or quarternary amine-Sepharose yielded overexpressed RNAP of an activity and purity comparable with that obtained by conventional purification (Fig. 2 and data not shown).

When the beta 'Delta SI3-intein-CBP fusion was expressed from a T7 promoter plasmid that did not encode the other RNAP subunits, essentially all tagged beta ' was found in inclusion bodies (data not shown). Therefore, co-overexpression with alpha , beta , and beta ' subunits facilitates RNAP assembly. Perhaps translation of beta  and beta ' from the same mRNA facilitates proper interaction, or the elevated level of the initially formed alpha 2beta complex simply allows plasmid-encoded beta ' to compete effectively with chromosomally encoded beta ' for assembly.

beta 'SI3 Stabilizes Open Initiation Complexes-- Using RNAPs with precise SI deletions obtained by co-overexpression, we first tested the effects of the E. coli SIs on open initiation complexes. Fully mature E. coli open complexes, which form on many but not all cellular promoters, exhibit extended contacts of RNAP with DNA from ~-55 to ~+20 relative to the transcription start site and melting of the DNA duplex between ~-12 and ~+2. These E. coli open complexes are long lived and collapse back to closed complexes at rates of <0.01 s-1 (28-31). In contrast, B. subtilis RNAP, which lacks insertion sequences, forms open complexes that are in rapid equilibrium with closed promoter complexes (32, 33). In B. subtilis RNAP open complexes, the DNA downstream of the start site is not efficiently protected against DNase I digestion (32, 34), and the melted region is shortened in the downstream direction (35). Interestingly, a deletion variant of the E. coli beta  subunit that includes part of beta SI1 but extends beyond its boundaries (Delta 186-433) forms open complexes that (at 37 °C) exhibit a melting pattern similar to those formed by the B. subtilis enzyme (35).

To measure the relative stability of open complexes formed by wild type or Delta SI RNAPs, we challenged open complexes formed on the T7 A1 promoter with the polyanion heparin. Although for many promoters heparin binds only free RNAP that is in equilibrium with closed initiation complexes and has no effect on open complexes, heparin directly attacks open complexes formed at the T7 A1 promoter and displaces RNAP from the promoter DNA in addition to binding free RNAP (36). Upon heparin addition to 14 µg/ml, transcriptionally competent open complexes (as assayed by the addition of NTPs and formation of an RNA transcript at different time intervals following heparin challenge) disappeared at a pseudo-first order rate of 0.006 s-1 for wild type and Delta beta SI2 RNAP, about twice as fast for Delta beta SI1 RNAP, and about 10 times faster for Delta beta 'SI3 RNAP and B. subtilis RNAP (Fig. 3A). We conclude that the characteristic instability of B. subtilis open complexes is mimicked by Delta beta 'SI3 E. coli RNAP and that beta SI1, but not beta SI2, also contributes to the stability of E. coli open complexes.

beta 'SI3 Promotes Abortive Initiation-- A second property that distinguishes B. subtilis and E. coli RNAPs is an ability of B. subtilis RNAP to escape promoters at which E. coli RNAP is trapped in abortive synthesis. We found this previously using a variant of the T7 A1 promoter whose initially transcribed sequence (5'-AUCCCACACC ... versus wild type 5'-AUCGAGAGGG ... ) appears to act cooperatively with strong contacts of E. coli RNAP to promoter DNA to trap the enzyme in abortive synthesis (24); B. subtilis RNAP is able to escape from the mutant T7 A1 promoter with relatively few abortive products made, and the patterns of abortive products also differ between the B. subtilis and E. coli enzymes (17).

To investigate whether any of the sequence insertions in E. coli RNAP might explain its characteristically poor escape from the mutant T7 A1 promoter, we measured the abortive to productive RNA product ratios for the various RNAPs on this promoter (Fig. 3B). Interestingly, deletion of beta 'SI3 reduced the abortive to productive product ratio, although only to a level still ~2-fold greater than found for B. subtilis RNAP. However, deletion of either SI in beta  dramatically increased the ratio. Thus, the E. coli sequence insertions profoundly affect promoter escape, although in different directions; beta SI1 and beta SI2 promote escape, whereas beta 'SI3 inhibits escape.

