Monoclonal antibodies that inhibit activation of transcription by the Escherichia coli cyclic AMP receptor protein

The properties of the two monoclonal antibodies which were found to inhibit cyclic AMP receptor protein (CRP)-stimulated abortive initiation without affecting cAMP binding (Li, X.-M., and Krakow, J. S. (1986) J. Biol. Chem. 260, 4378-4383) have been characterized. Binding of monoclonal antibody (mAb) 66C3 to CRP is stimulated by cAMP while CRP binding by mAb 63B2 is not affected by cAMP. Binding of cAMP-CRP-mAb 63B2 to the lac P+ DNA is completely inhibited. Whereas cAMP-CRP forms a stable complex only at the CRP site 1 of the lac P+ promoter fragment, cAMP-CRP-mAb 66C3 binds to both site 1 and site 2. DNase I footprinting using a HpaII fragment carrying only the lac site 2 does not show any protection by cAMP-CRP-mAb 66C3. With the lac L8UV5 promoter, binding is not seen at either the L8 site 1 or the unaltered site 2. In the presence of 25% glycerol, cAMP-CRP-mAb 66C3 binds to both L8 site 1 and site 2. RNA polymerase is unable to bind to the cAMP-CRP-mAb 66C3-lac P+ complex. In the presence of RNA polymerase, cAMP-CRP forms a stable complex at the L8 site 1, the subsequent addition of mAb 66C3 results in the release of CRP. The CRP present in the lac P+ open promoter complex is partially resistant to subsequent incubation with mAb 66C3. The results provide further evidence regarding possible contacts between CRP and RNA polymerase involved in establishing the open promoter complex.     

Characterization of nine monoclonal antibodies against the Escherichia coli cyclic AMP receptor protein

Nine hybridoma clones producing antibodies against the Escherichia coli cAMP receptor protein (CRP) have been isolated. Five of the monoclonal antibodies (Class I) had a much higher affinity for native CRP while the remaining four (Class II) bound equally well to native or denatured CRP. Using native N-terminal CRP cores, it was shown that none of the Class I monoclonal antibodies cross-reacted with the 15,000-Da CRP core, and only two bound to the 18,800-Da CRP core. The positions of the antigenic determinants for the Class II monoclonal antibodies were found by Western blotting analysis to reside in the N-proximal region of CRP. Only one monoclonal antibody strongly inhibited cAMP binding by CRP, and this was accompanied by a consequent strong inhibition of both lac DNA binding and abortive initiation by RNA polymerase. Each of the Class I monoclonal antibodies inhibited abortive initiation, and four of these antibodies also blocked the binding of cAMP X CRP to the lac DNA fragment. One Class I and one Class II monoclonal antibody bound to the cAMP X CRP X DNA complex. Two of the Class II monoclonal antibodies were without apparent effect on any of the assays used.     

Monoclonal antibodies as probes of the topological arrangement of the alpha subunits of Escherichia coli RNA polymerase

Three monoclonal anti-alpha antibodies were used to study the properties of the alpha subunit of Escherichia coli RNA polymerase. None of the monoclonal antibodies inhibited the d(A-T)n-directed synthesis of r(A-U)n. Reassembly of the RNA polymerase core was blocked by mAb 129C4 or mAb 126C6 while no effect was observed with mAb 124D1. The conversion of premature to mature core was partially inhibited by mAb 129C4 and almost totally inhibited by mAb 126C6. The data suggest that during the course of core assembly at least one of the alpha subunits undergoes conformational changes. The increase in affinity of mAb 126C6 for assembled alpha compared with free alpha also implies that alpha undergoes conformational changes during RNA polymerase assembly. Double antibody binding studies showed that the epitopes for mAb 124D1 and mAb 129C4 are available on only one of the alpha subunits in RNA polymerase. It would appear that the relevant domain on one of the alpha subunits in RNA polymerase is well exposed whereas this domain on the second alpha subunit is shielded by interaction with regions of the large beta and beta' subunits. The alpha domain in which the epitope for mAb 126C6 resides is not impeded by subunit interactions in the RNA polymerase. The data obtained also suggest that in the holoenzyme the sigma subunit may be positioned close to one of the alpha subunits, probably to the more exposed alpha. The alpha beta complex is the minimal stable subassembly since one of the alpha subunits dissociates from the alpha 2 beta complex following binding of any of the monoclonal antibodies studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and functional influence of enzyme-antibody interactions: effects of eight different monoclonal antibodies on the enzymatic activity of Escherichia coli tryptophan synthase

Twelve monoclonal antibodies directed against the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) were produced from hybridoma clones. These monoclonal antibodies are found to recognize at least eight different epitopes on beta 2, and eight classes of monoclonal antibodies are thus defined. The effects of these monoclonal antibodies on the enzymatic activities of beta 2 are studied. The monoclonal antibodies from three classes rapidly inhibit the serine deaminase activity catalyzed by the beta 2 subunit alone; two of them lead to an inhibition plateau under stoichiometric conditions, and their inhibitory effects are cumulative. With the antibodies from two of these three classes, the tryptophan synthase activity of the alpha 2 beta 2 complex is recovered, through a competition between the alpha subunit and the monoclonal antibody. On the contrary, the antibody from the third class is inhibitory even in the presence of an excess of alpha subunit. The antibodies from the five other classes, though binding easily to the coated antigen in the enzyme-linked immunosorbent assay, react only very slowly with beta 2 in solution and, only after a long time of incubation, inhibit the enzymatic activity at different levels.     

