Identification of a ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes.

A ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes has been identified using the following criteria: 1. A loss in the thiostrepton binding ability of the ribosome was correlated with the selective removal of ribosomal protein L11. This was achieved by a comparison of the thiostrepton binding ability of 50 S ribosomal subunits treated with 1 M NH4C1 and 50% ethanol at 37 degrees which still contained protein L11, and subunits treated successively at 0 degrees and 37 degrees in the same medium, from which protein L11 had been removed. 2. The thiostrepton binding ability of a ribosomal core containing only seven proteins, produced by treatment of 50 S subunits with 4 M LiC1, was fully restored by the rebinding of protein L11, obtained by Sephadex G-100 fractionation of the 1 M LiC1 split protein fraction from 50 S subunits. In addition, treatment of the 1 M LiC1 split protein fraction with an IgG specific for protein L11, uniquely inhibited the restoration of activity. 3. Thiostrepton binding to the 4 M LiC1 core, reconstituted with the 1 M LiC1 split protein fraction, was blocked by treatment with a monovalent antibody fragment (Fab) prepared against protein L11, but not by treatment with antibodies specific for the proteins of the 4 M LiC1 core. We conclude, therefore, that protein L11 is required for the ribosomal binding of thiostrepton.

Assembly of ribosomal proteins L7, L10, L11, and L12, on the 50 S subunit of Escherichia coli.

We have determined the in vitro assembly sequence of ribosomal proteins L7, L10, L11, and L12 on Escherichia coli 50 S subunits by reconstitution experiments with the use of various ribosomal core particles and split protein fractions produced by treatment of 50 S subunits with 1 m NH4Cl and 50% ethanol. Proteins L7, L10, L11, and L12 were removed by a two-step treatment, first at 0 degrees, then at 37 degrees. Small amounts of proteins L1, L5, and L6 were also removed under these conditions. A one-step extraction of 50 S subunits at 0 degrees removed only proteins L7 and L12, while a similar one-step extraction of intact 50 S subunits at 37 degrees removed proteins L7, L12, and L10. Two-dimensional gel electrophoresis of the protein components and measurement of the ribosome-elongation factor G-guanosine diphosphate complex formed with the various reconstituted particles showed that the binding of proteins L7 and L12 is dependent on the binding of protein L10 and in turn, that the binding of protein L10 is dependent on the binding of protein L11.

Coordinate inhibition of elongation factor G function and ribosomal subunit association by antibodies to several ribosomal proteins.

We previously showed that treatment of Escherichia coli ribosomes with antibodies specific for proteins L7 and L12 inhibits both EF-G.GDP binding and ribosome-dependent GTP hydrolysis (Highland et al. (1973) Proc. Nat. Acad. Sci. USA 70, 142-150; and Kischa et al. (1971) Nature New Biol. 233, 62-63). We now report that antibodies to six additional proteins also inhibit GTP hydrolysis, but do not inhibit EF-G.GDP binding. Moreover, inhibition by these antibodies is dependent on the state of association of the treated ribosomes. When 70S couples are treated, only antibodies to proteins L14 and L23 are inhibitory and then only partially. However, when separated ribosomal subunits are treated individually and then mixed with the complementary untreated subunits, inhibition by antibodies L14 and L23 is complete, and antibodies to proteins L19, L27, S9, and S11 now show an inhibitory effect. In addition, treatment of subunits with any of these six antibodies (but not those to L7 or L12) results in inhibition of reassociation, which is presumably responsible for the inhibition of hydrolysis. These data suggest that the area of interaction between EF-G and the ribosome is restricted to proteins L7 and L12, and that antibodies to proteins L14, L19, L23, L27, S9, and S11, but not L7 and L12, block the physical association of the subunits.

Identity of the ribosomal proteins involved in the interaction with elongation factor G.

Rabbit antibodies produced against 50 of the 55 individually purified ribosomal proteins of Escherichia coli were tested for their ability to interfere with the formation of the ribosome.EF-G.GDP complex. Only antibodies produced against proteins L7 and L12 inhibited complex formation, and they did so completely. These two proteins were previously shown to be immunologically indistinguishable and necessary for the interaction between ribosomes and EF-G. The present data are consistent with the view that the interaction between ribosomes and EF-G that results in GTP hydrolysis occurs on, and is limited to, proteins L7 and L12 on the surface of the 50S ribosomal subunit.