Preparation and Characterization of Two Monoclonal Antibodies against Different Epitopes in Escherichia coli Ribosomal Protein L7/L12*

Two monoclonal antibodies with specificities for Escherichia coli 50 S ribosomal subunit protein L7/ L12 were isolated. The antibodies and Fab fragments thereof were purified by affinity chromatography using solid-phase coupled L7/L12 protein as the immu- noadsorbent. The two antibodies were shown to recognize different epitopes; one in the N-terminal and the other in the C-terminal domain of protein L7/L12. Both intact antibodies strongly inhibited polyuridylic acid-directed polyphenylalanine synthesis, ribosome- dependent GTPase activity, and the binding of elongation factor EF-G to the ribosome. Ratios of antibody to ribosome of 4:l or less were effective in inhibiting these activities. Neither antibody prevented the asso- ciation of ribosomal subunits to form 70 S ribosomes. The Fab fragments showed similar effects.

The L7/L12 protein is essential for function. Its removal from the ribosome leads to the loss of all factor-dependent reactions of the protein synthesis cycle in which GTP hydrolysis occurs. The protein has been found to be associated with the binding of initiation factors 2 (15) and 3 (16); elongation factors EF'-Tu (17) and 19); release factor 2 (20); and stringent factor (21). It has been shown that 2 copies of L7/Ll2/ribosome are sufficient to support EF-G/ribosome-dependent GTPase activity, but that 4 copies of the protein are required for full activity in a polyphenylalanine-synthesizing system (6).
In order to understand fully the structure and function of this essential ribosomal protein, it will be necessary to determine precisely the location(s) of all 4 copies of L7/L12 on the ribosome and to identify the structurally and functionally important domains of the protein. Towards this goal, we set out to generate anti-L7/L12 monoclonal antibodies, to identify their epitopes, to test the functional properties of ribosomes that have these monoclonal antibodies or their Fab fragments bound to the known epitopes in L7/L12, and to locate the epitopes on the surface of the ribosome by means of immune electron microscopy. In this report, we present the isolation, purification, and the use as functional probes of two monoclonal antibodies specific for the N-terminal and Cterminal domains of protein L7/L12, respectively.