Characterization and epitope mapping of monoclonal antibodies directed against the beta' subunit of the Escherichia coli RNA polymerase.

Monoclonal antibodies (mAbs) raised against the beta' subunit of the Escherichia coli RNA polymerase were used to probe the structure and function of this subunit. Of the five anti-beta' monoclonal antibodies studied, only mAb 311G2 is a strong inhibitor of RNA polymerase activity. This antibody binds to an epitope which is exposed in both the assembled holoenzyme and isolated beta' subunit. In contrast, the null antibodies bind to the free beta' subunit but very weakly to native RNA polymerase. It would appear that the beta' domain in which their epitopes reside is either conformationally altered or blocked due to interaction with other subunits in native RNA polymerase. In order to locate the positions of the epitopes for these five monoclonal antibodies, a series of overlapping deletion mutants have been constructed by partial restriction and religation of the beta' gene present in pT7 beta' (Zalenskaya, K., Lee, J., Gujuluva, C. N., Shin, Y. K., Slutsky, M., nd Goldfarb, A. (1990) Gene 89, 7-12). The presence of the epitopes for each of the anti-beta' monoclonal antibodies was assessed by Western blotting. The results indicate that the epitopes for mAb 340F11, mAb 370F3, mAb 371D6, and mAb 372B2 are located between amino acids 817-876. This region may be important in enzyme assembly or subunit-subunit interaction. The epitope for the inhibitory antibody, mAb 311G2, is located between amino acids 1047-1093. This region may be involved in the catalytic function of RNA polymerase.     

Effects of an anti-beta monoclonal antibody on the interaction of the Escherichia coli RNA polymerase with the lac and TAC promoters

The effects of an inhibitory monoclonal antibody (mAb) raised against the beta subunit of the Escherichia coli RNA polymerase were determined on the kinetics and structural interactions during formation of the open promoter complex (RPo). Analysis of the kinetics of abortive initiation on linear and supercoiled templates of the lac and TAC16 promoters showed that abortive synthesis by mAb 210E8-RNA polymerase varied as a function of DNA topology. A kinetic analysis of RPl formation on the supercoiled lac UV5 promoter showed that mAb 210E8 effected a slight alteration in the isomerization rate and no effect on the initial rate of RNA polymerase binding to the promoter. The potent inhibition of initiation with linear promoters by mAb 210E8 was not apparent when the promoters were assayed in their supercoiled forms. Abortive synthesis with the TAC16 promoter was accompanied by an mAb 210E8 induced hindrance of ApUpU but not UpGpU synthesis. The data indicate that the inhibition by mAb 210E8 with the supercoiled TAC16 promoter is further alleviated when the spacer length is shifted from 16 base pairs (ApUpU formation) to 18 base pairs (UpGpU formation). When DNase I and dimethyl sulfate were used to probe DNA structure, mAb 210E8 was found to alter polymerase interactions with the lac promoter. DNase I footprinting indicated that the structural interactions for lac P+ promoter-RNA polymerase complexes were slightly altered in the presence of mAb 210E8. Treatment of the RNA polymerase-lac UV5 complex with dimethyl sulfate revealed an alternate mode of RNA polymerase interaction with essential guanine contacts which was intermediate between a fully protected and free promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.

We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication.     

Monoclonal antibodies directed against mammalian RNA polymerase I. Identification of the catalytic center

Mouse myeloma cells were fused with splenocytes from a mouse that had been immunized with RNA polymerase I purified from a rat hepatoma. Hybridoma cells were selected and colonies secreting antibodies directed against the enzyme were detected by analysis of cell culture supernatants in a solid phase radioimmunoassay. Two of these cell lines were grown on a larger scale and the interaction between the immunoglobulins obtained from them and RNA polymerase I was studied in detail. Antibodies from both of the hybridoma cell lines were able to inhibit DNA-dependent RNA synthesis catalyzed by RNA polymerases I and III, but not that catalyzed by polymerase II. The antibodies were also capable of reducing the RNA chain elongation reaction catalyzed either by RNA polymerase I associated with isolated nucleoli or by enzyme preinitiated in vitro on calf thymus DNA. Inhibition of RNA polymerase I activity by the monoclonal antibodies was inversely related to the nucleotide concentration. In contrast, the DNA concentration had relatively little effect on inhibition by the antibodies. Analysis of immune complex formation between the antibodies and isolated individual enzyme subunits demonstrated that the monoclonal antibodies were directed against the largest (Mr = 190,000) polypeptide of the polymerase I. These data indicate that the largest subunit of RNA polymerase I contains an immunological determinant in common with RNA polymerase III and suggest that the polymerase I polypeptide of Mr = 190,000 contains a catalytic center involved in RNA chain elongation.