JBC Transcription and Nuclear Factor Monoclonals

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J Biol Chem, Vol. 273, Issue 38, 24912-24920, September 18, 1998


Mutations in and Monoclonal Antibody Binding to Evolutionary Hypervariable Region of Escherichia coli RNA Polymerase beta ' Subunit Inhibit Transcript Cleavage and Transcript Elongation*

Natalya ZakharovaDagger , Irina Bass§, Elena Arsenieva§, Vadim Nikiforov§, and Konstantin SeverinovDagger parallel

From the Dagger  Waksman Institute, Piscataway, New Jersey 08854, the  Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, and the § Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

A 190 amino acid-long region centered around position 1050 of the 1407-amino acid-long beta ' subunit of Escherichia coli RNA polymerase (RNAP) is absent from homologues in eukaryotes, archaea and many bacteria. In chloroplasts, the corresponding region can be more than 900 amino acids long. The role of this hypervariable region was studied by deletion mutagenesis of the cloned E. coli rpoC, encoding beta '. Long deletions mimicking beta ' from Gram-positive bacteria failed to assemble into RNAP. Mutants with short, 40-60-amino acid-long deletions spanning beta ' residues 941-1130 assembled into active RNAP in vitro. These mutant enzymes were defective in the transcript cleavage reaction and had dramatically reduced transcription elongation rates at subsaturating substrate concentrations due to prolonged pausing at sites of transcriptional arrest. Binding of a monoclonal antibody, Pyn1, to the hypervariable region inhibited transcription elongation and intrinsic transcript cleavage and, to a lesser degree, GreB-induced transcript cleavage, but did not interfere with GreB binding to RNAP. We propose that mutations in and antibody binding to the hypervariable, functionally dispensable region of beta ' inhibit transcript cleavage and elongation by distorting the flanking conserved segment G in the active center.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

DNA-dependent RNA polymerases from eubacteria share a common subunit composition (1). The core RNAP1 enzyme (subunit composition alpha 2beta beta ') is catalytically proficient but is unable to initiate transcription on promoters. Binding of a sigma  subunit converts the core enzyme into a holoenzyme, which can recognize a specific set of promoters (2). The beta  and beta ' subunits together constitute more than 80% of the core RNAP mass and jointly form the catalytic center of the enzyme (3, 4). RNAPs from eukaryotes and archaea have subunits that are homologous to beta  and beta ' of eubacterial enzymes (5-7). The evolutionary conservation within the beta /beta ' lineages is limited to relatively short segments of primary sequence; each subunit has 8-10 highly conserved segments. The amino- to carboxyl-terminal order of the conserved segments is invariant.

The spacing between the conserved segments can vary even when subunits from closely related species are compared, due to an accumulation of insertions and deletions. There are several reasons why studies of such evolutionarily variable regions can shed light on RNAP structure and function. First, these regions may form docking sites for species-specific regulators of transcription (8, 9). Second, variable regions are likely to be surface-exposed and can therefore be used for affinity tagging of RNAP and transcription complexes (10). Third, variable regions often tolerate splits, allowing preparation of functional RNAP with relatively short beta  and/or beta ' subunit fragments, dramatically facilitating mapping of protein-protein and protein-nucleic acid contacts during transcription (11, 12).

The focus of this report is an evolutionary hypervariable region in the C-terminal portion of Escherichia coli beta ' (amino acids 1141-1131). This region is highly variable in proteobacteria and is absent from homologs from most other bacteria, archaea, and eukaryotes. Despite this apparent redundancy, numerous point mutations that altered the termination and elongation properties of E. coli RNAP were localized in the hypervariable region (25, 37, 38). In addition, mutations in the largest (beta '-like) subunit of yeast RNAP II that occurred very close to the hypervariable region dramatically decreased interaction of the enzyme with transcript cleavage factor TFIIS (33). Here, we probed the role of the hypervariable region and adjacent segments of E. coli beta ' by deletion mutagenesis of the cloned rpoC gene. Mutant RNAPs were assembled in vitro, and their elongation and termination properties, as well as their abilities to interact with the TFIIS analog GreB, were determined. A long deletion that completely removed the hypervariable region blocked enzyme assembly in vivo and in vitro. Short (40-60 amino acid) deletions, which together span the entire hypervariable region, did not prevent RNAP assembly and basic transcription function in vitro; however, the mutant enzymes had a dramatic defect in transcript elongation at low substrate concentrations. At high substrate concentrations, the mutants elongated and terminated transcription normally. The mutant enzymes were also defective in GreB-induced transcript cleavage, but the nature of the defect was complex, because both the ability to interact with GreB and the ability to support intrinsic transcript cleavage by the RNAP catalytic center were altered by mutations. A binding epitope for an inhibitory mAb, Pyn1, was mapped in the hypervariable region. Binding of Pyn1 to RNAP efficiently inhibited both the RNA synthesis and transcript cleavage reactions.

Our data thus demonstrate that the evolutionary hypervariable region of beta ', which is completely absent from homologues from eukaryotes, archaea, and many eubacteria, is unexpectedly important for E. coli RNAP assembly and is involved in transcript elongation and cleavage. Because this region is missing from beta ' homologues from most organisms, however, this involvement is probably indirect.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Deletion Mutagenesis of the Cloned rpoC Gene-- The pUC19-based pMKa201S793F rpoC expression plasmid was used to generate deletions in the rpoC gene. The plasmid is a derivative of pMKA201 (13) and carries a transdominant streptolydigin-resistance mutation, changing beta ' Ser793 to Phe (14). To generate nested Bal31 deletions in pMKa201S793F, a unique BamHI linker was inserted in the EcoRI site at codon 987 of the structural gene. pMKa201S793F was underdigested with EcoRI (cut twice at codons 175 and 987 of the structural gene and once in the vector), and ligated to the EcoRI fragment from phage mp17Km carrying the kanamycin resistance gene (11). Kanamycin-resistant transformants were screened for the appearance of inducible truncated fragments of the beta ' polypeptide using SDS-PAGE. Recombinant plasmids containing kanamycin cassette inserted at rpoC position 987 was treated with BamHI to remove the cassette and recircularized. The resultant plasmid pIBOmega 987 overproduced beta ' with 6 amino acids (IPRIRG) inserted between amino acids 987 and 988.

