The binding sites for tRNA on eukaryotic ribosomes.

We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes.     

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.     

Competition between trichodermin and several other sesquiterpene antibiotics for binding to their receptor site(s) on eukaryotic ribosomes.

1. Of the five sesquiterpene antibiotics tested and found to inhibit protein synthesis in yeast spheroplasts, trichothecin, trichodermol or trichodermin stabilized polyribosomes whereas, in contrast, verrucarin A or T-2 toxin induced 'run off' of polyribosomes with a corresponding increase in 80S monoribosomes. The effect of fusarenon X on the system could not be determined as the drug failed to enter the cells. 2. [acetyl-14C]Trichodermin bound to yeast polyribosomes with a dissociation constant of 2.10 muM and to yeast 'run off' ribosomes with a dissociation constant of 0.72 muM. 3. Trichothecin, trichodermol, fusarenon X, T-2 toxin and verrucarin A competed with [acetyl-14C]trichodermin for binding to its receptor site on 'run off' ribosomes. The observed competition was quantitatively similar for all drugs tested. In contrast, the five drugs competed to different extents with trichodermin for binding to its receptor site on polyribosomes. Thus trichothecin competed with relative efficiency, whereas verrucarin A competed poorly, and the other drugs occupied intermediate positions between these two extremes. 4. Studies were also carried out with yeast 'run off' ribosomes prepared from both a wild-type strain and a strain resistant to trichodermin. Competition experiments between verrucarin A and [3H]anisomycin indicated that verrucarin A bound to 'run off' ribosomes from the mutant strain less efficiently than to those from the wild-type.     

Zn(II) binding to Escherichia coli 70S ribosomes

Escherichia coli 70S ribosomes tightly bind 8 equiv of Zn(II), and EXAFS spectra indicate that Zn(II) may be protein-bound. Ribosomes were incubated with EDTA and Zn(II), and after dialysis, the resulting ribosomes bound 5 and 11 equiv of Zn(II), respectively. EXAFS studies show that the additional Zn(II) in the zinc-supplemented ribosomes binds in part to the phosphate backbone of the ribosome. Lastly, in vitro translation studies demonstrate that EDTA-treated ribosomes do not synthesize an active Zn(II)-bound metalloenzyme, while the as-isolated ribosomes do. These studies demonstrate that the majority of intracellular Zn(II) resides in the ribosome.     

Magnesium binding to yeast ribosomes

This paper describes a theoretical and experimental analysis of the binding of magnesium ions to yeast, ribosomes. In the theoretical considerations the interactions between charges located on a macroion are included. In the calculations these interactions result in a term, in which both the charge and the radius of the macroion are accounted for. It appears that on dissociation of the ribosomes both the charge and the radius change, but in such a way, that the term, which accounts for the electrostatic interactions, remains constant. As a consequence the dissociation can lie neglected in the analyses of the binding experiments. Our experiments indicate that two binding reactions between ribosomes and magnesium ions occur. The endpoints of these reactions correspond to about 0.40 and 1.0 equivalent magnesium per ribosomal phosphate, respectively. The pK values are about 3.8 and 2.2, respectively. The experimental results indicate that the effect, of monovalent cations can be explained as a pure ionic strength effect, though the binding of monovalent cations could not be excluded completely.     

The 5-S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes. Evolution of the eukaryotic 5-S RNA binding protein.

The ribonucleoprotein complex between 5-S RNA and its binding protein (5-S RNA . protein complex) of yeast ribosomes was released from 60-S subunits with 25 mM EDTA and the protein component was purified by chromatography on DEAE-cellulose. This protein, designated YL3 (Mr = 36000 on dodecylsulfate gels), was relatively insoluble in neutral solutions (pH 4--9) and migrated as one of four acidic 60-S subunit proteins when analyzed by the Kaltschmidt and Wittman two-dimensional gel system. Amino acid analyses indicated lower amounts of lysine and arginine than most ribosomal proteins. Sequence homology was observed in the N terminus of YL3, and two prokaryotic 5-S RNA binding proteins, EL18 from Escherichia coli and HL13 from Halobacterium cutirubrum: Ala1-Phe2-Gln3-Lys4-Asp5-Ala6-Lys7-Ser8-Ser9-Ala10-Tyr11-Ser12-Ser13-Arg14-Phe15-Gln16-Tyr17-Pro18-Phe19-Arg20-Arg21-Arg22-Arg23-Glu24-Gly25-Lys26-Thr27-Asp28-Tyr29-Tyr35; of particular interest was homology in the cluster of basic residues (18--23). Since the protein contained one methionine residue it could be split into two fragments, CN1 (Mr = 24700) and CN2 (Mr = 11300) by CNBr treatment; the larger fragment originated from the N terminus. The N-terminal amino acid sequence of CN2 shared a limited sequence homology with an internal portion of a second 5-S RNA binding protein from E. coli, EL5, and, based also on the molecular weights of the proteins and studies on the protein binding sites in 5-S RNAs, a model for the evolution of the eukaryotic 5-S RNA binding protein is suggested in which a fusion of the prokaryotic sequences may have occurred. Unlike the native 5-S RNA . protein complex, a variety of RNAs interacted with the smaller CN2 fragment to form homogeneous ribonucleoprotein complexes; the results suggest that the CN1 fragment may confer specificity on the natural 5-S RNA-protein interaction.     