The patterns of abortive products produced also are revealing (Fig. 3B). Both Delta beta SI1 and Delta beta SI2 RNAPs produce an altered pattern of abortive products relative to wild type or beta 'Delta SI3 RNAPs (compare the 60-min lanes for each RNAP in Fig. 3B). However, although beta 'Delta SI3 RNAP exhibited increased promoter escape, like B. subtilis RNAP, it does not recapitulate the distinctive pattern of B. subtilis abortive products. We conclude that the absence of the Proteobacterial sequence insertions substantially, although not completely, accounts for the distinctive initiation properties of B. subtilis RNAP.

beta 'SI3 Slows Escape from Pause Sites-- Another key difference between the E. coli and B. subtilis RNAPs is their responses to certain hairpin-dependent pause sites. Both enzymes can recognize hairpin-independent pause signals as well as a B. subtilis P RNA pause site at which a nascent RNA hairpin 12 nt upstream from the pause is important in the presence, but not in the absence, of NusA (17). However, B. subtilis RNAP fails to recognize the his pause site, at which a nascent RNA hairpin 11 nt upstream from the pause site contributes a factor of 5-10 to the delay in pause escape through an interaction with the beta  flap domain of the E. coli RNAP (17, 37).

The structural basis for this difference is not known; B. subtilis RNAP might be unable to respond to the RNA hairpin formation or to other components of this signal (the downstream DNA, the 3'-proximal RNA, and the nucleotides in the active site) that also slow pause escape independently of the RNA hairpin. We previously found that replacement of the E. coli beta  flap with the corresponding fragment of the B. subtilis beta  did not alter pausing (17), suggesting that the difference in their response to the his pause signal lay elsewhere. Thus, the sequence insertions in E. coli RNAP were the next logical possibility.

To ask if the E. coli sequence insertions play a role in pause site recognition, we first tested pausing on the his pause template. We found that, unlike B. subtilis RNAP, Delta beta SI1, Delta beta SI2, and Delta beta 'SI3 RNAPs all recognized the his pause site (Fig. 4). However, Delta beta 'SI3 RNAPs escaped from the pause site ~5 times faster than wild type and exhibited an ~2-fold decrease in the efficiency of pause site recognition. To ask if the residual pausing by Delta beta 'SI3 RNAPs was still hairpin-dependent, we tested the effect of an antisense oligonucleotide that pairs to nascent pause RNA including the two 5'-most nt of the pause hairpin stem (Fig. 4A) and that eliminates the 5-10-fold contribution of the hairpin to pausing by disrupting its structure (23). If beta 'SI3 somehow mediated the effect of the pause hairpin, despite their locations on opposite sides of the paused TEC, we would expect the antisense oligonucleotide to have little effect on pausing by Delta beta 'SI3 RNAP. However, the antisense oligonucleotide reduced the pause half-life of Delta beta 'SI3 RNAP by a factor of ~3 (Fig. 4D), suggesting that beta 'SI3 contributes to pausing through interactions that are largely independent of the hairpin interaction. We verified this conclusion by testing the effect of Delta beta 'SI3 on pausing at the hairpin-independent ops pause site, where the deletion had effects on half-life and efficiency nearly identical to the effects observed at the his pause site (Fig. 5). However, Delta beta 'SI3 did not fully recapitulate the pausing behavior of B. subtilis RNAP, which bypasses the his pause site without apparent delay (Fig. 4B) and transcribes through the ops pause site even more rapidly than does Delta beta 'SI3 RNAP (17).