To generate deletions, pIBOmega 987 was linearized with BamHI, treated with Bal31 for various times, and recircularized. After transformation in E. coli XL1-Blue cells, transformants were screened for the appearance of inducible beta ' polypeptides with increased mobility on SDS-gels.

pIBDelta (1145-1198) was constructed by treating pMKa201S793F with AsuI (cut twice at codons 1145 and 1198 of the structural gene) and religating. pIBDelta (1091-1130) was constructed from a plasmid expressing the C-terminal portion of rpoC (beta '877-1407, Ref. 12), containing two in-frame KpnI recognition sites corresponding to rpoC codons 1091 and 1130. The rpoC fragment containing the deletion was then recloned back into pMKa201S793F.

Plasmids expressing Delta (877-948), Delta (941-1130), Delta (1042-1091), and Delta (1131-1155)S were constructed by annealing a pair of corresponding "outward" primers, followed by polymerase chain reaction amplification of the whole pMKa201S793F plasmid (30 cycles of 1 min at 94 °C, 1 min at 56 °C, and 8 min at 72 °C). Each primer used in the amplification reaction contained an in-frame BamHI site. The amplified plasmid was treated with BamHI, recircularized, and transformed in XL1-Blue cells. Transformants were screened for the appearance of inducible beta ' polypeptides with increased mobility on SDS-gels, and deletions were confirmed by DNA sequencing. To minimize polymerase chain reaction-related errors, deletions were recloned as SalI-SacII (cut at rpoC codons 877 and 1321, respectively) fragments back into appropriately treated parental pMKa201S793F.

All of the overexpressed beta ' subunit variants were found in inclusion bodies when induced with 1 mM IPTG at 37 °C.

Antibodies-- mAbs Pyn1 and Pyn4 were described previously (15). mAbs were precipitated from ascites fluid by addition of (NH4)2SO4 to 50% saturation. Before use, aliquots of precipitated protein were dialyzed into transcription buffer. Some mAb preparations contained an RNase activity. Hence, transcription reactions were supplemented with 0.5 mg of tRNA to prevent the degradation of RNA in transcription complexes. The working concentration of mAb was determined by combining a fixed amount of RNAP with increasing amounts of mAb, followed by separation of protein complexes on native 4-15% Phast gels (Amersham Pharmacia Biotech). The amount of mAb used in transcription reactions was twice the amount needed to shift completely the band of RNAP on a native gel.

RNAP Reconstitution-- RNAP was reconstituted according to the published procedure (16). The molar ratio of alpha , beta , and mutant beta ' in the reconstitution reactions was 1:4:8. After reconstitution and thermoactivation in the presence of sigma 70, RNAP preparations were either used directly or further purified by FPLC/gel-filtration on a Superose-6 and Mono Q columns (Amersham Pharmacia Biotech) as described (17), concentrated by filtration through a C-100 concentrator (Amicon) to ~1 mg/ml, and stored in 50% glycerol storage buffer at -20 °C.

Affinity Cross-linking-- The synthesis of photoactive 4-azidotetrafluorobenzylamino-pApUpC derivative will be described elsewhere (18).2 A standard 20-µl cross-linking reaction contained 0.5-1 µg of RNAP holoenzyme immobilized on ~5 µl of Ni2+-NTA agarose (Qiagen), 0.5 µg of the 324-base pair DNA fragment containing T7 A1 promoter (template 1 of Ref. 18), and 10 µM ApUpC in standard transcription buffer containing 40 mM KCl, 40 mM Tris-HCl, pH 7.9, 10 mM MgCl2. Reactions were incubated for 5 min at 37 °C to form the open complexes and transferred to room temperature, and ATP and GTP were added to a final concentration of 25 µM. After a 10-min incubation, reactions were washed three times with 1.5 ml of transcription buffer as described (13) and left in a ~20-µl volume. alpha -[32P]CTP (3000 Ci/mmol, NEN Life Science Products) was added to a final concentration of 0.3 µM, and the incubation was continued for 10 min. After washing, the immobilized EC12 was UV-irradiated with a hand-held lamp at 254 nm for 3 min. The lamp was positioned flat at the top of open Eppendorf tubes that contained the reaction mixtures and that were kept on ice. After irradiation, EC12 was desorbed from the solid support by addition of imidazole to 100 mM, an equal volume of SDS-containing Laemmli loading buffer (20) was added, and proteins were separated by SDS-PAGE.

In protein-RNA cross-linking experiments in the presence of GreB, immobilized EC20 containing radioactively labeled RNA was prepared and used to prepare EC21 with 4-thio-UTP instead of UTP. Derivatized EC21 was washed with transcription buffer lacking MgCl2 and desorbed from Ni2+-NTA agarose by the addition of 100 mM imidazole. Recombinant GreB (a generous gift of Arun Malhotra, University of Miami) was added to reaction aliquots followed by UV-irradiation as described above. Reaction products were then resolved by SDS-PAGE and visualized by autoradiography.