Electric birefringence of eukaryotic ribosomes

Eukaryotic 60S ribosomal subunits were studied by transient electric birefringence. Conformations of subunits in active and inactive states upon changes in magnesium concentration were compared by electric birefringence and orientational relaxation time measurements. Active subunits exhibit a positive birefringence and a relaxation time of the order of 8 microseconds. In the presence of EDTA, inactive subunits show no birefringence. When Mg2+ is reverted in the cold to its initial level, the electro-optical properties of the subunits are partially restored although the particles remain biologically inactive.     

Dissociation of eukaryotic ribosomes by purified initiation factor EIF-3

Purified eukaryotic initiation factor, EIF-3, prepared from ascites cells dissociated rat liver 80S ribosomes into subunits. Ribosomes bearing endogenous mRNA and nascent peptide were not dissociated by EIF-3. When 80S ribosomes reconstituted from subunits were used as substrate the reaction had the following characteristics: Dissociation was rapid--the reaction being completed within 2 min at 30°. The extent of dissociation was directly proportional to the amount of EIF-3; with 21 μg of EIF-3 about 70% (or 10.5 μg) of the 80S monomers were dissociated. The dissociation of 80S monomers by EIF-3 decreased with increasing concentrations of magnesium. The reaction was not catalytic: 28 moles of EIF-3 were required to dissociate 1 mole of 80S ribosomes. The characteristic of the dissociation reaction promoted by EIF-3 and by $\text{E. coli}$ initiation factor IF-3 are remarkably similar. The dissociation reaction provides a practical assay for EIF-3 independent of complimentation of other initiating factors.     

Puromycin binding to the donor (P-) site of Escherichia coli ribosomes

Puromycin inhibits the interaction of peptidyl-tRNA analogues AcPhe-tRNA_ox-red^Phe, AcPhe-tRNA^Phe and fMet-tRNA_f^Met with the donor (P-) site of Escherichia coli ribosomes. affects almost equally both the rate of the binding and the equilibrium of the system. This means that the effect is due to direct competition for the P-site, but not due to the indirect influence via the acceptor (A-) site. The inhibition was observed also in 30 S ribosomal subunits, therefore the puromycin binding site is situated far from the peptidyl transferase center. Quantitative measurements show that the affinity of puromycin for its new ribosomal binding site is similar to its affinity for the acceptor site of the peptidyl transferase center.     

Nature of the EF G-dependent binding of 3H-GMPPCP to ribosomes

The EF G-dependent binding of ³H-GMPPCP to ribosomes was studied. The binding was dependent on temperature. Methanol, but not fusidic acid, stimulated the binding at 0°C to the level of that at 37°C. Kinetic studies of the binding suggested that two EF G were involved in the binding, and that one of the two of EF G could be replaced with fusidic acid.     

Acetone-sensitized inactivation of guanylyl-uridylyl-uridine (GUU) in the binding of valyl-tRNA to ribosomes

Summary UV-irradiation and acetone-sensitized irradiation inactivate the binding activity of GUU to valyl-t-RNA and ribosomes.     

Binding of (3H)7 alpha,17 alpha-dimethyl-19-nortestosterone (mibolerone) to progesterone receptors: comparison with binding of (3H)R5020 and (3H)ORG2058

The binding characteristics of (3H)7 alpha,17 alpha-dimethyl-19-nortestosterone [3H)DMNT) to progesterone receptors (PgR) of calf uterine tissue cytosol were determined and compared to those of the synthetic progestins (3H)ORG2058 and (3H)R5020. Scatchard plot analysis of the equilibrium binding data showed that (3H)DMNT binds to calf uterine PgR with a KD of 2.35 +/- 1.1 nM. This value is slightly higher than that of (3H)R5020 (KD = 1.16 +/- 0.4 nM) and (3H)ORG2058 (KD = 1.04 +/- 0.65 nM). Analysis of dissociation kinetics showed that (3H)DMNT dissociates much more rapidly from the receptor than the other two ligands. Competition experiments showed that ORG2058 has a lower inhibition constant (Ki) than DMNT. Sucrose density gradient (SDG) analysis of PgR showed that (3H)ORG2058-PgR complexes sediment as 8S, (3H)R5020-PgR complexes sediment as 8S and 4S, and (3H)DMNT-PgR complexes sediment as 8S entities along with dissociated (3H)steroid. These data suggest that (3H)DMNT binds to PgR with lower affinity than (3H)ORG2058 and (3H)R5020. The number of binding sites detected with (3H)DMNT are significantly lower than those measured with (3H)ORG2058.     

Preparation of spin-labeled poly(U) and its activity in assays with eukaryotic ribosomes

ESR studies on the interaction of spin-labeled polynucleotides with ribosomes require a sufficient label-to-nucleotide ratio. Using three different spin labels (SL) we have elaborated a technique to label poly(U) up to a ratio of 1 SL per 30 uridine residues. This ratio is much higher than maximal values obtained by other authors. The SL-poly(U) was shown to have the same activity as unlabeled poly(U) to direct synthesis of poly(Phe). SL-poly(U) binds to rat liver ribosomes in the presence of Mg2+ as shown by ESR. Titration with EDTA leads to a release of SL-poly(U) from ribosomes.