A codon sugar structure strongly influences tRNA binding to programmed ribosomes.

To study the role of a codon sugar-phosphate backbone in aminoacyl-tRNA selection on the ribosome a comparison of tRNA(Phe) affinity for pdTpdTpdT and prUprUprU in solution, and for correspondingly programmed 30S ribosomal subunits has been performed. In solution the tRNA(Phe) affinity for pdTpdTpdT appeared to be even slightly higher than for prUprUprU, whereas deoxyribocodon was significantly less efficient in the stimulation of Phe-tRNA(Phe) binding to the 30S ribosomal subunit. Some difference in neomycin action in both systems was revealed.     

The involvement of 5S RNA in the binding of tRNA to ribosomes

The tRNA fragment TpψpCpGp was found to bind to 5S RNA. This binding is ten times increased when a specific 5S RNA-protein complex is used. The ability of TpψpCpGp to bind to the complex could be abolished by selective chemical modification of two adenines in 5S RNA. Such 5S RNA, when incorporated into 50S ribosomal subunits, yielded particles with greatly reduced biological activities. From the results presented we conclude that 5S RNA is most likely part of a site with which the TψC-loop of tRNA interacts on the ribosome.     

tRNA binding sites on the subunits of Escherichia coli ribosomes

Programmed 30 S subunits expose only one binding site, to which the different classes of tRNA (deacylated tRNAPhe, Phe-tRNAPhe, and N-acetylphenylalanyl (AcPhe)-tRNAPhe) bind with about the same affinity. Elongation factor Tu within the ternary complex does not contribute to the binding of Phe-tRNA. Binding of acylated or deacylated tRNA to 30 S depends on the cognate codon; nonprogrammed 30 S subunits do not bind tRNA to any significant extent. The existence of only one binding site/30 S subunit (and not, for example, two sites in 50% of the subunits) could be shown with Phe-tRNAPhe as well as deacylated tRNAPhe pursuing different strategies. Upon 50 S association the 30 S-bound tRNA appears in the P site (except the ternary complex which is found at the A site). Inhibition experiments with tetracycline demonstrated that the 30 S inhibition pattern is identical to that of the P site but differs from that of the A site of 70 S ribosomes. In contrast to 30 S subunits the 50 S subunit exclusively binds up to 0.2 and 0.4 molecules of deacylated tRNAPhe/50 S subunit in the absence and presence of poly(U), respectively, but neither Phe-tRNA nor AcPhe-tRNA. Noncognate poly(A) did not stimulate the binding indicating codon-anticodon interaction at the 50 S site. The exclusive binding of deacylated tRNA and its dependence on the presence of cognate mRNA is reminiscent of the characteristics of the E site on 70 S ribosomes. 30 and 50 S subunits in one test tube expose one binding site more than the sum of binding capacities of the individual subunits. The results suggest that the small subunit contains the prospective P site and the large subunit the prospective E site, thus implying that the A site is generated upon 30 S-50 S association.     

Chemical synthesis of capped RNA fragments and their ability to complex with eukaryotic ribosomes

In other to examine the binding ability of the 5'-terminal part of eukaryotic mRNA to 80S ribosome, several kinds of oligoribonucleotides, pA-U-G, m7G5'pppA-U-G, m7G5'pppG-U, m7G5'pppA-U-G-A-C-C, were synthesized chemically. The binding experiments of oligonucleotides to 80S ribosome showed that the capped structure as well as AUG are essential for ribosome binding, and the efficiency is enhanced by the 5'-leader sequence if it would include complementary sequence to the 3'-terminal part of 18S rRNA.     

The effect of acridine orange on the structure of ribosomes

Exposure of Escherichia coli MRE-600 ribosomes to acridine orange (AO) at low ionic strength (1mM Tris-acetate pH 7.4) results in quantitative binding of the dye. Under our experimental conditions about a few hundred dye molecules can be bound to any one of the 30, 50, or 70-S particles. AO causes the 30 and the 50-S subunits to form ribosomal aggregates of approximate sedimentation constants of 70 and 100-S.     