In Vitro Transcription-- To determine transcription elongation rates, elongation complexes stalled at position +20 were prepared in 50 µl of transcription buffer containing 25 µl of Ni2+-NTA agarose, 20 nM of T7 A1 promoter DNA fragment described above, 40 nM RNAP, 0.5 mM ApU, 50 µM CTP and GTP, 2.5 µM [alpha 32P]ATP (300 Ci/mmol). Reactions were incubated for 15 min at 23 °C and were washed as described above. Washed EC20 was synchronously started by making reactions 2.5 µM with NTPs. Reactions proceeded for 0-600 s at 23 °C. Reactions were terminated by the addition of formamide-containing loading buffer. To determine transcription termination efficiencies, EC20 was restarted by the addition of 250 µM NTPs. Reactions proceeded for 1 min at 23 °C. Products were analyzed by urea-PAGE electrophoresis (7 M urea, 6% polyacrylamide), followed by autoradiography and PhosphorImager analysis.

Transcript Cleavage-- Purified immobilized elongation complexes were briefly washed with 1 ml of transcription buffer, pH 9.0. Reactions were left in ~50 µl and incubated at room temperature. At various time points, reaction aliquots were withdrawn, combined with an equal volume of formamide-containing loading buffer, and analyzed by denaturing gel-electrophoresis and autoradiography. Cleavage with GreB was done as described earlier (21).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Deletion Mutagenesis of the Hypervariable Region of E. coli rpoC and Mapping of mAb Binding Epitopes-- Alignment of the amino acid sequence of E. coli beta ' with homologues from other organisms reveals a long region of primary sequence (E. coli residues 941-1131) that is present in proteobacteria but is totally absent in homologues from other eubacteria, archaea, and eukaryotes (Fig. 1). In contrast, a much longer sequence (up to 900 amino acids) is found in the corresponding region of beta ' subunits from cyanobacteria and chloroplasts. On the N terminus of this hypervariable region is highly conserved segment G (residues 912-940). C-terminal to the hypervariable region is a short segment of conserved sequence that we will refer to as segment G' (residues 1132-1148; Fig. 1). Segments G and G' form one continuous stretch of conserved amino acids in eukaryotes, archaea, and most bacteria. Sequence C-terminal to G' is not conserved and is followed by the C-terminal conserved segment H (residues 1324-1362).


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  		Fig. 1.   Genetic context of the hypervariable region in E. coli beta '. The 1407-amino acid-long beta ' subunit is represented by the heavy bar, with shaded areas corresponding to the highly conserved sequence segments, designated A-H. The locations of the streptolydigin-resistant mutations (13), residues involved in coordination of the catalytic Mg2+ ion (4), residues contacting the template DNA downstream of the catalytic center (29), and sites of cross-linking to the 3'-end of the nascent RNA are also shown (31). The hypervariable region under study is shown by the white box and is expanded underneath. In the alignment, the top E. coli (E. c.) sequence is aligned with the corresponding regions from P. putida (P. p.), Bacillus subtilis (B. s.), M. leprae (M. l.), chloroplasts from tobacco (T.), Halobacterium halobium (H. h.), and yeast RNA polymerases I, II, and III (YP1, YP2, and YP3, respectively). Deletions obtained in this work are shown as black bars. In addition, point mutations altering E. coli RNAP termination properties in vivo (24) (plain text), suppressing the nusA134 allele (33) (boldface type), and affecting chromosomal replication control (38) (underlined), as well as insertional mutations in the homolog from yeast RNAP II altering interactions with TFIIS (35) (italicized) are also shown above the E. coli sequence.

We probed the functional role of the hypervariable region by deletion mutagenesis of the cloned E. coli rpoC gene. A linker insertion mutation and a set of limited deletions were obtained as described under "Materials and Methods" and are shown above the alignment in Fig. 1. Delta (941-1130) removed all of the E. coli hypervariable region and thus resembles the beta ' subunit from Mycobacterium leprae (22). Delta (1131-1155)S removed all of segment G' and replaced it with a Ser residue; Delta (877-948) (not shown in Fig. 1) deleted conserved segment G. Delta (1145-1198) removed 54 amino acids immediately C-terminal to G'. Delta (1145-1198) removed coding sequences between in-frame naturally occurring recognition sites of restriction endonuclease AsuI and is referred to as Delta Asu throughout the text. Similarly, Delta (1091-1130) removed coding sequences between the in-frame naturally occurring KpnI sites and is referred to as Delta Kpn.

Previously, one of us reported the isolation of two anti-beta ' mAbs, Pyn1 and Pyn4, using RNAP core enzyme as an antigene (15). Pyn1 efficiently inhibited transcription by E. coli RNAP as well as by the Pseudomonas putida enzyme,3 whereas Pyn4 had no effect on transcription (15). We localized Pyn1 and Pyn4 binding epitopes using the beta ' deletion mutants described above. To locate mAb binding epitopes, cells expressing different plasmid-borne variants of rpoC were induced and lysed, and proteins were separated by SDS-PAGE, followed by Western blotting with Pyn1 or Pyn4. As shown in Table I, all deletions in the hypervariable region abolished Pyn1 binding and thus must have destroyed the binding epitope. In contrast, Delta (877-948), Delta (1131-1155)S, and Delta Asu did not affect Pyn1 binding. These data suggest that beta ' amino acids 949-1091 contain the Pyn1 binding epitope. However, because both Delta (941-1000) and Delta Kpn---Delta (1091-1130), which are 90 amino acids apart, disrupt Pyn1 binding, the antibody epitope may be conformational rather than linear. Thus, we can not exclude that beta ' sequences outside the hypervariable region may also participate in the binding of this inhibitory antibody.