RESULTS
Two monoclonal antibodies reacting with different epitopes in ribosomal protein L7/L12 were isolated. One of the antibodies, designated Ab-NTF, was randomly chosen from a series of anti-L7/L12 antibody-producing hybridomas and subsequently shown to recognize an epitope in the N-terminal portion of L7/L12 (see results below). Ab-NTF was induced by immunization of BALB/c mice with uncleaved, purified L7/L12 protein. In order to obtain a monoclonal antibody with predetermined specificity for the C-terminal portion of protein L7/L12 (Ab-CTF), a fragment comprising amino acid l The abbreviations used are: EF, elongation factor; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid. * Portions of this paper (including "Experimental Procedures" and additional references) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-3775, cite the authors, and include a check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. 6522 residues 74-120 (L7/L12-CTF) was used for immunization. The L7/L12-CTF fragment was obtained by cleavage of citraconylated L7/L12 protein with trypsin and further purified by reverse-phase high performance liquid chromatography. The material eluted at 5296, acetonitrile migrated as a single band. Amino acid composition analysis of the material confirmed its identity as homogeneous L7/L12-CTF.
Highly purified monoclonal antibodies to be used as functional probes in protein synthesis assays were obtained by affinity chromatography of ascites fluid using solid-phase coupled L7/L12 protein as immunoadsorbent. The material that eluted from the column after lowering the pH to 3.0 was analyzed further under nonreducing conditions by high performance size exclusion chromatography with a TSK 3000 column. The material eluted as a single peak with an apparent molecular weight of 155,000, consistent with the size of an IgG molecule.
The fractions obtained from the affinity chromatography of ascites fluid were also characterized by SDS electrophoresis under reducing conditions. The electrophoretic patterns ob- tained from purified Ab-NTF and Ab-CTF showed the presence of heavy and light chains of the IgG molecules, but no detectable contaminants from the ascites fluid. Purified antibodies were digested with papain and the resulting Fab fragments were purified by affinity chromatography. Size exclusion chromatography under nonreducing conditions of the affinity chromatogrpahy flow through material and of material eluted after lowering the pH of the elution buffer to 3.0 revealed the presence of the Fc portion of the IgG molecule and the Fab fragments. Electrophoretic analysis of the two peak fractions under reducing conditions also identified the Fc fragment and the heavy and light chain portions of the Fab fragments. Both the Ab-NTF and Ab-CTF were found to belong to the IgG, k subclass.
The reactivity of the isolated antibodies with 30 S and 50 S ribosomal subunit proteins was assessed by immunoblot analysis. The antibodies were tested for their capacity to interact with intact ribosomes and ribosomal subunits. The results are shown in Fig. 2. Wells of microtiter plates were coated with 70 S ribosomes, 50 S and 30 S subunits, prior to reaction with Ab-NTF ( Fig. 2A) or Ab-CTF (Fig. 2B). The bound antibodies were assayed by the standard enzyme-linked immunosorbent assay procedure. Both antibodies recognize their respective epitope when L7/L12 is part of intact ribosomes. The Ab-NTF was less reactive than the Ab-CTF with the immobilized particles; moreover, it reacted more readily with 50 S subunits than with 70 S couples, suggesting decreased accessibility of the N-terminal epitope upon association of 50 S and 30 S subunits. As expected from the immunoblot analysis, neither antibody reacted with the 30 S ribosomal subunit.
Peptide fragments of L7/L12 were prepared in order to define the epitope specificity of the antibodies, in particular that of Ab-NTF for which intact L7/L12 was used as the immunogen. Citraconylated L7/L12 was cleaved with trypsin at Arg 73; intact L7/L12 was partially digested with V8 protease. The polypeptides were separated by electrophoresis in SDS, blotted onto nitrocellulose, and analyzed for reactivity with the antibodies. The results are shown in Fig. 3. Panel A shows that the antibody raised against intact L7/L12 reacts with fragment 1-73 and not 74-120. Furthermore, it does not react with fragment 29-120. Either cleavage at position 28-29 destroyed the epitope because it spans 1-28 and 29-73, or it lies within residues 1-28. Panel B summarizes the results. The solid lines represent L7/L12 protein or L7/L12 fragments that reacted with the antibody while the broken lines indicate fragments that were tested, but did not react with Ab-NTF. C, analysis with Ab-CTF. The numberedpanels are as described in the legend to Fig. 6A. 0, summary of the reactivity of Ab-CTF with L7/L12 fragments.
The antibody was designated Ab-NTF, indicative of its reactivity with the N-terminal fragment. Panel C shows that the antibody raised against fragment 74-120 reacts specifically with this fragment and not with fragment 1-73. Fragments shorter than 74-120 were not generated. Panel D summarizes the results for this antibody which was designated Ab-CTF, indicative of its reactivity with the C-terminal fragment. Both Ab-NTF and Ab-CTF were tested for their effect on in vitro poly(U)-directed polyphenylalanine synthesis. Synthesis was strongly inhibited by both antibodies when 50 S ribosomal subunits were preincubated with a 2-fold molar excess of either antibody prior to their use in the assay (Fig.   4A). A similar result was obtained when 70 S ribosomes were preincubated with antibody (Table I). On the other hand, if the antibodies were exposed to a 10-fold molar excess of purified protein L7/L12 prior to their preincubation with ribosomes, their inhibitory action was neutralized (Table I). Ribosomes preincubated with nonimmune mouse IgG were found to be as active as untreated ribosomes when tested in the polyphenylalanine synthesis assay (Table I). Fab fragments prepared from Ab-NTF and AB-CTF were also found inhibitory in the polyphenylalanine synthesis assay (Fig. 4B).
Preincubation of 50 S ribosomes with a 5-8-fold molar excess of Fab fragments over ribosomal subunits resulted in a degree of inhibition comparable to that produced by a 2-4-fold molar excess of intact antibody over ribosomal subunits.
Poly(U)-directed polyphenylalanine synthesis is dependent on the association of the ribosomal subunits to form 70 S ribosomes in the reaction mixture. Inhibition of subunit association or, alternatively, dissociation of 70 S ribosomes caused by the antibodies would result in inhibition of polyphenylalanine synthesis. An effect of Ab-NTF on subunit association would be consistent with the lower reactivity of 70 S versus 50 S shown in Fig. 2. To test this possibility, antibodies were preincubated with 50 S ribosomal subunits followed by addition of a 3-fold excess of 30 S ribosomal subunits over 50 S subunits. The mixtures were then analyzed by sucrose gradient centrifugation. The presence of neither Ab-NTF nor Ab-CTF prevented the formation of 70 S ribosomes: the sucrose gradient profiles were identical to those obtained from control ribosomes in the absence of antibody. In addition, the antibodies, despite their bivalent nature, did not cause the formation of any detectable 50 S ribosomal subunit dimers.
To investigate further the inhibitory effect of the antibodies in the polyphenylalanine synthesis system, the ribosomedependent GTPase activity, also a requirement for the protein synthesis-elongation cycle, was assayed. Fig. 5 shows that this activity was inhibited by Ab-NTF and Ab-CTF, while the presence of nonimmune mouse IgG had little effect. The antibodies, when preincubated with L7/L12 showed no significant inhibition (results not shown). Subsequent analysis of the binding of elongation factor EF-G to 70 S ribosomes revealed that both Ab-NTF and Ab-CTF prevented the factorribosome complex formation in the presence of GTP and fusidic acid (Fig. 6). This was also found when a noncleavable analog of GTP (in the absence of fusidic acid) was used in the assay (result not shown).

