The binding of initiator and elongator tRNAs to 70-S ribosomes and the 30-S subunits was followed by velocity sedimentation in the analytical ultracentrifuge. Met-tRNAy" binds to A-U-G-programmed 30-S subunits, but not to free or misprogrammed particles. Both the formylmethione residue and the initiation factors increase the stability of the 30-S . A-U-G . Met-tRNAy" complex. M e t -t R N A p ' is bound only to the P site of the 70-S ribosome even in the absence of A-U-G. Two copies of tRNAPh' or Phe-tRNAPhe are bound to the ribosome with similar affinity. Incontrast to a recent report [Rheinbergeret al. (1981) Proc. NutlAcad. Sci. U S A , 78,5310-53141, it is shown that three copies of tRNA cannot be bound simultaneously to the ribosome with binding constants higher than 2 x lo4 M-'. Phe-tRNAPhe when present as the ternary complex Phe-tRNAPhe . EF-Tu . guanosine 5'-[P,y-methyleneltriphosphate binds exclusively to the A site. The peptidyl-tRNA analogue, acetylphenylalaninetRNA, can occupy both ribosomal centers, albeit with a more than tenfold higher affinity for the P site. The thermodynamic data obtained under equilibrium conditions confirm the present view of two tRNA binding sites on the ribosome. The association constants determined are discussed in relation to the mechanism of ribosomal protein synthesis.The two-site model for the interaction of tRNA with the ribosome has served to rationalize the individual steps occurring during protein synthesis. Both ribosomal centers interact with two types of tRNA each. The aminoacyl-tRNA binding site (A site) binds the ternary complex aminoacylt R N A . EF-Tu . GTP and the peptidyl-tRNA, whereas the peptidyl-tRNA binding site (P site) holds the peptidyl-tRNA and the deacylated tRNA [1,2].In a recent publication Rheinberger et al. reported three tRNA binding sites on the ribosome [3]. They showed that poly( U)-programmed 70-S ribosomes can retain three tRNA molecules on a nitrocellulose filter. The additional site is functionally defined as the exit site for the deacylated tRNA. This finding revived the discussion on the numbers and substrate specificity of the active centers of the ribosome. Thus Lake et al. postulated the existence of the recognition site (R site) for the incoming aminoacyl-tRNA on the basis of immune electron microscopy data [4]. The earlier literature on this topic is reviewed by Matthaei and his colleagues [5].Recently we introduced velocity sedimentation in the analytical ultracentrifuge as a new method to study tRNAribosomes complexes [6]. These experiments were carried out under conditions of thermodynamic equilibrium without any assumption on the rates of the binding process. In a continuation of this work we report here the binding characteristics of the initiator and elongator tRNAs to the 30-S subunit and 70-S ribosomes and discuss the influences of the Abbreviations. A site, aminoacyl-tRNA binding site of the ribosome; P site, peptidyl-tRNA binding site; AcPhe-tRNAPh', acetylphenylalanine-tRNAPhe; GuoPP[CH2]P, guanosine 5'-[j,y-methylene...
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