                              
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Table I
Properties of rpoC mutants

Delta Asu was the only deletion that abolished the binding of Pyn4. We conclude that beta ' amino acids 1145-1198 contribute to Pyn4 binding epitope.

Properties of rpoC Mutants in Vivo-- All rpoC deletions were obtained in pMKa201S693F rpoC expression plasmid. In this plasmid, a transdominant streptolydigin-resistant S693F allele of rpoC is placed under the control of the inducible lac promoter (14). Therefore, the function of mutant, plasmid-borne rpoC can be tested in vivo by registering its ability to confer streptolydigin resistance to the sensitive host cells CAG 14064 (13). When CAG 14064 cells overproducing the WT and mutant beta ' subunits were plated on a medium containing 12.5 µg/ml streptolydigin, only cells overproducing parental S693F beta ', and the linker insertion Omega 987 formed colonies (Table I).

In another test of the in vivo function of mutant rpoC genes, the RL602 E. coli cells were transformed with pMKa201S693F and its derivatives, and plated at 42 °C. RL602 cells harbor an amber mutation in the chromosomal copy of rpoC that is suppressed by a temperature-sensitive suppressor (23). RL602 grows at 30 °C but fails completely to grow at 42 °C. Cells overproducing S693F beta ', as well as Omega 987, Delta (1042-1091), Delta (941-1000), Delta (987-1042), Delta Kpn, and Delta Asu formed colonies at the elevated temperature (data not shown). Cells overproducing S693F beta ', Omega 987, and Delta Asu grew robustly at 42 °C, whereas the others formed minute colonies (Table I).

We do not know whether the mutant beta ' subunits were the only source of beta ' at high temperature. Landick et al. (24) encountered similar "partial" phenotypes when testing in vivo function of mutant, plasmid-borne rpoB genes. These authors suggested that growth at restrictive temperature may occur when the plasmid-borne rpo gene provides enough partially functional RNAP to allow the growth of the host, which has a low level of the wild-type, chromosomally encoded RNAP even at restrictive temperature. In contrast, administering streptolydigin completely inhibits wild-type, chromosomal RNAP, and thus prevents the cell growth.

In Vitro Reconstitution of Mutant RNAPs-- Because the results of functional assays in vivo were inconclusive, we assembled mutant beta ' into RNAP in vitro. Assembled RNAP forms characteristic peaks during chromatography on Superose-6 and Mono Q columns (17). No such peaks were observed in the course of purification of reconstitution mixtures containing Delta (877-948), Delta (1131-1155)S, and Delta (941-1130) and WT alpha , beta , and sigma  subunits; neither crude reconstitution mixtures nor chromatographic fractions contained any RNAP activity (Table I and data not shown).

The beta ' subunits expressed from pMK201 plasmid are fused to the C-terminal His6 tag, allowing easy purification of RNAP harboring plasmid-borne beta ' (13). Our repeated attempts to purify RNAPs harboring the two largest beta ' deletions from cells using affinity chromatography failed (data not shown). We conclude that deletions of conserved segments G and G', as well as removal of the entire hypervariable region, completely abolish the ability of beta ' to enter E. coli RNAP core in vitro and in vivo.

5 smaller deletions (Delta (941-1000), Delta (987-1042), Delta (1042-1091), Delta Kpn and Delta Asu) assembled into RNAP in vitro as judged by the appearance of characteristic chromatographic peaks in the course of purification. The catalytic proficiency of RNAP mutants was demonstrated by template-dependent affinity labeling of shortened beta ' polypeptides with derivatized nascent RNA. In this reaction, Ni2+-NTA agarose-immobilized RNAP was used to form open complexes with the T7 A1 promoter-containing DNA fragment. Transcription was primed with a derivatized, photoactive ApUpC, complimentary to positions +1-+3 of the promoter. In the presence of unlabeled GTP and ATP, transcription proceeded to position +11 and was halted because of the absence of CTP, specified by the position +12 of the template. After extensive washing, elongation complexes stalled at position 11 (EC11) were extended with radioactive CTP to obtain EC12. EC12 containing radioactive and photoactive RNA was washed and UV-irradiated to induce protein-RNA cross-link formation, and the reaction products were separated by SDS-PAGE. Irradiation of RNAPWT elongation complexes resulted in radioactive labeling of both beta  and beta ' with equal efficiency (Fig. 2A). As can be seen from the autoradiogram presented on Fig. 2A, labeled, full-sized beta ' is absent from lanes containing mutant enzymes, establishing that little or no contaminating RNAPWT was present. Instead, lanes containing mutant enzymes have a labeled band with the mobility of beta , which represents a mixture of labeled full-sized beta  and mutant beta ' subunits (full-sized beta ' and beta  are 1407 and 1342 amino acids long, respectively; deletions in beta ' make it impossible to separate the two proteins by SDS-PAGE). Control experiments established that (i) subunit labeling was template-dependent, and (ii) subunit labeling did not occur when photoactive, radioactive RNA 12-mer was added to RNAP in trans. Furthermore, cleavage of the RNA-product adduct with formic acid demonstrated that the RNA moiety of the adduct was 12 nucleotides in length (data not shown).2 On the basis of these results, we conclude that the mutant enzymes are assembled, active, and free of contaminating RNAPWT.