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Fig. 4.   Pausing at the his pause site. A, schematic of his pause template showing sequence in the region of the his pause. B, electrophoretic separation of RNAs formed on the his pause template as a function of time. For each panel, samples were taken at 5, 10, 20, 30, 45, 60, 120, and 240 s; the last lane in each panel (chase) contains sample that was incubated for an additional 5 min with 250 µM each NTP after completion of the time course. Preformed [alpha -32P]CMP-labeled A29 complexes were incubated with 20 µM GTP, 150 µM ATP, CTP, and UTP on the his pause template. Prominent transcripts are pause RNA transcript (hisP; 71 nt) and run-off RNA transcript. C, a plot of pause RNA fraction versus time for the wild type E. coli RNAP and beta 'Delta SI3 enzyme. The reactions were performed as in A, except that the chase was at 10 µM GTP to allow for more accurate measurements of the pause efficiency and half-life. D, effect of the antisense oligonucleotide (shown in A) competition on the half-life of his paused transcription complexes.


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Fig. 5.   Pausing at the ops pause site. A, schematic of ops pause (underlined) template showing sequence in region of the ops pause and electrophoretic separation of RNAs formed on the ops pause template as a function of time. Preformed [alpha -32P]CMP-labeled G37 complexes were allowed to elongate at 10 µM GTP, 150 µM ATP, CTP, and UTP, and the samples were taken at 5, 10, 15, 20, 40, 90, and 180 s. The last lane in each panel contains chase sample that was treated as described in the legend to Fig. 4. Prominent transcripts are pause RNA transcript (opsP; 43 nt) and run-off RNA transcript (RO). B, the fractions of paused RNA were plotted against time; pause half-lives and pause efficiencies were determined as described under "Experimental Procedures" and in Ref. 25. C, a plot of pause RNA fraction versus time for the wild-type E. coli RNAP and beta 'Delta SI3 enzyme determined from the samples shown in panel A.

We conclude that beta 'SI3 plays a central role in the strong pausing behavior of E. coli RNAP but that differences between E. coli and B. subtilis RNAP in addition to beta 'SI3 must contribute to reduced pausing by the latter enzyme. Interestingly, a partial deletion in beta 'SI3 (Delta 1091-1130) displays the opposite effect on pausing as the precise beta 'SI3 deletion that we studied; i.e. it increases rather than decreases pausing (14). We return to the implications of this discrepancy under "Discussion."

Multiple Deletions of E. coli RNAP Sequence Insertions-- Given the distinct effects of beta SI1 and beta 'SI3 on open complex longevity, abortive initiation, and pausing, we wondered how these effects would combine in an enzyme lacking both SIs. All attempts to produce such an enzyme failed, however (data not shown). Derivatives of pIA423 that expressed beta Delta SI1 and beta 'Delta SI3 never yielded assembled RNAPs bearing the deletions, even when we included on the co-expression plasmid rpoZ, the gene encoding the dispensable RNAP subunit omega  that is reported to promote RNAP assembly (38).

Deletions of the Sequence Insertions Impair Growth of Bacteria-- Finally, we wished to test the dispensability of sequence insertions in RNAP for bacterial growth. Studies conducted prior to the availability of high resolution RNAP structures on RNAPs lacking portions of beta SI1, beta SI2, or beta 'SI3 led to the idea that these were dispensable regions, at least for core RNAP functions (9, 12-14, 39). Now that we could define precise deletions of the sequence insertions and express functional enzymes containing these deletions in vivo, we wished to revisit this idea and ask to what extent the sequence insertions are required for bacterial growth.

To test the requirement for sequence insertions for bacterial growth, we expressed the wild type or Delta SI subunits from plasmids on which their expression either singly or in combination with the other RNAP subunits could be regulated by IPTG and lac repressor encoded on the same plasmids (Table I). We transformed the plasmids into strains that express wild type RNAP from the chromosome (rich medium, wild type subunit from chromosome expressed) (Table II) or in which expression of the corresponding subunit from the chromosome could be inactivated at 39 or 42 °C (for rpoC and rpoB, respectively; wild type subunit from chromosome not expressed, Table II).


