Binding of tRNA to Escherichia coli Ribosomes as Measured by Velocity Sedimentation

Abstract

We followed the binding of initiator and elongator tRNA to 70-S ribosomes and its subunits by velocity sedimentation in the analytical ultracentrifuge. This technique shows the advantage over the previously used methods (adsorption of the complexes to nitrocellulose filters or fluorescence titrations) in that no kinetic effects obscure the equilibrium data and that none of the components has to be chemically modified. The concentrations of the macromolecular compounds are kept constant and the binding equilibria are shifted by varying the Mg2+ concentration in a range which is accessible to experimental analysis.Free 30-S ribosomes bind no tRNA, whereas one tRNA molecule is bound to 50-S ribosomal subunits. In thc presence of the cognate codon one tRNA can be associated with the small subunit. Free, programmcd, or misprogrammed 70-S ribosomes bind exactly two elongator tRNAs. Only the initiator t RNA does discriminate significantly between the two ribosomal sites when bound to a ribosome . A-U-G complex.The current picture of the mechanism of protein synthesis is still consistent with the original proposal by Watson [l]. The 70-S ribosome contains two tRNA binding sites, the socalled aminoacyl-tRNA binding site (A site) and the peptidyltRNA binding site (P site). Both centers are capable of interacting with two types of tRNA each, the A site with the ternary complex aminoacyl-tRNA . EF-Tu . GTP and the peptidyl-tRNA; the P site with peptidyl-tRNA and the deacylated tRNA. In both instances the first type of tRNA represents the substrate, the latter the product for the respective binding sites. The experimental distinction between the two sites relies on the fact that the peptidyl group of the tRNA when bound to the P site is transferable to puromycin but not when bound to the A site.However, additional tRNA binding sites with functional roles in protein synthesis have been suggested [2,3]. Matthaei and his colleagues postulated from Phe-tRNA binding to poly(U)-programmed ribosomes three functionally defined sites on a 70-S ribosome [4]. The same authors showed with phage RNA that in addition to m e t -t R N A two aminoacyl tRNAs can be bound to the ribosome [5]. Kirillov et al. postulated two binding sites on the 30-S ribosomal subunit and two sites on the 70-S ribosome, albeit with different affinities [6,7]. Lake derived from immune electron microscopy data the existence of the so-called recognition site (R site) as an entry site for aminoacyl tRNA [8]. This site shares the anticodon region with the A site, but the remaining parts of the tRNA are bound to the exterior of the 30-S subunit. Recently Rheinberger and Nierhaus presented evidence for a third site as well which they defined as an exit site for the deacylated tRNA. The existence of the E site was concluded from the binding of two tRNAPh" molecules to a ribosome . poly(U) . AcPhe-tRNA complex 191.Ahbrcviations. A site, aminoacyl-tRNA binding site of the ribosome; P sitc, peptidyl-tRNA binding site; t R N A y and tRNAM,", the methionine tRNAs which can a...

The Ribosomal Elongation Cycle: tRNA Binding, Translocation and tRNA Release

“…even at a 37-fold molar excess of tRNA over 70s ribosomes, non-programmed ribosomes do not bind more than one deacylated t RNA molecule/ribosome in agreement with our previous report [2]. In contrast, Gassen and coworkers found two t RNA molecules bound/ribosome using velocity sedimentation [16]. If in fact a second tRNA can be detected by the centrifugation technique, then this tRNA must dissociate rapidly, thus escaping detection by the filtration technique.…”

“…Alternatively (although less probable), tRNA could be released and, due to the high affinity of the P site, rebind to another ribosomal P site. Thus, previous experiments (for review see [16]) and the one in Fig. 4 cannot definitively distinguish between a lack of release and a release plus rebinding.…”

The peptidyl‐substituent within peptidyl‐tRNA increases the stability of tRNA—mRNA association

“…The differences in biochemical behaviour of a peptidyl-tRNA and of a deacylated tRNA are shown by the characteristics of their binding to the ribosome. Here, we used velocity sedimentation in the analytical ultracentrifuge and altered the affinity of the tRNA towards the programmed ribosome by varying the [Mg 2÷] [10]. Free tRNA Met does not discriminate between the A and the P site of the ribosome and two tRNA molecules are bound with equal affinity to both sites.…”

“…Velocity sedimentation experiments using a 'Spinco model E' analytical ultracentrifuge have been described in [10]. The optical cells contained in 450 gl: 50 mM Tris-HCl (pH 7.5), 150 mM NH4CI, 10 mM mercaptoethanol, 8-35 mM MgCI2, 90 pmol 70 S ribosomes, 450 pmol tRNA, and 30 nmol AUG.…”

Binding of tRNA in Different Functional States to Escherichia coli Ribosomes as Measured by Velocity Sedimentation

“…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.…”