Energetic contribution of tRNA hybrid state formation to translocation catalysis on the ribosome.

Upon transpeptidylation, the 3' end of aminoacyl-tRNA (aa-tRNA) in the ribosomal A site enters the A/P hybrid state. We report that transpeptidylation of Phe-tRNA to fMetPhe-tRNA on Escherichia coli ribosomes substantially lowers the kinetic stability of the ribosome-tRNA complex and decreases the affinity by 18.9 kJ mol(-1). At the same time, the free energy of activation of elongation factor G dependent translocation decreases by 12.5 kJ mol(-1), indicating that part of the free energy of transpeptidylation is used to drive translocation kinetically. Thus, the formation of the A/P hybrid state constitutes an important element of the translocation mechanism.

Thiostrepton inhibits the turnover but not the GTPase of elongation factor G on the ribosome.

The region around position 1067 in domain II of 23S rRNA frequently is referred to as the GTPase center of the ribosome. The notion is based on the observation that the binding of the antibiotic thiostrepton to this region inhibited GTP hydrolysis by elongation factor G (EF-G) on the ribosome at the conditions of multiple turnover. In the present work, we have reanalyzed the mechanism of action of thiostrepton. Results obtained by biochemical and fast kinetic techniques show that thiostrepton binding to the ribosome does not interfere with factor binding or with single-round GTP hydrolysis. Rather, the antibiotic inhibits the function of EF-G in subsequent steps, including release of inorganic phosphate from EF-G after GTP hydrolysis, tRNA translocation, and the dissociation of the factor from the ribosome, thereby inhibiting the turnover reaction. Structurally, thiostrepton interferes with EF-G footprints in the alpha-sarcin stem loop (A2660, A2662) located in domain VI of 23S rRNA. The results indicate that thiostrepton inhibits a structural transition of the 1067 region of 23S rRNA that is important for functions of EF-G after GTP hydrolysis.

The "allosteric three-site model" of elongation cannot be confirmed in a well-defined ribosome system from Escherichia coli.

For the functional role of the ribosomal tRNA exit (E) site, two different models have been proposed. It has been suggested that transient E-site binding of the tRNA leaving the peptidyl (P) site promotes elongation factor G (EF-G)-dependent translocation by lowering the energetic barrier of tRNA release [Lill, R., Robertson, J. M. & Wintermeyer, W. (1989) EMBO J. 8, 3933-3938]. The alternative "allosteric three-site model" [Nierhaus, K.H. (1990) Biochemistry 29, 4997-5008] features stable, codon-dependent tRNA binding to the E site and postulates a coupling between E and aminoacyl (A) sites that regulates the tRNA binding affinity of the two sites in an anticooperative manner. Extending our testing of the two conflicting models, we have performed translocation experiments with fully active ribosomes programmed with heteropolymeric mRNA. The results confirm that the deacylated tRNA released from the P site is bound to the E site in a kinetically labile fashion, and that the affinity of binding, i.e., the occupancy of the E site, is increased by Mg2+ or polyamines. At conditions of high E-site occupancy in the posttranslocation complex, filling the A site with aminoacyl-tRNA had no influence on the E site, i.e., there was no detectable anticooperative coupling between the two sites, provided that second-round translocation was avoided by removing EF-G. On the basis of these results, which are entirely consistent with our previous results, we consider the allosteric three-site model of elongation untenable. Rather, as proposed earlier, the E site-bound state of the leaving tRNA is a transient intermediate and, as such, is a mechanistic feature of the classic two-state model of the elongating ribosome.

Puromycin reaction of the A-site bound peptidyl-tRNA.

AcPhe2-tRNA(Phe) which appears in ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. One could readily explain this fact to be the consequence of spontaneous translocation. However, a detailed study of kinetics of puromycin reaction carried out with the use of viomycin (inhibitor of translocation) and the P-site test revealed that, apart from spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction triggers conformational changes in the A-site ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl site of peptidyltransferase center. This is detected functionally as a highly pronounced ability of such a peptidyl-tRNA to react with puromycin.

Interaction of tRNA with the A and P sites of rabbit-liver 80S ribosomes and their 40S subunits.

The interaction between tRNA and rabbit liver 80S ribosomes and 40S subunits was studied using a nitrocellulose membrane filtration technique. Binding of the different tRNA forms (aminoacyl-, peptidyl- or deacylated) to poly(U)-programmed 40S subunits and 80S ribosomes was found to be a cooperative process. The association constants of AcPhe-TRNA(Phe) for the A and P sites of 80S ribosomes and the cooperativity constant were measured at different temperature and Mg2+ concentration. The AcPhe-tRNA(Phe) association constant for the P site was shown to be between 2 x 10(7) M-1 and 2 x 10(8) M-1 at 25-37 degrees C and 5-20 mM Mg2+, while the affinity for the A site was 10-100-fold lower. The cooperativity constant was shown to decrease with the increase of incubation temperature and the decrease of Mg2+ concentration. The affinity of AcPhe-tRNA(Phe) for the A site of 80S ribosomes was shown to depend upon the codon specificity of tRNA at the P site. The cooperativity of the tRNA interaction with 80S ribosomes was suggested to be mostly contributed by the association with the 40S subunit and result from the correct codon-anticodon pairing at the P site. The data presented imply a codon-anticodon interaction at the P site of eukaryotic 80S ribosomes.