                              
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Table II
Effect of expressing plasmid-encoded RNAP subunits lacking sequence insertions on the growth of E. coli

We found that plasmid-encoded expression of mutant beta  subunit lacking SI1 or SI2 in the presence of wild type beta  encoded on the chromosome had little effect on cell growth However, beta Delta SI1 was unable to substitute for wild type beta  at 42 °C, and it compromised growth at 30 °C when it contained a rifampicin-resistant amino acid substitution (SF531) not present in the chromosomal copy of rpoB and when rifampicin was added to the medium (Table II). Growth on rifampicin at 30 °C was not restored when beta SF531Delta SI1 was co-expressed with beta ' and alpha  (conditions that yielded assembled beta Delta SI1 for purification). Thus, beta Delta SI1 appears to be incorporated into RNAP but unable to support cell growth. beta Delta SI2 was able to support growth on rich medium even at 42 °C or in the presence of rifampicin at 30 °C when combined with SF531. However, beta SF531Delta SI2 was unable to support growth on minimal medium containing rifampicin, when expressed either singly or together with alpha  and beta '. Thus, both sequence insertions in E. coli beta  appear necessary for growth on minimal medium, suggesting that they play some regulatory role in RNAP function that becomes essential in stringent growth conditions.

We found that beta 'SI3 also was required for growth in some conditions; however, the requirements for this sequence insertion are complex. A beta 'Delta SI3 plasmid could not be transformed into RL602 (Table I) in which expression of chromosomally encoded beta ' is temperature-sensitive, even in the absence of IPTG (probably because low, uninduced expression of beta 'Delta SI3 produced too much defective enzyme when combined with the lowered expression of beta ' in RL602). The beta 'Delta SI3 co-expression plasmid could be transformed into the conditional rpoC strain but blocked growth when induced at the permissive temperature and failed to support growth at the nonpermissive temperature. Although induction of beta 'Delta SI3 in a strain producing wild type beta ' lowered plating efficiency by 102, it actually raised plating efficiency by >103 relative to wild type beta ', when plated in the presence of an antibiotic (microcin J25) that inhibits RNAP upon binding at a site near the beta 'SI3 in the RNAP secondary channel (62). Co-expression beta 'Delta SI3 with beta  and alpha  was not inhibitory in a strain expressing wild type RNAP (Table I) and conferred nearly 100% plating efficiency in the presence of microcin J25 (62). However, the growth rate of these colonies in the presence of microcin J25 is drastically slower than strains carrying the prototypical microcin J25 resistance mutation (rpoCT931I). On balance, it appears that beta 'SI3 is essential for normal growth of E. coli but that, at least in the presence of microcin J25, RNAP lacking beta 'SI3 can still support slow growth on rich medium.

We conclude that sequence insertions, although dispensable for basic RNAP function at all stages of the transcription cycle, nonetheless are important to growth of the bacterium. This may reflect effects of the sequence insertions on the basic steps in transcription by RNAP that become crucial for expression of certain genes or in some growth conditions but could also reflect roles in yet undescribed but essential interactions of transcription factors similar to the previously described interaction of the T4 Alc protein with beta SI1 (15).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have described a co-overexpression system for mutant E. coli RNAPs and its use to investigate whether sequence insertions in the beta  and beta ' subunits could explain the different initiation and elongation properties of E. coli RNAP compared with B. subtilis RNAP, which lacks these insertions. We found that co-overexpression facilitates assembly of otherwise difficult to assemble or toxic mutant RNAPs and that the sequence insertions, most notably beta 'SI3, confer some of its distinctive biochemical properties on E. coli RNAP and partially account for its differences from B. subtilis RNAP. We will discuss the implications of these findings for the study of mutant RNAPs, the function of the E. coli sequence insertions with focus on beta 'SI3, and the evolution of sequence insertions in RNAP.