DISCUSSION
Two antibodies against mutually exclusive epitopes of protein L7/L12 have been prepared and used as probes of ribosome function. The epitope of one antibody (Ab-CTF) is located within residues 74-120, the C-terminal portion of the molecule. That of the other (Ab-NTF) is located within residues 1-73. Attempts to define the epitopes more precisely have been inconclusive, but suggest that the Ab-NTF epitope is located within the N-terminal one-third since the antibody fails to react with fragment 29-120. The Ab-NTF was obtained by immunization with the intact protein and the Ab-CTF by immunization with the 74-120 residue fragment. The latter result shows the feasibility of generating monoclonal antibody probes of predetermined specificity for ribosomal proteins.
The monoclonal antibodies have been tested for their effects on ribosome function. The results refine and extend those obtained previously with polyclonal antibodies of undetermined epitope specificity (22). Polyclonal antibodies to L7/L12 were found to inhibit polyphenylalanine synthesis (23), EF-G-dependent GTP hydrolysis (24), and EF-G binding to the ribosome (25). Our results agree with these earlier findings. Both NTF and CTF antibodies prevent association of elongation factor EF-G with the ribosome. Consequently, both EF-G-dependent GTP hydrolysis and polyphenylalanine synthesis are inhibited. A 2-4-fold excess of antibodies over ribosomes led to strong inhibition of the functions assayed, while in earlier studies, using polyclonal antibodies, a 20-100fold molar excess of antibody over ribosomes was required to

TABLE I Effect of antibodies against L7/L12 and mnimmune immunoglobulin on polyphenylalanine synthesis
Polyphenylalanine synthesis was assayed as described under Experimental Procedures ("Miniprint"). Particles were preincubated with antibodies for 10 min before initiating the reaction. The antibodies were in equimolar (1 and 2) or in 2-fold excess (3 and 4). The antibodies were preabsorbed with a 10-fold excess of L7/L12 prior to mixing with ribosomes. The activity of control 70 S ribosomes and 50 S subunits was 52 and 9 phenylalanine residues polymerized per particle, respectively.  at the tip of the stalk. 3 The mechanism by which this antibody inhibits EF-G binding is not clear at this time. The result suggests that the C termini may not always occupy a fixed location distal from the body of the ribosomal subunit at the tip of the stalk.
Preliminary studies on the effect of Ab-NTF show that incubation of 50 S subunits with these antibodies leads to the appearance of particles without stalks? On the other hand, 2 copies of L7/L12 remain on the ribosome as shown by radioactive labeling: The appearance of stalks has previously been studied as a function of the ratio of L7/L12 to 50 S core particles in reconstitution experiments and the results suggest that only one dimer is required for the appearance of a stalk (9). The possible specificity of the antibody induced release H. Olson and D. Glitz, unpublished results.  with respect to the multiple copies of L7/L12 and the locations of all the copies of L7/L12 are being investigated.