WO1999047650A2

WO1999047650A2 - PURIFIED RNA POLYMERASE FROM $i(ENTEROBACTER)

Info

Publication number: WO1999047650A2
Application number: PCT/US1999/006158
Authority: WO
Inventors: Kelvin T. Lam
Original assignee: Scriptgen Pharmaceuticals, Inc.
Priority date: 1998-03-16
Filing date: 1999-03-16
Publication date: 1999-09-23
Also published as: AU3359699A; WO1999047650A3

Abstract

The present invention provides a purified Enterobacter DNA-dependent RNA polymerase. In particular, RNA polymerase haloenzyme and core enzyme from E. cloacae are disclosed. The invention further provides methods of screening for antibacterial agents that bind to or modify the activity of an Enterobacter DNA-dependent RNA polymerase.

Description

PURIFIED RNA POLYMERASE FROM ENTEROBACTER

Field of the Invention

This invention relates to purified bacterial DNA-dependent RNA polymerases, particularly those from Enterobacter species, which are useful as targets for antibacterial drugs.

Background of the Invention

Enterobacteriaciae are Gram-negative rods that are the most common cause of urinary tract infections and are important etiological agents of diarrhea (Farmer III, 1995). Spread of Enterobacteriaciae to the bloodstream leads to Gram-negative sepsis. The Enterobacteriaciae are a large, diverse family and include E. coli, Salmonella, Yersinia,

Klebsiella, Enterobacter, Serratia, Proteus, Providencia and Morganella (Farmer III, 1995). Many are found in normal flora and are opportunistic pathogens. Enterobacteriaciae are often resistant to a variety of antimicrobial agents such as penicillin G, erythromycin and clindamycin. Thus, there is a need in the art for diverse antibacterial agents that are directed against different bacterial target proteins.

Regulation of gene expression in bacteria is controlled by DNA-dependent RNA polymerase. RNA polymerase holoenzyme consists of four subunits. The core enzyme (containing two α subunits, a β subunit and a β' subunit) binds to a σ subunit to form a holoenzyme. The primary σ factor, originally identified as a stimulating factor (Burgess, 1969), determines promoter specificity. The core enzyme can initiate transcription only in a non-specific manner.

RNA polymerase has been purified from several bacterial species, including Escherichia coli (Burgess, 1969; Burgess & Jendrisak, 1975; Engbaek et al, 1976; Hager et al., 1990), Bacillus subtilis (Avila et al., 1971; Davison et al., 1979; Hager, et al., 1990), Pseudomonas aeruginosa (Allan & Kropinski, 1987), Pseudomonas putida (Johnson et al.,

1971), Micromonospora echinospora (Lin & Rothstein, 1992), Streptomyces lividans (Lin and Rothstem, 1992), Salmonella typhimurium (Young BS et al , 1976) and Staphylococcus aureus (Deora & Misra, 1996) Prior to the present invention, however, little information was available regarding RNA polymerase from Enterobacter Thus, there is a need in the art for purified RNA polymerase from Enterobacter Purified RNA polymerase can be used, e g , to identify antibacterial agents for prevention and treatment of enterobacterial infections

Summary of the Invention

The present invention provides purified RNA polymerase from Enterobacter species, preferably E. cloacae The invention encompasses purified RNA polymerase core enzyme (i e , containing α, β, and β' subunits), purified holoenzyme (i e , containing, α, β, and β'subunits, and σ factor), and individually purified subunits The invention also provides recombinant α, β, and β' subunits and σ factor, all of which may further compπse sequences useful as purification tags, such as, e g , polyhistidine sequences

In another aspect, the invention encompasses methods for purification of Enterobacter RNA polymerase holoenzyme, core enzyme, and subunits In one embodiment, the method is carried out using the steps of

(i) harvesting and lysis of the bacterial cells,

(ii) precipitation with polyethylenimine,

(iii) separating the RNA polymerase by gel filtration chromatography, and

(iv) separating the RNA polymerase by affinity chromatography using a heparin column In another embodiment, purification of histidine-tagged subunits is achieved using affinity chromatography on, e g , Ni⁺²-NTA-agarose

In yet another aspect, the invention encompasses screening methods to identify candidate antibacterial agents In one embodiment, the method comprises

(i) providing a transcription mixture comprising one or more purified RNA polymerase subunits, core enzyme, or holoenzyme and components necessary for in vitro transcription,