An RNAP Overexpression System That Promotes Efficient Assembly-- Previous studies of toxic RNAP mutants have relied either on conditional expression of a tagged mutant subunit followed by tag affinity separation from wild type RNAP (or tagging and removal of the wild type enzyme from the RNAP preparation) (40) or in vitro reconstitution of RNAP by gradual renaturation of mixtures of the denatured subunits (19, 41-43). beta 'Delta SI3 illustrates the limitation of these approaches. When expressed as an individual subunit in vivo, beta 'Delta SI3 fails to out-compete wild type beta ' for assembly into useful amounts of mutant enzyme; when used in in vitro reconstitution protocols, it fails to assemble into RNAP (see "Results" and Ref. 14).

Co-overexpression of wild type and mutant tagged subunits solved these problems for beta 'Delta SI3 and has allowed us to isolate substantial quantities of many mutant RNAPs, including notably toxic mutants such as the beta Delta (900-909) flap mutant RNAP (37). Thus, the co-overexpression plasmid provides a powerful tool for genetic and biochemical analysis of RNAP. The resulting enzymes are substantially pure after two steps, primarily because chitin affinity chromatography is much more selective than the hexahistidine-Ni2+-nitrilotriacetic acid-agarose method that has been widely used to date (44), and has the advantage of being assembled by the normal pathway in vivo. We anticipate that the co-overexpression plasmid will also prove useful for selection of RNAP suppressor mutants of inviable amino acid substitutions in the enzyme. Finally, because RNAP is pure enough for in vitro transcription assays after just the single chitin column step, the method should facilitate rapid screening of sets of closely related alterations to RNAP.

In using the co-overexpression method for mutant RNAP assembly and purification, we encountered a few problems that require care to avoid. First, the chitin matrix apparently can break down when batch binding and elution are used, which prevents retention of the tagged enzyme on the solid chitin matrix. We used slow passage of lysates through a column of chitin matrix to avoid this problem. Second, the efficiency of DTT-mediated cleavage of the Sce VMA intein that connects beta ' to the CBP was usually less than 100%, and both the binding capacity and the cleavage efficiency appeared to vary among lots of chitin matrix. It is advisable to test new batches of matrix before committing valuable samples; some loss of material due to inefficient cleavage appears unavoidable. Third, we sometimes observed loss of expression of one or more of the RNAP subunits when the co-expression plasmid was maintained in recA+, T7 RNAP-expressing strains, apparently due to plasmid rearrangements, mutations, or recombination with the chromosomal copies of rpoA, rpoB, or rpoC. The last problem was substantially eliminated by maintaining the plasmids in recA strains such as DH5alpha except during expression. Finally, we emphasize that not all RNAP mutants can be successfully recovered by the co-overexpression approach. For instance, a deletion slightly larger than beta 'Delta SI3 (Delta 932-1137 versus Delta 943-1130 in beta 'Delta SI3) failed to assemble and yielded only insoluble beta 'Delta (932-1137) aggregates in vivo.

Role of beta 'SI3 in RNAP Function-- Although both SI1 and SI2 in beta  exhibited some effects on the biochemical properties of RNAP (and were essential for full viability in vivo), the most dramatic effects on RNAP properties were observed for beta 'SI3. beta 'SI3 profoundly affected initiation and pausing by E. coli RNAP, in both cases accounting for many but not all of its differences from B. subtilis RNAP. We will consider the effects on initiation and pausing separately, although the underlying changes in RNAP's properties could be related.