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  		Fig. 2.   In vitro transcription by mutant enzymes. A, RNA-protein photo-cross-linking in EC12 formed by mutant RNAPs. Immobilized EC12 formed by the indicated RNAPs and containing photoactivatable, cross-linkable nascent RNA was UV-irradiated, and the reaction products were resolved on the 4% SDS-polyacrylamide gel, stained, and visualized by autoradiography. B, transcript elongation at subsaturating NTP concentration. EC20 were prepared using template 1 of Nudler et al. (19), containing the T7 A1 promoter and lambda  tR2 terminator. Transcription was resumed by the addition of NTP to 2.5 µM. Reactions proceeded for the times indicated, and reaction products were analyzed by 6% urea-PAGE followed by autoradiography. RNA in the indicated paused complexes was sized using artificially stalled elongation complexes with RNA of defined length as markers.

Transcription by Mutant RNA Polymerases-- We decided to concentrate on RNAPDelta Kpn and RNAPDelta Asu because preliminary experiments showed that RNAPDelta (941-1000), RNAPDelta (987-1042), and RNAPDelta (1042-1091) are very similar to RNAPDelta Kpn.

Weilbaecher et al. (25) reported that point mutations in the beta ' region under study altered the elongation and termination properties of RNAP in vivo and in vitro. Accordingly, we checked the transcription elongation and termination properties of mutant RNAPs. Immobilized elongation complexes stalled at position 20 of the T7 A1 promoter-driven transcription unit were prepared. To determine elongation rates, transcription was resumed by the addition of NTPs at subsaturating (2.5 µM) concentration. At various time points, reaction aliquots were withdrawn, and the products were analyzed by denaturing PAGE (Fig. 2A). As can be seen from Fig. 2, RNAPDelta Asu did not differ significantly from RNAPWT in this assay, but RNAPDelta Kpn was dramatically "slower." The apparent slow rate of transcript elongation by RNAPDelta Kpn was caused by extended (t[ifrax;1/2] ~ 2.5 min) pausing at template positions 26 and 27, and later at positions 37 and 56. The same pausing pattern was obtained when transcription by immobilized RNAPDelta Kpn was resumed from positions 12, 23, and 25 (data not shown).

Extended pausing by RNAPDelta Kpn at positions 26, 27, and 56 is intriguing, because transcription complexes artificially stalled at these positions are efficiently converted into an arrested conformation (26-30). During arrest at EC26 or EC27, the catalytic center of the enzyme disengages from the 3'-end of the nascent RNA, and RNAP slides backward. As a result, the catalytic center repositions to about position 20. This intermediate complex is still able to continue transcription by sliding forward to the active conformation; it can also slide back even further, to position 11, and become permanently arrested. The permanently arrested, dead-end complex can only be rescued by transcript cleavage. In the presence of the transcript cleavage factor GreB, the catalytic center hydrolyses the RNA at position 11. The newly generated 3'-end of the 5'-end-proximal, 11-mer cleavage product can then be elongated by RNAP; the 3'-end-proximal cleavage products are lost from the complex.

To investigate the nature of the RNAPDelta Kpn pause at position 26, immobilized EC20 was incubated for 2 min in the presence of low (2.5 µM) concentrations of NTPs, followed by a brief (30 s) incubation with GreB (Fig. 3A). As can be seen, addition of GreB to transcription reaction resulted in the appearance of new products. These new cleavage products were stably associated with transcription complexes (data not shown) and had the electrophoretic mobility of 20- and 22-mers. Control lanes of Fig. 3A demonstrate that the addition of GreB to stalled, arrested RNAPDelta Kpn EC27 resulted in cleavage, generating a 5'-terminal 11-mer, as expected. Based on these data, we conclude that in paused RNAPDelta Kpn EC26 and EC27, the transcript has reversibly slid out of the active site, and therefore, the paused complexes correspond to intermediate of the arrested complex formation pathway (28-30).


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  		Fig. 3.   The mechanism of pausing defect of RNAPDelta Kpn. A, transcription elongation in the presence of GreB. Immobilized RNAPDelta Kpn EC20 (lane 1) was restarted by the addition of 5 µM NTP, and reaction was allowed to proceed for 2 min (lane 2). At this point, 400 ng of recombinant GreB was added, followed by additional 30 s of incubation (lane 3). Stalled EC27 (lane 4) was supplemented with 5 µM NTP, incubated for 2 min (lane 5), and treated with GreB for additional 30 s (lane 6). An autoradiograph of 15% urea polyacrylamide gel is presented. B, arrested complex formation by RNAPDelta Kpn and RNAPWT. Stalled EC27 was prepared (lanes 1 and 4) and immediately restarted by the addition of 10 µM of ATP and CTP (chase) to allow the formation of EC32 (lanes 2 and 5). Alternatively, EC27 was incubated for 5 min at 37 °C, followed by the addition of NTP (lanes 3 and 5). Reaction products were analyzed as in A. C, transcription termination at lambda  tR2 terminator. Elongation by purified EC20 was resumed by the addition of 250 µM NTP. Reactions proceeded for 1 min at 23 °C. Products were analyzed on a 6% urea gel.

Increased pausing by RNAPDelta Kpn could be explained by its increased ability to slide backward at certain parts of the template. Although the ability to slide back is difficult to test, we investigated the efficiency of EC27 arrest complex formation by RNAPDelta Kpn. In this experiment, shown in Fig. 3B by the extent of transcription arrest by artificially stalled, purified RNAPDelta Kpn EC27 was determined by the ability to be chased into EC32 upon the addition of ATP and CTP. Freshly purified EC27 formed by both RNAPWT and RNAPDelta Kpn were elongation competent (Fig. 3B, lanes 2 and 5). However, after 5 min of incubation at 37 °C in the absence of nucleotides only 10% of initial EC27 could be chased upon addition of nucleotides. Importantly, there was no difference between RNAPDelta Kpn and RNAPWT in the extent of arrest (Fig. 3B, lanes 3 and 6).