(ii) contacting the mixture with a plurality of test compounds,

(iii) incubating the mixture formed in (ii) under conditions appropπate for in vitro transcription and measuring the transcriptional activity of the mixture, and (iv) selecting as candidate antibacterial compounds those compounds that modify the transcriptional activity of the mixture

In another embodiment, the method comprises (i) contacting purified RNA polymerase core enzyme, holoenzyme, or one or more individual subunits with a plurality of test compounds, and

(ii) measuring the binding of the compounds to the RNA polymerase or subunit, and (iii) selecting as candidate antibacterial agents those compounds that bind to the RNA polymerase or subunit

Brief Description of the Drawings

Figure 1 A is a graphic illustration of the elution profile of E. cloacae RNA polymerase from a Bio-Gel Al 5 column Fifty μl aliquots of each fraction were used to assay promoter-specific transcriptional activity (solid line) The absorbance at 280 nm was measured

(dotted line)

Figure IB is a graphic illustration of the elution profile of E. cloacae RNA polymerase from a Heparin agarose column Elution was carried out using a linear gradient of 0 1- 0 6 M KC1, starting at fraction #24 Fifty μl aliquots were used to assay promoter- specific transcription (solid line with squares) and non-specific transcription (solid line with open circles) The absorbance at 280 nm was measured for every other fraction (dotted line)

Figure 2 is a photographic illustration of SDS-PAGE analysis of fractions obtained during purification of the E. cloacae RNA polymerase The proteins were separated on a 4-15% gradient acrylamide gel Lane 1, molecular weight markers, lane 2, ammonium sulfate precipitate of the supernatants of soluble fractions, lane 3, Bio-Gel A 1 5 pooled fractions, lane 4, purified E. cloacae RNA polymerase (RNAP), and lane 5, purified E. coli RNA polymerase

Figure 3 A is a graphic illustration of the effect of KC1 (rnM) on enzymatic properties of purified E. cloacae RNA polymerase Both promoter-specific and non-specific in vitro transcription activity were measured (using pTac and CT, respectively, as indicated)

Figure 3B is a graphic illustration of the effect of MgCl₂ (mM) on enzymatic activity, as with Figure 3 A Figure 3C is a graphic illustration of the effect of DTT (mM) on enzymatic activity, as with Figure 3A Figure 3D is a graphic illustration of the effect of temperature on enzymatic activity Figure 4 is a graphic illustration of the broad-spectrum activity of rifamycin against purified RNA polymerases derived from Eschenchia coli (solid squares), Pseudomonas aeruginosa (solid circles), Enterobacter cloacae (solid triangles), Staphylococus aureus (open squares), Enterococcus faecium (open circles), and Bacillus subtilhs (open triangles)

Figure 5 is a photographic illustration of an immunoblot of RNA polymerases purified from different source organisms The blots were stained using polyclonal antibodies against E. coli σ⁷⁰ factor Lane 1, prestained molecular weight markers, lane 2, Escherichia coli, lane 3, Enterobacter cloacae, lane 4, Pseudomonas aeruginosa, lane 5, Staphylococcus aureus, lane 6, Bacillus subtilhs, lane 7, Enterococcus faecium

Figures 6A and 6B are a graphic illustration of RNA polymerase activity of E. cloacae core polymerase reconstituted in vitro with E. coli σ factor, in the presence (A) or absence (B) of tac promotor RNA polymerase activity was expressed as the counts per minute of [α-₃₂P] UTP incorporated after 30 minutes of polymerase activity at 37 °C

Figure 7A is a photographic illustration of promoter-specific transcπption products displayed on PAGE Lane 1, in vitro transcription using purified E. cob RNA polymerase, lane 2, in vitro transcription using purified E. cloacae RNA polymerase Figure 7B shows the DNA template used in the reaction

Detailed Description of the Invention

The present invention advantageously provides purified Enterobacter DNA-dependent RNA polymerase, and methods for purifying the core enzyme and the holoenzyme The purified RNA polymerase (RNAP) is useful for screening for inhibitors, which may provide anti-bacterial drugs that avoid or overcome antibiotic resistance problems

In practicing the present invention, many techniques in molecular biology, microbiology, recombinant DNA, and protein biochemistry such as these explained fully in, for example, Sambrook et al , 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, O gonucleotide Synthesis, 1984, (M L Gait ed ), Ausubel et al. , Current Protocols in Molecular Biology,

1997 (John Wiley & Sons), the series, Methods in Enzymology (Academic Press, Inc ), and Protein Purification- Principles and Practice, Second Edition (Springer- Verlag, N Y ), may be used