beta 'SI3 stabilized open complexes formed at the T7 A1 promoter against disruption by heparin. This stabilization could reflect either direct contact with the downstream DNA, as previously proposed (16), or it could reflect indirect stabilization of the open complex if folding of beta 'SI3 is coupled to structural transitions in RNAP that occur upon open complex formation. Although direct DNA interaction is an attractive hypothesis, no cross-linking studies to date unambiguously document interaction of beta 'SI3 with the downstream DNA in open complexes. In the recently published crystal structure of T. thermophilis RNAP, the loop in beta 'G to which beta 'SI3 connects is positioned near the downstream DNA channel (11), also favoring its possible interaction with downstream DNA. However, this structure is of an enzyme that lacks beta 'SI3 and was obtained in the absence of DNA. The beta 'G loop is poorly ordered in both the yeast RNAPII and T. aquaticus RNAP crystal structures and was found in different conformations in the ordered portions of the two structures, suggesting that it is capable of significant motion. Further, beta 'SI3 itself is not visible in a recent electron microscopy crystal structure of E. coli RNAP, suggesting that it is even more mobile than the beta 'G loop (10). Thus, beta 'SI3 folding could be coupled either directly or indirectly to interactions of RNAP with downstream DNA. Many DNA-binding proteins undergo folding transitions in protein segments distant from the actual DNA binding surface (45). These indirect but coupled protein folding events are thought to contribute significantly to the avidity of protein-DNA interactions. Thus, stabilization of open complexes by beta 'SI3 could arise not via its direct interaction with the downstream DNA but via an indirect coupling of its folding to open complex formation. Further study will be required to distinguish these possibilities.

Both the direct and indirect explanation of beta 'SI3 function also could apply to its effect on transcriptional pausing. Both formation of paused transcription elongation complexes and their slow escape are enhanced by an interaction of downstream DNA with RNAP (46-49). Thus, beta 'SI3 could strengthen the pause-enhancing downstream DNA interaction either directly or by the indirect mechanism described above. Interestingly, the effects on pausing of deleting beta 'SI3 are quite similar to the effects of deleting the beta ' jaw domain of E. coli RNAP, for which a variety of data suggest a direct DNA interaction (49).

In the case of pausing, however, a third explanation is possible. beta 'G, in which beta 'SI3 is inserted, appears to cross-link to the RNA 3' nt in the his paused transcription complex3 as well as to the RNA 3' nt in arrested complexes (50) and in certain artificial transcription complexes that may mimic paused transcription complexes (51). Further, amino acid substitutions in beta 'G, near the site of beta 'SI3 insertion, strongly affect chain elongation and transcriptional pausing and termination (40, 51). If movements of the beta 'G loop are involved in transcriptional pausing, it could readily explain why deletion of beta 'SI3 has such a strong effect on pausing. This also might explain why a partial deletion in beta 'SI3 (Delta 1091-1130) as well as monoclonal antibody binding to beta '1091-1130 in wild type RNAP greatly increase pausing (14), whereas complete deletion of beta 'SI3 (Delta 943-1130) decreases pausing (Figs. 4 and 5). By altering the structure of beta 'SI3, both beta 'Delta (1091-1130) and antibody binding could interfere with movements of the beta 'G loop necessary for rapid nucleotide addition, whereas removal of beta 'SI3 could favor such movements.

Roles of beta SI1 and beta SI2 in Abortive Initiation-- beta SI1 and beta SI2 both exhibited effects on initiation complexes, either in open complex longevity, abortive initiation, or both. Interestingly, both SIs increased abortive initiation significantly, although they decreased open complex longevity modestly (beta SI1) or had no effect on it (beta SI2). This suggests that RNAP must be exceptionally sensitive to changes in its structure during abortive initiation. This could be related to the sequential rearrangements of contacts between core RNAP and sigma  that are thought to occur during the initial stages of RNA synthesis (52). Perhaps beta SI1 and beta SI2 somehow facilitate the core-sigma rearrangement during initial transcript synthesis. Alternatively, they could indirectly affect the conformation of the main channel of RNAP such that their removal increases the rate of abortive transcript release.