Extensive pausing by RNAPDelta Kpn could be caused by an unusually high apparent Km for the incoming UTP (corresponding to position 27), accompanied by reversible sliding to position 20. Indeed, increasing NTP concentration decreased pausing by RNAPDelta Kpn. At high (250 µM) substrate concentrations, RNAPDelta Kpn completed a single round of transcription in 1 min and terminated transcription on the lambda  tR2 terminator with nearly the same efficiency as RNAPWT and RNAPDelta Asu (Fig. 3C).

Transcript Cleavage by Mutant RNAPs-- A genetic screen of yeast RPO21 (an evolutionary homologue of rpoC) identified mutations that altered the ability of RNAP II to interact with transcript cleavage factor TFIIS (31). Seven mutations were isolated and they all clustered between conserved segments G and H (32). It was subsequently shown that these mutations inhibited interaction of mutant RNAP II with TFIIS by as much as 50-fold (33). Because these mutations occurred very close to hypervariable region studied here (see Fig. 1), we investigated the transcript cleavage activity of mutant E. coli RNAPs. As can be seen from Fig. 4A, EC21 formed by RNAPDelta Kpn required approximately 10 times more GreB to achieve the same extent of cleavage as complexes formed by RNAPWT and RNAPDelta Asu. The same result was obtained when the GreB homolog GreA was used to induce the cleavage reaction (data not shown).


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  		Fig. 4.   Transcript cleavage by mutant RNA polymerases. A, EC21 containing RNA radioactively labeled at U21 was desorbed from Ni2+-NTA agarose with 50 mM imidazole (lanes 1, 5, and 9) and supplemented with GreB (lanes 2, 6, and 10, 0.04 ng; lanes 3, 7, and 11, 0.4 ng; lanes 4, 8, and 12, 4 ng). Reactions were incubated for 5 min, and products were resolved by denaturing PAGE in 10% urea-gel. B, intrinsic transcript cleavage at elevated pH. Immobilized EC20 was transferred into a pH 9.0 transcription buffer, and reactions were incubated for indicated times, followed by electrophoresis and autoradiography. C, RNA-protein photo-cross-linking. EC21 formed by RNAPWT and RNAPDelta Kpn was UV-irradiated in the absence (lanes 1 and 7) or in the presence of decreasing concentrations of GreB (lanes 2 and 8, 400 ng; lanes 3 and 9, 40 ng; lanes 4 and 10, 4 ng; lanes 5 and 11, 0.4 ng; lanes 6 and 12, 0.04 ng). Reaction products were resolved on the 4-20% gradient SDS-gel and visualized by autoradiography. D, binding of RNAPWT and RNAPDelta Kpn to [32P[HMPK-GreB. 1 µg of RNAP of the indicated core enzymes was combined with increasing concentrations of [32P]HMKP-GreB. Reactions were incubated for 10 min to allow the complex formation, resolved by native PAGE on a 4-15% Phast gel, and visualized by autoradiography.

When EC20 formed by RNAPWT was incubated in pH 9.0 buffer (a condition known to stimulate the intrinsic nucleolytic activity of RNAP (21)) transcript cleavage was observed (Fig. 4B). After 5 min incubation in pH 9.0 buffer, >75% of RNAPWT and RNAPDelta Asu EC20 had undergone cleavage (Fig 4B, lanes 2 and 6, respectively). In contrast, only 10% of RNAPDelta Kpn EC20 had undergone cleavage after a 5-min incubation, and less than 60% was cleaved after a 20-min incubation in pH 9.0 buffer (lanes 10 and 12). To estimate GreB binding to RNAPDelta Kpn, we performed UV-photo-cross-linking of GreB to derivatized 3'-end of nascent RNA in RNAPWT and RNAPDelta Kpn EC21 (Fig. 4C). In agreement with previous data (36, 39), beta ' and GreB were efficiently cross-linked to derivatized, radioactive RNA. Lowering the amount of GreB in the reaction resulted in decreased cross-linking of GreB to RNA, but the amount of beta '-RNA cross-link remained constant. As can be seen from Fig. 4C, RNAPDelta Kpn complexes required approximately 10 times more GreB to achieve the same extent of GreB-RNA cross-links as complexes formed by RNAPWT. This effect was especially evident at low GreB concentrations (compare Fig. 4C, lanes 6 and 12). From this result, we conclude that Delta Kpn either directly decreases GreB binding to RNAP or alters the relative position of GreB and the RNA 3'-end, thus decreasing the efficiency of GreB-RNA cross-linking near the catalytic center.

To estimate GreB binding to mutant RNAP directly, we used GreB protein tagged with an N-terminal heart muscle protein kinase (HMPK) recognition site.4 Different amounts of 32P-end-labeled HMPK-GreB were combined with RNAPWT or RNAPDelta Kpn core enzymes, and after a short incubation to allow complex formation, the reaction products were resolved by native PAGE and GreB-containing complexes were visualized by autoradiography (Fig. 4D). As can be seen, both enzymes formed complexes with [32P[HMPK-labeled GreB with the same efficiency.

Effects of mAb Binding to RNAP on Transcription in Vitro-- In agreement with our previous data (15), mAb Pyn1 efficiently inhibited elongation of the nascent RNA from EC20 to EC23 by RNAPWT (Fig. 5A, top, lane 3). Transcription by RNAPDelta Kpn was unaffected by Pyn1 (Fig. 5A, top, lane 7), because Pyn1 did not bind RNAPDelta Kpn (Fig. 5A, bottom, lane 5). In contrast, Pyn4 had no effect on EC20 to EC23 conversion by both enzymes, even though more than 50% of elongation complexes were bound to mAb at the experimental conditions used (lanes 3 and 6).


