Biochemistry zyxwvu 1988, 27, zyxwvu 5755-5762 5755 Structure and Function of the a-70 Subunit of Escherichia coli RNA Polymerase. Monoclonal Antibodies: Localization of Epitopes by Peptide Mapping and Effects on Transcriptiont Marie S. Strickland,* Nancy E. Thompson, and Richard R. Burgess* McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 450 North Randall Avenue, Madison, Wisconsin 53706 Received January 21, 1988 ABSTRACT: Murine monoclonal antibodies reactive with the major zyxwv u subunit (a-70) of Escherichia zyxw coli RNA polymerase were obtained by standard hybridoma techniques. Western blot analyses established that seven antibodies had unique specificities after various chemical and enzymatic methods were used to fragment u. Peptides were purified by HPLC using size-exclusion, reverse-phase, or ion-exchange chromatography. The epitopes for six of these antibodies have been localized to specific peptides. These peptides were further characterized by amino acid composition and N-terminal sequencing. u, which has a molecular weight of 70.2K, runs as 83K on SDS gels in this study. This anomalous behavior has been localized to the very acidic N-terminal half of the molecule. One antibody is unable to bind to native u. Two others do not bind well to u when it is contained in holoenzyme, indicating that their epitopes are in regions of u which are inaccessible in the holoenzyme complex. All three of these antibodies fail to inhibit in vitro transcription by holoenzyme. The other four antibodies all can inhibit in vitro transcription. Escherichia coli DNA-dependent RNA polymerase consists of a core polymerase zyxwvutsr (a2&3’w) and a u subunit which deter- mines the specificity for promoter sites. The major u factor (a-70) was originally isolated as a factor that stimulated in vitro transcription (Burgess et al., 1969). Many properties of the protein have been reported previously (Lowe et al., 1979), but the relatively small amount of protein available has slowed the study of the structural and functional properties. The DNA sequence of rpoD, the gene encoding a-70, has been determined (Burton et al., 1981) and a plasmid constructed that overexpresses the protein (Gribskov zyxwvuts & Burgess, 1983). Milligram quantities can now easily be purified, making possible the present study. As part of a detailed study of the structure and function of 070, we report the isolation of seven anti-a-70 monoclonal antibodies and localization of their ep- itopes. Studies of the effects of monoclonal antibodies to E. coli RNA polymerase /3 and zyxwvutsrq p’ subunits have been reported (Rockwell et al., 1985). We hope to use the antibodies re- ported here to help elucidate the function of u-70. MATERIALS AND METHODS All reagents were from Sigma Chemical unless otherwise specified. Milli-Q water (Millipore) was used throughout. Purification of zyxwvuts a-70. u was purified from an overproducing strain as described previously (Gribskov & Burgess, 1983) with the exception that a Mono Q column (5 mm X 5 cm, Phar- macia) was used instead of the Sephacryl S-200 column in the final purification step. The 0.3 M NaCl elution peak from the DE-52 column was loaded onto the Mono Q column which had been equilibrated with 10 mM tris(hydroxymethy1)- aminomethane hydrochloride (Tris-HCl),’ pH 8.9, 0.1 mM EDTA, 0.1 mM DTT, and 0.3 M NaCl, and the u was eluted with a 0.3-0.6 M NaCl, 30-mL gradient. Two peaks con- taining u elute from this column at about 0.42 and 0.54 mM NaCl, respectively. The earlier peak (which is the major ‘Supported by N I H Research Grants GM 28575, CA 23076, and CA * Address correspondence to this author. *Present address: Biotechnology Center, University of Wisconsin- Madison, 1710 University Ave., Madison, WI 53705. 09230. fraction) was used for all studies in this paper. The concen- tration of u was determined using El:& = 8.4 (Lowe et al., 1979). Preparation of Hybridomas. Adult female Balb/c ByJ mice (Jackson Laboratories, Bar Harbor, ME) were injected with 20 pg of purified u contained in Freund’s complete adjuvant (Difco), administered subcutaneously; 1 month later, the in- jection was repeated with Freund’s incomplete adjuvant. Approximately 1 month later and 3 days prior to the fusion, mice with titers greater that 1:2400, as determined by ELISA (Voller et al., 1978), were injected intraperitoneally with 20 pg of u contained in PBS. Spleen cells were fused with either NS1 or SP2/0 myeloma cells, using 40% poly(ethy1ene glycol) 1000 (Baker) according to standard methods (Fazekas de St. Goth & Scheidegger, 1980). Fusions were screened for specific antibody-producingcells by ELISA, using 96-well polystyrene plates coated with 0.8 Mg of u/mL and blocked with either 3% BSA or 1% BLOTTO (Carnation non-fat dry milk; Johnson et al., 1984). Culture fluid from each well was reacted with the immobilized u, and the presence of a-specific antibody was detected by using a horseradish peroxidase labeled goat antibody prepared against the mouse zyx y heavy chain (Zymed, South San Francisco, CA). Cells were cloned twice by limiting dilution, using spleen cells from nonimmunized Balb/c ByJ mice as helper cells. Stable cell lines were injected into Pristane-primed Balb/c ByJ mice for production of ascites fluid. Purification of Antibodies. Each antibody was precipitated from about 10 mL of cell-free ascites fluid by the addition of Abbreviations: BSA, bovine serum albumin; DTT, dithiothreitol; EIA, enzyme immune assay; ELISA, enzyme-linked immunosorbent assay; Gdn-HC1 (GuHC1 in figures), guanidine hydrochloride; HFBA, heptafluorobutyric acid; kDa, kilodalton(s); MAb, monoclonal antibody; NTCB, 2-nitro-5-thiocyanobenzoate; PBS, 10 mM phosphate (pH 7.4) buffered saline (150 mM NaC1); TBS, 10 mM Tris (pH 7.4) buffered saline (150 mM NaC1); TBST, TBS + 0.1% Tween-20 [poly(oxy- ethylene) sorbitan monolaurate]; TGED, 10 mM Tris (pH 7.9), 5% glycerol, 0.1 mM EDTA, and 0.1 mM DTT; TFA, trifluoroacetic acid; Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenediaminetetra- acetic acid. 0006-2960 f 8810427-5755%01.50 f0 0 1988 American Chemical Society