Identity of the Ribosomal Proteins Involved in the Interaction with Elongation Factor G

Abstract

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 ribosomceEF-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.Establishment of the structure-function relationships of ribosomes and the mechanisms of GTP involvement in ribosome function are two of the major goals in defining the process of translation of the genetic code (for recent reviews, see refs. 3 and 4).Elucidation of ribosomal structure-function relationships has been thwarted by the complexity and cooperativity of ribosomal structure (the ribosome is composed of more than 50 distinct macromolecules), as well as the complexities of its functions. The availability of antibodies produced against individually purified ribosomal proteins now provides a potentially powerful tool with which to investigate the functional topology of ribosomes. Already, experiments involving the use of antibodies have successfully defined the specific ribosomal proteins involved in streptomycin and spectinomycin binding (5)**,tt, yielded information on the identity of the ribosomal proteins that bind ribosomal RNA (6, 7), and implicated proteins L7 and L12 in the interaction between ribosomes and EF-G (8).Similarly, elucidation of the roles of GTP in ribosome function has been hampered by the complexities of the re-* This is paper no. XLII in the series "Ribosomal Proteins" and paper no. XIII in the series "Studies on Translocation." The preceding papers in these series are refs. 1 and 2, respectively.

The Requirement for Ribosomal Proteins L7 and L12 in Peptide-Chain Termination

“…It has been suggested that L7 and L12 constitute a universal GTPase region on the ribosome which may recognize GTP bound to the various soluble factors (37). To date, GTP involvement has been only indirectly implicated in peptidechain termination in vitro using E. coli extracts, and no E. coli ribosomal GTPase activity of RF has been demonstrated.…”

Elongation factor‐dependent reactions on ribosomes deprived of proteins L7 and L12

“…It has been shown that the selective removal of acidic proteins L7 and L12 results in the inhibition of elongation factor-dependent reactions on the ribosome [2-81. Treatment of ribosomes with antibodies against proteins L7 and L12 gave a similar effect [9]. The use of bifunctional cross-linking agents has shown that proteins L7 and L12 are neighbours of the elongation factors when they interact with the ribosome, both for EF-G [lo] and EF-T, [ 111.…”

“…As seen in fig.2, the polyphenylalanine synthesis rate with the original (30 S t 50 S) and the 8 Time, min To exclude the possibility of the partial presence of the proteins L7 and L12 in some small active fraction of the extracted ribosomes, a special experiment was done in which the ribosomes were treated with antibodies against proteins L7 and L12. A 50-fold molar excess of the antibodies over the ribosomes was used which induces, according to Highland et al, a 50% inhibition of the translating acitivty of normal ribosomes [9]. The results of the EF-G-dependent poly(U) translation in the system where the 50 S subparticles were treated with antibodies against proteins L7 and L12, and when the antibodies continued to be present in the reaction mixture, are given in table 2.…”

“…At the sanie time, if antibodies against any other ribosomal proteins except L7 and L12 do not affect the binding of EF-G with the ribosome [9], then there is nothing left but to assume that ribosomal RNA could play the key role in the interaction of the elongation factors with the ribosome.…”

Acidic ribosomal proteins from the extreme halophile,Halobacterium cutirubrum

“…The functions of the ribosome are, to a large degree, invested in the protein components of the macrostructure [1][2][3][4][5]. Ribosomal proteins in most procaryotic and eucaryotic cells are basic in ionic character [6].…”