Sequence Insertions Are Ubiquitous in Bacterial RNAPs-- Finally, we note that sequence insertions are not limited to those found in the RNAPs from enteric and thermophilic bacteria. A search of sequences now available for beta  and beta ' subunits in bacteria revealed that sequence insertions are ubiquitous and that the positions of these insertions are not restricted to those observed in the enteric and thermophilic bacteria (Figs. 1 and 6). Rather, the sequence insertions differ in size, sequence, and location but are predicted to be surface-exposed (Fig. 6). Such a distribution of sequence insertions is consistent with the idea that they confer species-specific properties on core RNAP, either by modulating its enzymatic activity directly or through interactions with transcription factors. The relatively low degree of divergence among SIs in closely related proteobacterial species (see Introduction) also lends support to this idea.


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Fig. 6.   Locations of sequence insertions in E. coli RNAP and in other bacterial RNAPs. E. coli RNAP is depicted in paired stereo images as observed by electron crystallography by Darst et al. (10) using the atomic coordinates of T. aquaticus RNAP adjusted to match the electron density of E. coli RNAP (the main channel of RNAP is more open in the E. coli structure than in the T. aquaticus structure). Pink, beta '; cyan, beta ; white, alpha  and omega ; magenta, Mg2+ at catalytic center. (Note that the omega  subunit is included based on the T. aquaticus RNAP structure; it was not seen in the E. coli RNAP electron microscopy structure.) The locations of beta SI1 and beta SI2 are depicted in red space fill (the density for beta SI1 represents only a portion of the space expected to be occupied by this insertion; the remainder is disordered in the electron crystal structure (10)). The location of beta 'SI3, which is completely disordered and not visible in the electron crystal structure, is depicted based on its junction to the beta 'G loop as red balls. The locations of sequence insertions shown in Fig. 1 are marked by green letters and lines. A, view into the main channel of RNAP with the enzyme oriented for transcription from left to right. B, view into the NTP entry channel of RNAP.

This pattern of surface-exposed sequence insertions is reminiscent of the small subunits of eukaryotic RNAPs, which also are surface-exposed (at least in RNAPII); vary in composition to some extent among RNAPI, RNAPII, and RNAPIII; and can affect the biochemical properties of the enzyme yet are dispensable for core function. The RNAPII subunits 4 and 7, for instance, are readily dissociated from RNAP and affect its biochemical properties but are dispensable for nucleotide addition (53, 54); deletion of RNAPII subunit 9, like deletions in beta 'SI3, affects responses of the RNAPs to their respective RNA cleavage factors, GreB (14) and TFIIS (55). Perhaps the bacterial sequence insertions are like these small eukaryotic RNAP subunits, conferring distinctive properties on the RNAPs of different bacterial species much as the small RNAP subunits may confer distinctive properties on RNAPI, RNAPII, and RNAPIII.

    ACKNOWLEDGEMENTS

We thank Seth Darst for help with molecular modeling and Josefine Ederth, Kati Geszvain, Rachel Mooney, Ruth Saecker, and Kesha Toulokhonov for helpful comments on the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM38660 and Department of Agriculture Grant WIS04022 (to R. L.).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.

§ Supported by National Institutes of Health Grant GM 28575 (to Richard R. Burgess, Department of Oncology, University of Wisconsin, Madison, WI).

** To whom correspondence should be addressed. Fax: 608-262-9865; E-mail: landick@bact.wisc.edu.

Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M211214200

2 Sequence insertions in E. coli RNAP previously have been designated DR, to signify dispensable regions (see Ref. 7 and references therein). We prefer the less restrictive abbreviation SI to signify lineage-specific sequence insertions, because E. coli cells containing SI deletions are inviable in at least some conditions and exhibit altered biochemical properties, suggesting that these features of RNAPs play important functional roles. Further, the SI nomenclature is applicable to other bacterial RNAPs for which functional tests have not been performed.

3 K. Toulokhonov and R. Landick, unpublished results.

    ABBREVIATIONS

The abbreviations used are: RNAP, RNA polymerase; SI, lineage-specific sequence insertion; ORF, open reading frame; CBP, chitin-binding protein; nt, nucleotide(s); aa, amino acid(s); IPTG, isopropyl-1-thio-beta -D-galactopyranoside; DTT, dithiothreitol.

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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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