Enterobacter RNA Polymerase The present invention provides purified and isolated RNA polymerase derived from Enterobacter species, including, e g , E. cloacae, E. aerogenes, E. hafniae, and E. quifaciens An RNA polymerase subunit that is "derived from" a particular organism is a polypeptide encoded by the genome of that organism

"Purification" of an RNA polymerase complex or subunit refers to the isolation of the complex or subunit in a form that allows its enzymatic activity to be measured without interference by other components of the cell from which the complex or subunit was purified A purified complex or subunit contains less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated As used herein, RNA polymerase "core enzyme" refers to a complex containing two α, one β, and one β' subunit RNA polymerase "holoenzyme" contains, in addition to the α, β, and β' subunits, a σ factor polypeptide The invention encompasses purified core enzyme, purified holoenzyme, and individually purified subunits RNA polymerase holoenzyme according to the invention may comprise subunits derived from different species, such as, e g , a holoenzyme having a σ subunit derived from a different species than the core enzyme subunits In a specific embodiement, a E. cloacae core enzyme contains an Escherdchia coli σ factor polypeptide

According to the present invention, Enterobacter RNA polymerase may be purified from wild-type or mutant Enterobacter cells, or from heterologous organisms or cells (including, but not limited to, bacteria, fungi, insect, plant, and mammalian cells) into which an

Enterobacter-dQTwed RNA polymerase-encoding sequence has been introduced and expressed Furthermore, the polypeptides may be part of recombinant fusion proteins

Various well-known methods can be used to obtain Enterobacter-άeήved RNA polymerase-encoding sequences Genes encoding one or more subunits of Enterobacter RNAP can be isolated from any Enterobacter source Methods for obtaining Enterobacter

RNAP genes are well known in the art, e.g., as described above (see, e.g., Sambrook et al., 1989, supra) The DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library"), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired bacterium (See, for example, Sambrook et al., 1989, supra, Glover, D M (ed ), 1985, DNA Cloning A Practical

Approach, MRL Press, Ltd , Oxford, U K Vol I, II) Whatever the source, the gene or genes should be molecularly cloned into a suitable vector for propagation of the gene Identification of the specific DNA fragment containing the desired Enterobacter RNAP gene may be accomplished in a number of ways. For example, a portion of an Enterobacter RNAP gene can be purified and labeled to prepare a labeled probe, and the generated DNA may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, Science 196: 180, 1977; Grunstein and Hogness, Proc. Natl. Acad. Sci. U.S.A. 72:3961, 1975). Alternatively, degenerate probes derived from the Enterobacter RNAP amino acid sequences can be prepared and used for hybridization to possible cloned sequences. Those DNA fragments with substantial homology to the probe will hybridize.

In yet another embodiment, expression cloning methods can be used to identify the Enterobacter RNAP coding sequence, including detecting expression from an expression library using an -Enterobacter RNAP antibodies (described infra). Thus, selection can be carried out on the basis of the properties of the gene, e.g., if the gene encodes a protein product having the isoelectric, electrophoretic, amino acid composition, partial or complete amino acid sequence, antibody binding activity, or ligand binding profile of Enterobacter RNAP protein as disclosed herein. The presence of the gene may be detected by assays based on the physical, chemical, immunological, or functional properties of its expressed product.

The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.

Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated. Preferably, the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired. For example, a shuttle vector, which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. co and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2m plasmid

Various methods known in the art can be used to reconstitute holoenzyme activity (see, e.g., US Patent No 5,583,026 and Patent No 5,668,004)

In a preferred embodiment, Enterobacter RNA polymerase holoenzyme is purified from a bacterial culture by the following steps

(i) Harvesting, analysis: Typically, a bacterial paste of E. cloacae is lysed using, e g , a French press, and a cleared supernatant is prepared (ii) Precipitation: The cleared lysate is subjected to precipitation using polyethylenimine, and the pellet is recovered and solubilized

(iii) Gel filtration chromatography: The material is size-fractionated and fractions are assayed for RNA polymerase activity

(iv) Affinity chromatography: RNA polymerase-containing fractions are adsorbed to, and specifically eluted from, a heparin column (See, e g , Example 1 below)

The invention also provides recombinant α, β, and β' subunits and σ factor, all of which may further comprise sequences useful as purification tags, such as, e g , polyhistidine sequences Purification of histidine-tagged subunits is achieved using affinity chromatography on, e g , Ni⁺²-NTA-agarose (Qiagen) Purified RNA polymerase holoenzyme, core enzyme, or subunits may be modified in any manner known in the art, such as, for example, phosphorylation, sulfation, acylation, or other protein modifications They may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds