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  		Fig. 5.   Effects of mAb binding on transcription in vitro. A, EC20 containing radioactively labeled RNA were desorbed from Ni2+-NTA agarose with 50 mM imidazole (lanes 1 and 5, top panel, and lanes 1 and 4, bottom panel). Reactions were then supplemented with the indicated mAbs and 10 µM ATP and CTP (chase) to allow elongation to position 23. Reaction products were resolved by denaturing PAGE in 10% urea-gel (top panel) or by native PAGE in 4% gel (bottom panel). B, intrinsic transcript cleavage at elevated pH. Immobilized EC20 was transferred into a pH 9.0 transcription buffer, and reactions were incubated for indicated times in the presence or in the absence of Pyn1, followed by electrophoresis and autoradiography. C, EC20 formed by RNAPWT was treated with increasing concentrations of GreB in the presence or in the absence of Pyn1, and reaction products were resolved on a 15% denaturing gel, visualized by autoradiography. In B and C, lanes 1 and 2 are controls, demonstrating that Pyn1 efficiently inhibited transcript elongation at the conditions used. D, binding of RNAP to [32P]HMPK-GreB. RNAP core enzyme was combined with increasing concentrations of [32P]HMKP-GreB in the presence (lanes 4-6) or the absence (lanes 1-3) of Pyn1. Reactions were incubated for 10 min to allow the complex formation and resolved by native PAGE on a 4-15% Phast gel.

Pyn1 completely inhibited intrinsic transcript cleavage by RNAPWT at pH 9.0 (Fig. 5B, lanes 6 and 7). Transcript cleavage by RNAPDelta Kpn was unaffected at these conditions (data not shown). When GreB was added to RNAPWT, EC20 transcript cleavage occurred even in the presence of Pyn1 (Fig. 5C). Still, an ~10-fold excess of GreB was required in the presence of Pyn1 to achieve the same extent of cleavage as in its absence (compare Fig. 5C, lanes 5 and 7). GreB did not cause dissociation of Pyn1 from RNAP, as the experiment shown in Fig. 5D demonstrates. In this experiment, different amounts of 32P-end-labeled HMPK-GreB were combined with RNAPWT core in the presence or the absence of Pyn1. After a short incubation to allow complex formation, the reaction products were resolved by native PAGE, and GreB-containing complexes were visualized by autoradiography. In the absence of Pyn1, [32P[HMPK-GreB formed a complex with RNAP (Fig. 5D, lanes 1-3). Addition of Pyn1 resulted in the appearance of a radioactive complex with a slower electrophoretic mobility (lanes 4-6). We conclude that GreB and Pyn1 can simultaneously bind to the RNAP core molecule.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

A set of deletions in the cloned E. coli rpoC gene was constructed to evaluate the role of an evolutionarily hypervariable region of the RNAP beta ' subunit (amino acids 941-1130) in enzyme function. This region is completely absent from beta ' subunits of most bacteria and is highly divergent within proteobacteria (Fig. 1). Suprisingly, deletion of the entire hypervariable region completely prevented RNAP assembly both in vivo and in vitro. Obviously, similar deletions (relative to E. coli beta ') naturally found in the beta ' subunits from other bacteria do not prevent RNAP assembly in these organisms. Apparently, compensating differences in the alpha , beta , and/or beta ' subunits of these bacteria allow RNAP assembly to proceed.

The five functional deletions obtained in this work together remove 243 amino acids (>17%) of beta ' and prove that, as in beta , long regions of beta ' sequence are dispensable for basic functions of E. coli RNAP in vitro. These functional deletions clearly fall into two classes. Delta Asu removes 54 amino acids N-terminal of the hypervariable region and does not change RNAP transcription in vitro. In contrast, deletions of similar size spanning the hypervariable region (Delta 941-1000, Delta 987-1042, Delta 1042-1091, and Delta 1091-1130 (Delta Kpn)) cause a dramatic defect in transcription elongation at subsaturating substrate concentrations and inhibit transcript cleavage.

The functional difference between the two classes of mutants is also evident from different effects of mAb binding. Pyn1 binds to the hypervariable region (amino acids 948-1130) and efficiently inhibits RNA synthesis and intrinsic transcript cleavage. In contrast, Pyn4, the binding epitope of which is destroyed by Delta Asu, has no effect on transcription. The hypervariable region is highly immunogenic: in an independent study Luo and Krakow (34) isolated several mAbs that interacted with this region of E. coli beta '. mAb 311G2 inhibited RNA synthesis by interfering with substrate binding, and its binding epitope was within beta ' amino acids 1047-1093. Thus, 311G2 must be very similar to Pyn1.

