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...

Oxidative injury contributes to cellular damage during and after cerebral ischemia. However, the downstream catabolic pathways of damaged cellular components in neurons are largely unknown. In the current study, the authors examined the formation of oxidized proteins and their active degradation by the proteasome. In near-pure rat primary cortical neurons, it was found that protein-bound carbonyls as markers for oxidized proteins are increased after oxygen-glucose deprivation (OGD). During and after OGD, degradation of proteins metabolically radiolabeled before OGD increases two-to threefold compared with the normal protein turnover. Proteolysis after reoxygenation was attenuated by the presence of dimethylthiourea, a radical scavenger, and was blocked by lactacystin, a specific proteasome inhibitor. Lactacystin also increased the amount of protein carbonyls formed. In contrast, the activity of the proteasome complex itself after OGD was not different from sham-washed controls. The authors suggest that oxygen-glucose deprivation increases free radicals, which, in turn, oxidize proteins that are recognized and actively degraded by the proteasome complex. This protease itself is relatively resistant against oxidative injury. The authors conclude that the proteasome may be an active part of the cellular defense system against oxidative stress after cerebral ischemia.

Oligonucleotides such as G-A-G-G, which are complementary to the C-U-C-C region at the 3' end of 16-S RNA, inhibit the R17-RNA-dependent binding of the initiator tRNA (fMet-rRNA) to 30-S ribosomal subunits. However, if phage RNA is replaced by A-U-G, the same oligonucleotides stimulate the binding of fMet-tRNA to the 30-S subunits. This indicates that the formation of the RNA x RNA hybrid acts as a positive control signal for the selection of the initiator tRNA by the 30-S-subunit x mRNA complex. Tetranucleotides of the type A-U-G-N (where N = A, G, C or U) stimulated the IF-2-dependent binding of fMet-tRNA to the 30-S subunit more effectively than A-U-G, with A-U-G-R better than A-U-G-Y (where R is a purine nucleoside and Y is a pyrimidine nucleoside). Since the 3'-terminal adenosine in A-U-G-A can be replaced by 6-deamino-adenosine, a stacking type of interaction between U-33 of tRNA and N of A-U-G-N should additionally stabilize the codon-anticodon complex. The situation is strictly reversed for 70-S ribosomes where A-U-G is the best codon followed by A-U-G-U, A-U-G-C, A-U-G-G and A-U-G-A. Replacement of GTP by guanosine 5'-[beta, gamma-methylene]triphosphate (GuoPP[CH2]P] results in A-U-G-A becoming more efficient than A-U-G as the codon for the binding of fMet-tRNA to 70-S ribosomes. This indicates that IF-2 and GTP hold the anticodon of the fMet-tRNA in a conformation capable of binding to a tetranucleotide codon. GTP hydrolysis and release of IF-2 from the 70-S ribosome results in a change of the tertiary structure of fMet-tRNA as a consequence of which the initiator tRNA reassumes the conformation which preferentially binds to A-U-G.

No abstract

The involvement of nucleotides adjacent to the termination codons in tRNA during the suppression of termination has been formulated as the ‘context theory’ by Bossi and Roth (1980) [Nature (Lond.) 286, 123–127], The finding that U‐U‐G functions as an initiator codon has revived the discussion on the participation of the nucleotides flanking the initiator triplet in the decoding of initiator tRNA (context theory of initiation by the ribosome). We compared the capacity of oligonucleotides cognate to the anticodon loop of formylmethionine tRNA, such as A‐U‐G, A‐U‐G‐A and U‐A‐U‐G‐A, to enhance the formation of the 30‐S and 70‐S ribosomal initiation complexes. Three different methods were used to determine the apparent binding constants and the stoichiometries of the respective complexes : adsorption of the complexes to nitrocellulose filters, equilibrium dialysis, and velocity sedimentation. We found that in the 30‐S ribosomal initiation complex and in the presence of initiation factor 2 and GTP, formylmethionyl‐tRNA is preferentially decoded by more than three mRNA bases. With the 70‐S ribosome, however, once initiation factor 2 had been released, A‐U‐G represented the most effective codon to direct the formylmethionyl‐tRNA to the peptidyl site. An extended initiator sequence may either give additional stability to the 30‐S initiation complex or may allow for an ambiguity by one base pair in the decoding of the initiator tRNA.