Antibodies to Enterobacter RNAP

Antibodies to Enterobacter RNAP are useful, /«ter aha, for diagnostics, detecting expression of Enterobacter RNAP (e.g., for cloning), and for screening According to the invention, Enterobacter RNAP polypeptides may be used as an lmmunogen to generate antibodies that recognize the Enterobacter RNAP or a polypeptide thereof Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library

Various procedures known in the art may be used for the production of polyclonal antibodies to Enterobacter RNAP polypeptides. For the production of antibody, various host animals can be immunized by injection with the Enterobacter RNAP polypeptide, or a derivative (e.g., fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, the Enterobacter RNAP polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin

(BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG

(bacille Calmette-Gueriή) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the Enterobacter RNAP polypeptide, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used (Kohler and Milstein, Nature 256:495-497, 1975; Kozbor et al.. Immunology Today 4:72, 1983; Cote et al, Proc. Natl. Acad. Sci. U.S.A.

80:2026-2030, 1983; Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985; International Patent Publication No. WO 89/12690, published 28 December 1989). According to the invention, techniques described for the production of single chain antibodies (U.S. Patent Nos. 5,476,786 and 5,132,405 to Huston; U.S. Patent 4,946,778) can be adapted to produce Enterobacter RNAP polypeptide-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., Science 246:1275-1281, 1989). Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab¹ fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

In the production and use of antibodies, screening for or testing with the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay,

ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, Western blots, precipitation reactions, etc. In one embodiment, antibody binding can be detected by detecting a label on the primary antibody. In another embodiment, the primary antibody can be detected by detecting binding of a secondary antibody or reagent to the primary antibody In a further embodiment, the secondary antibody is labeled Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the Enterobacter RNAP polypeptide, e.g., for Western blotting, imaging Enterobacter RNAP polypeptide in situ, measuring levels thereof in appropriate physiological samples, etc using any of the detection techniques mentioned above or known in the art Such antibodies can also be used in assays for ligand binding, e.g., as described in US Patent No 5,585,277 and Patent No 5,679,582 In addition, such antibodies can be used to affinity purify Enterobacter RNAP or components thereof

Screening and Chemistry

According to the present invention, nucleotide sequences derived from the gene encoding a polymorphic form oϊ a Enterobacter RNAP, and Enterobacter RNAP protein (particularly the core enzyme or the holoenzyme), are useful targets to identify drugs that are effective in treating Enterobacter infections, particularly infection with antibiotic resistance bacteria Drug targets include without limitation (i) isolated nucleic acids derived from the gene encoding a Enterobacter RNAP, (ii) isolated derived from Enterobacter RNAP, and (iii) Enterobacter RNAP (core enzyme or holoenzyme)

Knowledge of the primary sequence of the, and the similarity of that sequence with proteins of known function, can provide an initial clue as the inhibitors or antagonists of the protein Identification and screening of antagonists is further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination These techniques provide for the rational design or identification of agonists and antagonists

Purified Enterobacter RNA polymerase holoenzyme, core enzyme, or individual subunits according to the invention can be used in screening methods to identify candidate antibacterial agents In one embodiment, in vitro transcription is carried out using one or more purified Enterobacter RNA polymerase subunits, core enzyme, or holoenzyme, in a mixture containing components necessary for in vitro transcription A plurality of test compounds are added to the mixture prior to initiation of the reaction, and the effect of the compounds on transcription is monitored. Candidate antibacterial compounds are those compounds that modify, preferably decrease, the transcriptional activity of the mixture.

In another embodiment, the screening method comprises contacting purified RNA polymerase core enzyme, holoenzyme, or one or more individual subunits with a plurality of test compounds; and measuring the binding of the compounds to the RNA polymerase or subunit. Candidate antibacterial compounds are those compounds that bind to the RNA polymerase or subunits thereof.

Preferably, the screening methods of the present invention are adapted to a high-throughput format, allowing a multiplicity of compounds to be tested in a single assay. Such inhibitory compounds may be found in, for example, natural product libraries, fermentation libraries (encompassing plants and microorganisms), combinatorial libraries, compound files, and synthetic compound libraries. For example, synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich Chemical Company, Inc.