Effect of spermine on the efficiency and fidelity of the codon-specific binding of tRNA to the ribosomes

Binding of the yeast Tyr-tRNA and Phe-tRNA to the A site, and the binding of their acetyl derivatives to the P site of poly(U11,A)-programmed Escherichia coli ribosomes was studied. Spermine stimulated the rate of binding of both tRNAs at least threefold, enabling more than 90% final saturation of both ribosomal binding sites. The effect is observed when the tRNAs, but not ribosomes or poly(U11,A), are preincubated with polyamine. Regardless of the binding site, optimal saturation was reached at spermine/tRNA molar ratios of 3 for tRNA(Phe) and 5 for tRNA(Tyr). The same low spermine/tRNA ratios were previously reported to stabilize the conformation of these tRNAs in solution. On the other hand, the messenger-free, EF-Tu- and EF-G-dependent polymerization of lysine from E. coli Lys-tRNA is drastically reduced, while the poly(A)-directed polymerization is stimulated by spermine through a wide range of Mg2+ concentrations. Misreading of UUU codons as isoleucine, assayed by the A-site binding of E. coli Ile-tRNA, is also inhibited by spermine. All these results demonstrate that spermine increases the efficiency and accuracy of a series of macromolecular interactions leading to the correct incorporation of an amino acid into protein, at the same time preventing some unspecific or erroneous interactions. From the analogy with its known structural effects, it can be inferred that spermine does so by conferring on the tRNA a specific biologically functional conformation.     

Number of tRNA binding sites on 80 S ribosomes and their subunits

The ability of rabbit liver ribosomes and their subunits to form complexes with different forms of tRNAPhe (aminoacyl-, peptidyl- and deacylated) was studied using the nitrocellulose membrane filtration technique. The 80 S ribosomes were shown to have two binding sites for aminoacyl- or peptidyl-tRNA and three binding sites for deacylated tRNA. The number of tRNA binding sites on 80 S ribosomes or 40 S subunits is constant at different Mg2+ concentrations (5-20 mM). Double reciprocal or Scatchard plot analysis indicates that the binding of Ac-Phe-tRNAPhe to the ribosomal sites is a cooperative process. The third site on the 80 S ribosome is formed by its 60 S subunit, which was shown to have one codon-independent binding site specific for deacylated tRNA.     

Conformational change of 50 S ribosomes on enzymatic binding of phenylalanyl-tRNA

Tight couple 70 S ribosomes are converted to loose couple ones on enzymatic binding of phenylalanyl-tRNA. Enzymatic binding at 0 degree C as well as nonenzymatic binding does not lead to any change. Further, no change takes place when the P site is occupied by N-acetylphenylalanyl-tRNA. Loose couple 70 S ribosomes are not affected by either enzymatic or nonenzymatic binding of phenylalanyl-tRNA.     

Translation initiation by factor-independent binding of eukaryotic ribosomes to internal ribosomal entry sites

Two exceptional mechanisms of eukaryotic translation initiation have recently been identified that differ fundamentally from the canonical factor-mediated, end-dependent mechanism of ribosomal attachment to mRNA. Instead, ribosomal 40S subunits bind in a factor-independent manner to the internal ribosomal entry site (IRES) in an mRNA. These two mechanisms are exemplified by initiation on the unrelated approximately 300 nt.-long Hepatitis C virus (HCV) IRES and the approximately 200 nt.-long cricket paralysis virus (CrPV) intergenic region (IGR) IRES, respectively. Ribosomal binding involves interaction with multiple non-contiguous sites on these IRESs, and therefore also differs from the factor-independent attachment of prokaryotic ribosomes to mRNA, which involves base-pairing to the linear Shine-Dalgarno sequence. The HCV IRES binds to the solvent side of the 40S subunit, docks a domain of the IRES into the ribosomal exit (E) site and places the initiation codon in the ribosomal peptidyl (P) site. Subsequent binding of eIF3 and the eIF2-GTP/initiator tRNA complex to form a 48S complex is followed by subunit joining to form an 80S ribosome. The CrPV IRES binds to ribosomes in a very different manner, by occupying the ribosomal E and P sites in the intersubunit cavity, thereby excluding initiator tRNA. Ribosomes enter the elongation stage of translation directly, without any involvement of initiator tRNA or initiation factors, following recruitment of aminoacyl-tRNA to the ribosomal aminoacyl (A) site and translocation of it to the P site.