The most striking feature of RNAPDelta Kpn-like enzymes is prolonged pausing at sites of transcriptional arrest (26-30), suggesting that the hypervariable region is involved in transcript elongation and cleavage. However, this involvement is probably indirect, because the hypervariable region is completely absent from RNAP of most organisms. We therefore propose that the deletions in beta ' studied here act indirectly, by affecting function(s) of adjacent conserved segments. There is good evidence that segment G, which is located immediately to the N terminus of the hypervariable region, participates directly in transcript cleavage and transcriptional arrest. Borukhov et al. (35) demonstrated that an 80-amino acid-long beta ' fragment containing the 8 C-terminal-most amino acids of conserved segment G and the 74 N-terminal-most amino acids of the hypervariable region contains the site that is cross-linked to the 3'-end of the nascent RNA in EC20. It was later found that this protein-RNA contact is indicative of the arrested conformation of the elongation complex (40). Further protein-RNA cross-linking experiments established that segment G and GreB proteins could both be cross-linked to the nascent RNA 3'-terminus and thus must be within several Å of each other in the complex (36). Thus, it is likely that segment G can form an alternative, unproductive RNA binding site during transcript elongation. The defect in RNAPDelta Kpn-like enzymes could be the result of increased interactions between region G and the RNA 3'-end in the mutant complex at the potential arrest sites, causing backsliding, and elevated apparent Km for the incoming nucleoside triphosphate. This defect is only observed at subsaturating substrate concentrations, because at high substrate concentrations, addition of NMP to the 3'-end of the nascent RNA occurs faster than backsliding, and mutant enzymes elongate RNA normally. Close inspection of the gel shown in Fig. 2B reveals that RNAPWT also pauses at the sites of transcriptional arrest, but clears the pause sites rapidly. Thus, reversible backsliding may play a role in RNAPWT pausing as well.

One of the goals of this study was to obtain RNAP mutants defective in interactions with transcript cleavage factors. Data of Wu et al. (33) suggested that the region of the largest (beta '-like) subunit of yeast RNAP II between conserved segments G and H may form the primary site of TFIIS interactions with its target RNAP. Seven tightly clustered linker insertion mutants studied by these authors showed little or no effect in intrinsic transcript cleavage and transcription elongation at high NTP concentrations but were defective in the TFIIS-dependent transcript cleavage, and did not form a complex with TFIIS in vitro. Three of the seven yeast mutations occurred immediately to the right of conserved segment G'. In E. coli, the corresponding amino acids are removed by Delta (1145-1198) (Delta Asu) mutation. Suprisingly, Delta Asu is the least defective of the five functional RNAP mutants obtained in this work. In contrast, Delta Kpn-like enzymes with deletions in hypervariable region itself are defective in Gre-dependent transcript cleavage, but the defect is relatively mild. Unlike the situation with yeast RNAP, the interactions between the mutant enzymes and the transcript cleavage factor were not affected as measured by the native gel binding assay. The nature of the cleavage defect is complex, because both the intrinsic transcript cleavage activity of the catalytic center and GreB-RNA cross-linking are affected by mutations. The latter defect could be due to repositioning of segment G, which is known to be close to GreB in the elongation complex (see above). Thus, our data suggest that TFIIS and GreB, which are functional analogs, but not homologous to each other, may interact with their respective RNA polymerases differently.

Published alignments of beta '-like RNAP subunits (7, 24) differ significantly from the alignment presented in Fig. 1. The main feature of our alignment is the existence of an additional conserved segment, G' (E. coli positions 1031-1040). Our alignment offers clues to the striking differences in the biochemical properties of mutant enzymes obtained in this work and is supported by the pattern of evolutionary variation in this region. In archaea, eukaryotes, and most bacteria, segments G and G' are fused, forming one continuous stretch of conserved amino acids. In contrast, in proteobacteria, cyanobacteria, and chloroplasts, a long insertion occurred at the G/G' boundary (referred to as the hypervariable region in this work). The sequence of this insert appears to be unrelated between proteobacteria and cyanobacteria, and is highly divergent within each group. Interestingly, the site of the insertion relative to the G/G' boundary is the same in both groups, which are far apart from each other phylogenetically (41). Thus, insertions at the G/G' boundary may have occurred at least twice in evolution. The inserted region is highly immunogenic, and may therefore loop out at the surface of the RNAP. Deletions in or mAb binding to the hypervariable region may alter the relative position of G and G', resulting in the observed defects in transcription. The importance of G' is highlighted by the fact that the smallest deletion obtained in this work, Delta (1131-1155)S, which removed G', failed to assemble in RNAP in vitro. On the other hand, it is possible that removal of approximately 50 amino acids C-terminal to G' in the Delta Asu mutant did not alter the relative positions of G and G' and thus had no major impact on the transcription properties of RNAP. Ongoing site-specific mutagenesis should clarify the functional role of this segment in RNA polymerase assembly, catalytic function, and transcript cleavage.

    ACKNOWLEDGEMENTS

We are grateful to Arkady Mustaev for reagents and advice on protein-RNA cross-linking, Nick Loizos for the gift of plasmid overproducing HMPK-GreB, and Robert Landick and Cathleen Chan for useful comments and critically reading the manuscript. Part of this work was done by N. Z. during her time at Alex Goldfarb's laboratory (Public Health Research Institute, New York, NY).

    FOOTNOTES

* This work was supported by The Borroughs Wellcome Fund for Biomedical Research Career Award, by the start-up funds from Rutgers University (to K. S.), and by Research Grants 96-04-49019 and 96-1598076 from the Russian Foundation for Basic Research and INTAS-RFBR Grant 9501150 (to V. N.).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.

parallel To whom correspondence should be addressed. Tel.: 732-445-6095; Fax: 732-445-5735; E-mail: severik{at}waksman.rutgers.edu.

The abbreviations used are: RNAP, RNA polymerase; EC, elongation complex stalled at position +n of the template, mAb, monoclonal antibodyPAGE, polyacrylamide gel electrophoresisUTP, uridine triphosphateWT, wild-typeHMPK, heart muscle protein kinase.

2 A. Mustaev and A. Goldfarb, manuscript in preparation.

3 A. Lebedev and V. Nikiforov, unpublished results.

4 N. Loizos, unpublished results.

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Abstract
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Materials & Methods
Results
Discussion
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