(Milwaukee, WI). Useful synthetic libraries have been described (Needels et al., Proc. Natl. Acad. Sci. USA 90:10700, 1993; Ohlmeyer et al. , Proc. Natl. Acad. Sci USA 90: 10922, 1993; Lam et al, WO 92/00252; and Kocis et al., WO 94/28028). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from, for example, Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., TibTech, 14:60, 1996), preferably using automated equipment, to allow for the simultaneous screening of a multiplicity of test compounds. Compounds identified as RNA polymerase inhibitors using the methods of the present invention may be modified to enhance potency, efficacy, uptake, stability, and suitability for use in pharmaceutical formulations, etc. These modifications are achieved and tested using methods well-known in the art.

The following examples are intended as non-limiting illustrations of the present invention.

Example 1: Purification of RNA Polymerase Holoenzyme and Core Enzyme From Enterobacter Cloacae Enterobacter cloacae RNA polymerase was purified as follows

A culture ofE. cloacae strain ATCC 13047 (obtained from the American Type Culture Collection) was grown to early log phase (A^ - 0 4-0.6) in LB medium and the cells were harvested by centrifugation The bacterial paste was weighed (100 g) and lysed (approximately 1 1 weight/volume) using a French Press (10,000-15,000 p s i) in 0.2 M KCl in TGΕD (50 mM Tris-HCl, pH 7 5, 10% glycerol, 1 mM ΕDTA 10 mM DTT and 10 mM MgCl₂) The crude extract was centrifuged at 10,000 x g for 30 min at 4°C

The supernatant solution was collected and 3 5 ml of 10% polyethylenimine (PΕI) (Sigma Chemical Co ) was added per 100 ml of supernatant The solution was stirred gently for 30 min at 4°C and centrifuged at 10,000 x g for 30 min at 4°C The pellet was resuspended with 30 ml of 0 5 M KCl in TGΕD and centrifuged at 10,000 x g for 30 min at 4°C The pellet was resuspended with TGΕD containing 1 M KCl and centrifuged at 10,000 x g for 30 min at 4°C The supernatant was collected and 35 g of ammonium sulfate was added per

100 ml supernatant The solution was stirred at 4°C for 3 hr and the pellet was collected by centrifugation at 20,000 x g for 60 min at 4°C and resuspended in TGΕD containing 0.2 M KCl

The solution was loaded onto a Bio-Gel A 1 5M (Bio-Rad, Hercules, CA) gel filtration column (bed volume 350 ml) equilibrated with TGΕD containing 0 1 M KCl The flow rate was 0 5 ml/min and 8 ml fractions were collected and analyzed by in vitro transcription reactions

In vitro transcription reactions utilized either pTac-promoter-containing DNA or calf thymus DNA as a template, to measure promoter-specific and non-specific transcription, respectively The pTac plasmid was constructed by cloning the following pTac promoter sequence

5'-TCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGT GTGGAATTGTGAGCG-3' (SΕQ LD NO- 1) into the BamHI and ΕcoRI sites of pBSKII plasmid (Straiagene, La Jolla, CA) Supercoiled plasmid DNA was used as the template for these reactions

Transcription reactions were performed essentially as described previously (Shorenstein 8c Losick, 1973) [α- ²P] UTP-incorporated RNA was synthesized in 50 μl reaction volumes containing transcription buffer (50 mM Tris-HCl, pH 8 0, 200 mM KCl, 10 mM MgCl₂, 10 mM DTT and 1 5 μM BSA), 1 μg of DNA template, 4 μM UTP containing 5 μCi of [α-³²P] UTP, and 400 μM each of ATP, GTP, and CTP The transcription reactions were incubated at 37°C for 30 min, after which newly transcribed RNA was precipitated onto glass fiber filter membrane using TCA (trichloroacetic acid) and radioactivity determined by scintillation counting

Figure 1A shows the size separation of RNA polymerase activity on the Bio- Gel column. A single peak of promoter-specific activity was observed

Transcriptionally active fractions from the Bio-Gel column were pooled and loaded onto a heparin agarose column (bed volume 30 ml) pre-equilibrated with TGED containing 0 1 M KCl The column was washed with three bed volumes of TGED containing

0 1 M KCl and eluted with a linear gradient of 0 1 M to 0 6 M KCl (150 ml). Fractions were assayed for promoter-specific and non-specific transcriptional activity

Most of the RNA polymerase activity eluted from the heparin column at 0 4M KCl Two peaks of promoter-specific activity were detected (Figure IB), the second of which overlapped with the peak of non-specific activity This suggests that the first and second major peaks for pTac DNA are due to the transcriptional activity of the holo and core enzymes, respectively The presence of sigma factor in the holoenzyme was confirmed by immunoblot analysis using polyclonal antibodies against the σ⁷⁰ from E. coh (see, Example 2 below) Fractions containing predominantly promoter-specific transcriptional activity and those containing predominantly non-specific transcriptional activity were pooled separately, dialyzed against TGED containing 50% glycerol and 0 5 M KCl, and stored in small aliquots at -80°C This procedure efficiently separated the holoenzyme from the core enzyme One hundred fifty g of bacterial pellet typically yielded 15 mg of pure RNA polymerase and purification quality was determined by SDS-PAGE analysis, as shown in Figure 2A. Purified E. cloacae RNA polymerase (lane 4) contains four polypeptides having molecular masses of approximately 156, 151, 82 and 45 kDa Without wishing to be bound by theory, based on their similarity in size to the corresponding E. coh. subunits, these polypeptides are identified as the β', β, σ, and α subunits, respectively, of E. cloacae RNA polymerase

To purify sigma factor and core enzyme, holoenzyme was dialyzed against TGED containing 0 1 M KCl The sample was loaded onto a 1 5 ml Bio-Rex70 column (BioRad, Hercules CA) pre-equilibrated with TGED containing 0 1 M KCl The column was washed with TGED containing 0 1 M KCl, and the core polymerase was eluted with 0 6M KCl TGED buffer The flow through contained the σ factor and the eluted peak contained core enzyme, as determined by SDS-PAGE analysis The enzymes were concentrated and dialyzed against TGED containing 50% glycerol and 0 5M KCl This procedure efficiently separated the σ factor from the core enzyme

Example 2: Characterization of E. Cloacae RNA Polymerase

E. cloacae RNA polymerase holoenzyme purified as described in Example 1 above was characterized as follows 1 Reaction conditions The optimum KCl. MgCl_?, and DTT concentrations, as well as the optimum temperature for E. cloacae RNA polymerase activity, were determined using in vitro transcription reactions containing pTac DNA as a template The KCl concentration was varied from 10 mM to 400 mM in the presence of 10 mM MgCl₂ and 10 mM DTT The optimum KCl concentration under these conditions was between 200 and 300mM (Figure 3 A) In a similar manner, the optimum MgCl₂ concentration was determined to be between 5 and 15 mM (Figure 3B) With respect to DTT, RNA polymerase retained full activity in buffers containing up to 100 mM DTT (Figure 3C) The optimum temperature was determined to be 40°C (Figure 3D)

By contrast, the concentrations of KCl, MgCl₂, and DTT and the temperature have very little effect on transcriptional activity when calf thymus DNA is used as a template

2 Drug sensitivity To test whether the E. cloacae RNA polymerase was sensitive to known transcription inhibitors, in vitro transcription reactions were performed using pTac DNA as a template in the presence of varying concentrations of rifampicin Three Gram-positive (S. aureus, E. facieum, B. subti s) and two other Gram-negative (E. coli and P. aeruginosa) RNA polymerases were tested in parallel Rifampicin inhibited the activites of the six polymerases with similar efficiency (Figure 4)

Example 3: Comparison of E. cloacae and E. coli RNA Polymerases

The following experiments were performed to evaluate the structural and functional homology between E. cloacae and E. co RNA polymerases

1 Immunoreactivitv Antibodies were obtained against E. coh σ factor as follows The full-length E. coli σ⁷⁰ gene (rpoD) was cloned in a plasmid containing a sequence encoding a polyhistidine tag (Qiagen, Chatsworth, CA) using the manufacturer's instructions; this plasmid was designated pKL210. N-terminally histidine-tagged σ⁷⁰ polypeptide was purified from E. coli strain BL21(DΕ3) (Novagen Inc., Madison WI) transformed with pKL210. Transformed bacteria were grown at 37° C in 1 L of LB containing ampicillin (200μg/ml) until the culture reached an OD₆₀₀ of 0.5. σ⁷⁰ expression was induced by the addition of isopropylthio-β-D-galactoside (LPTG) to a concentration of 2 mM, after which the culture was shaken for an additional 3 hr at 37° C. The culture was harvested by centrifugation (3000 x g; 30 min at 4°C), and the cell pellet was resuspended in 20 ml buffer A (20 mM Tris- HCl (pH 8.0), 500 mM NaCl, 5 mM imidazole). The cells were lysed by sonication, and the lysate was cleared by centrifugation (16,000 x g; 30 min at 4°C). N-terminally histidine- tagged σ⁷⁰ was precipitated by addition of ammonium sulfate to 60% saturation and collected by centrifugation (16,000 x g; 20 min at 4°C). The pellet was redissolved in buffer A containing 6M guanidine hydrochloride. The sample was adsorbed onto 1 mL of Ni²⁺-NTA agarose (Qiagen, Chatsworth, CA) equilibrated with buffer A, washed three times with buffer A followed by three washes with buffer B (50 mM Tris-HCl (pH 8), 200 mM KCl, 10 mM

MgCl₂, 10 μM ZnCl₂, 1 mM EDTA 5 mM 2-mercaptoethanol, 20% (v/v) glycerol) containing 30 mM imidazole. The protein was eluted with buffer A containing 500 mM imidazole. The eluted fraction was dialyzed for 16 hr at 4°C against two 1 L changes of buffer B. Following dialysis, the protein solution was incubated for 45 min at 30 °C and cleared by centrifugation (16,000 x g; 15 min at 4°C). The supernatant was adjusted to a final concentration of 50% glycerol (v/v) and stored at -20 °C. Polyclonal antibodies were raised in rabbits against the overexpressed and purified σ⁷⁰ gene product (Research Genetics, Huntsville, Alabama).

The anti-E. coli σ⁷⁰ antibodies prepared as described above were used to detect cross-reactivity between E. cloacae and E. coli RNA polymerases using immunoblots. Proteins were resolved by SDS-PAGΕ analysis and transferred to nitrocellulose membrane

(Bio-Rad Laboratories, Richmond, CA) as directed by the supplier. Membranes were treated with 1% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST) for one hour. Membranes were incubated with antibodies diluted with TBST for 2 hours. After washing, the membranes were treated with goat anti-rabbit alkaline phosphatase-conjugated antibody (Amersham, Arlington Heights, IL) for 60 minutes. After washing, antigen-antibody complexes were detected by soaking the membrane in NBT/BCLP solution for 10 mininutes.

As shown in Figure 5, antibody directed against E.coli σ⁷⁰ specifically recognized a single polypeptide species in the purified RNA polymerases of E. coli (lane 2), E. cloacae (lane 3) and P. aeruginosa (lane 4) At the antibody dilution used in Figure 5, cross- reacting bands were not observed for purified RNA polymerases from S. aureus, B. subti s and E. faecium. However, cross-reacting bands were observed when the primary antibody concentration was raised 25-fold 2 Functional reconstitution In vitro reconstitution assays were performed to determine whether E. cloacae σ factor can be substituted with the E. coh σ factor Core polymerases from E. coli and E. cloacae were purified which exhibited no detectable σ factor immunoreactivity Purified E. co σ factor was reconstituted in vitro with core polymerase to form holoenzyme by incubating for 30 minutes on ice prior to the transcription assay Efficient transcription was observed using both core enzymes, but only in the presence of E. coh σ factor (Figure 6 A) Furthermore, the transcriptional activity measured in these reactions was promoter-specific, as evidenced by a lack of non-specific activity (Figure 6B)

3 Promoter selectivity The promoter selectivity of E. cloacae and E. co RNA polymerases was compared by in vitro transcription using a plasmid containing a tac promoter and a rho-independent terminator as a template (Figure 7B) This plasmid was constructed by purifying the ribosomal gene terminator-containing Hindlll-Sspl fragment from plasmid pKK233-3 (Pharmacia LKB Technology), blunt-ending the Hindlll site, and cloning the resulting fragment into the Sspl site of the pTac-containing pBSKII plasmid described above

Both polymerases were able to initiate transcription from the tac promoter and produced a specific 527 nucleotide transcript (Figure 7A)

References

Allan, B. & Kropinski, A. M. (1987). DNA-dependent RNA polymerase from Pseudomonas aeruginosa Biochem. Cell. Biol 65 776-782

Avila, J., Hermoso, J. M., Vinuela, E. & Salas, M. (1971). Purification and properties of DNA-dependent RNA polymerase from Bacillus subtihs vegetative cells Eur. J. Biochem. 21 526-535

Burgess, R. R. (1969). Separation and characterization of the subunits of ribonucleic acid polymerase J. Biol. Chem. 244 6168-6176 Burgess, R. R. & Jendrisak, J. J. (1975). A procedure for the rapid, large-scall purification oϊ Escherichia coh DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography Biochem. 14 4634-4638

Davison, B. L., Leighton, T. & Rabinowitz, J. C. (1979). Purification oϊ Bacillus subtihs RNA polymerase with heparin-agarose In vitro transcription of phi 29 DNA J. Biol. Chem.

254 9220-9226

Deora, R. & Misra, T. K. (1996). Purification and characterization of DNA dependent RNA polymerase from Staphylococcus aureus. Biochem. Biophy. Res. Com. 208 610-616

Engbaek, F., Gross, C. & Burgess, R. R. (1976). Biosynthesis oϊ Escherichia coli RNA polymerase subunits upon release of rifampicin inhibition Mol. Gen. Genetics 143 297-299

Farmer EH, J. J. (1995). Enterobacteπaceae Introduction and Identification Clinical Microbiology, Murray, P.R.(ed) (ASM Press, Washington, D.C.) , 438-449

Hager, D. A., Jin, D. J. & Burgess, R. R. (1990). Use of Mono Q high-resolution ion- exchange chromatography to obtain highly pure and active Escherichia co RNA polymerase Biochem. 29 7890-7894

Johnson, J. C, DeBacker, M. & Boezi, J. A. (1971). Deoxyribonucleic acid-dependent ribonucleic acid polymerase of Pseudomonas putida J. Biol. Chem. 246 1222-1232

Lin, L.-S. & Rothstein, D. M. (1992). Micromonospora RNA polymerase activity changes dunng stationary phase J. Gen. Microbiol 138 1881-1885 Lonetto, M., Gribskov, M. & Gross, C. A. (1992). The sigma 70 family sequence conservation and evolutionary relationships J. Bacteriol. 174 3843-3849

McClure, W. R. (1985). Mechanism and control of transcription initiation in prokaryotes Ann. Rev. Biochem. 54 171-204

Shorenstein, R. G. & Losick, R. (1973). Comparative size and properties of the sigma subunits of ribonucleic acid polymerase from Bacillus subtihs and Escherichia coli. J. Biol

Chem. 248 6170-6173

Young BS, Guterman SK & A, W. (1976). Temperature-sensitive ribonucleic acid polymerase mutant oϊ Salmonella typhimurium with a defect in the beta' subunit J. Bacteriol.

127 1292-1297

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values are approximate, and are provided for description.

All patents, patent applications, publications, and other materials cited herein are hereby incorporated herein reference in their entireties.

SEQUENCE LISTING

<110> Scriptgen Pharmaceuticals Lam, Kevin

<120> PURIFIED RNA POLYMERASE FROM ENTEROBACTER

<130> 0342/2E133-WO

<140> Pending <141>

<150> 60/078,203 <151> 1998-03-16

<160> 1

<170> FastSEQ for Windows Version 3.0

<210> 1 <211> 59 <212> DNA

<213> "Artificial Sequence"

<400> 1 tctgaaatga gctgttgaca attaatcatc ggctcgtata atgtgtggaa ttgtgagcg 59

Claims

WHAT IS CLAIMED IS;

1 1 Purified RNA polymerase holoenzyme derived from Enterobacter species

1 2 A holoenzyme as defined in claim 1, wherein said species is E. cloacae

X 3 Purified RNA polymerase core enzyme derived from Enterobacter

2 species

1 4 A core enzyme as defined in claim 3, wherein said species is E. cloacae

X 5 A method for purification oϊ Enterobacter RNA polymerase, said method comprising

3 (i) harvesting and lysis of the bacterial cells and preparation of a cleared lysate,

5 (ii) precipitation of the cleared lysate with polyethylenimine,

6 followed by recovery and solubilization of the precipitate, (iii) separating the RNA polymerase by gel filtration chromatography

8 of the solubilized precipitate, and

9 (iv) separating the RNA polymerase by affinity chromatography using a hepann column

1 6 A screening method for identification of candidate antibacterial agents, said method comprising (i) providing a transcription mixture comprising Enterobacter RNA polymerase core enzyme or holoenzyme and components necessary for in vitro transcription, (ii) contacting the mixture with a plurality of test compounds, (iii) incubating the mixture formed in (ii) under conditions appropriate for in vitro transcription and measuring the transcriptional activity of the mixture, and (iv) selecting as candidate antibacterial compounds those compounds that modify the transcriptional activity of the mixture.

7. A screening method for identification of candidate antibacterial agents, said method comprising: (i) contacting purified Enterobacter RNA polymerase core enzyme, holoenzyme, or one or more individual subunits with a plurality of test compounds; and (ii) measuring any binding of the compounds to the RNA polymerase or subunit; and (iii) selecting as candidate antibacterial agents, those compounds that bind to the RNA polymerase or subunit.