Localization of 5' and 3' ends of the ribosome-bound segment of template polynucleotides by immune electron microscopy.

Poly(U) with an average chain length of 40-70 nucleotides was modified at the 5'- or 3'-terminal residues with 2,4-dinitrophenyl derivatives. The modified poly(U) was used to form 30S.poly(U) or 70S.poly(U).Phe-tRNA complexes. Localization of the 5' and 3' ends of the template polynucleotide on the 30S subunit and the 70S ribosome was performed by immune electron microscopy using antibodies against dinitrophenyl haptens. The 5' and 3' ends of poly(U) (putative entry and exit sites of the message) were found in the same region both on the 30S subunit and the 70S ribosome. They were located on the dorsal side of the 30S subunit between the head and the body near the groove bordering the side ledge (platform). Comparison of the size of this region with the possible length of the polynucleotide chain covered by the ribosome allowed us to suggest that the message makes a 'U-turn" (or forms a 'loop') as it passes through the ribosome.

Mechanism of codon-anticodon interaction in ribosomes. Direct functional evidence that isolated 30S subunits contain two codon-specific binding sites for transfer RNA.

30S subunits were isolated capable to bind simultaneously two molecules of Phe-tRNAPhe (or N-Acetyl-Phe-tRNAPhe), both poly(U) dependent. The site with higher affinity to tRNA was identified as P site. tRNA binding to this site was not inhibited by low concentrations of tetracycline (2 x 10(-5)M) and, on the other hand, N-Acetyl-Phe-tRNAPhe, initially prebound to the 30S.poly(U) complex in the presence of tetracycline, reacted with puromycin quantitatively after addition of 50S subunits. The site with lower affinity to tRNA revealed features of the A site: tetracycline fully inhibited the binding of both Phe-tRNAPhe and N-Acetyl-Phe-tRNAPhe. Binding of two molecules of Phe-tRNAPhe to the 30S.poly(U) complex followed by the addition of 50S subunits resulted in the formation of (Phe)2-tRNAPhe in 75-90% of the reassociated 70S ribosomes. These results prove that isolated 30S subunits contain two physically distinct centers for the binding of specific aminoacyl- (or peptidyl-) tRNA. Addition of 50S subunits results in the formation of whole 70S ribosomes with usual donor and acceptor sites.

Comparative study of the interaction of polyuridylic acid with 30S subunits and 70S ribosomes of Escherichia coli.

Fractionated polyuridylic acid with an average chain length of 55 nucleotides forms binary complexes with 30S subunits with a stoichiometry of I:I. These complexes are heterogeneous in stability. The more stable one is characterized by an association constant K2 - 5.5xI09 M-I, and the less stable-by KI = I06xM-I, at 20 mM Mg2+, 200 mM NH4(+) and 0 degrees C. The main reason for this heterogeneity is the presence or absence of the ribosomal protein SI in the presence or absence of the ribosomal protein SI in the subunits. Decrease of Mg2+ concentration down to 5 mM hardly changes the K2 values but reduction of the NH4(+) concentration to 50 mM results in a 25-fold increase of K2. Association constants K2 for the stable complex, i.e. in the presence of SI protein, were measured at different temperatures (0 - 30 degrees C) and the thermodynamic parameters of binding (delta H degrees, delta S degrees, delta G degrees) were determined. Analogous experiments were made with 70S ribosomes. K2 values as well as delta H degrees, delta S degrees, delta G degrees appeared the same both for 30S and 70S ribosomes in all conditions examined. This is strong evidence that the 50S subunits do not contribute to the interaction of poly(U) with the complete 70S ribosomes.

Separation of ribosomal subunits of Escherichia coli by Sepharose chromatography using reverse salt gradient.

A mixture of 30 S and 50 S subunits quantitatively absorbs on a column of Sepharose--4B from the buffer: 0.02 M Tris--HCl, pH 7.5, containing 1.5 M (NH4)2SO4. During elution by reverse gradient of ammonium sulphate (1.5--0.05 M) the subunits are eluted at different salt concentrations. Complete separation of subunits is attained in the absence of Mg2+ ions. The 30 S subunits prepared from 70 S ribosomes according to this procedure are fully active in the codon--dependent binding of a specific aminoacyl--tRNA. After their reassociation with 50 S subunits isolated by zonal centrifugation, the resulting 70 S ribosomes are active in polypeptide synthesis at the same degree as control 70 S ribosomes in which both types of subunits were prepared by zonal centrifugation. The initial 70 S ribosomes for the chromatographic separation into subunits can be obtained by their pelleting from a crude extract with subsequent washing with concentrated solutions of NH4Cl in the ultracentrifuge, or by salt fractionation of the crude extract according to a slightly modified procedure of Kurland.

The mechanism of codon-anticodon interaction in ribosomes. Quantitative study of codon-dependent binding of tRNA to the 30-S ribosomal subunits of Escherichia coli.

The formation of a ternary complex 30-S-subunit . poly(U) . tRNAPhe is discussed and the conditions for its correct description by Langmuir's isotherm are deduced. The affinity constant of the binary complex 30-S-subunit . poly(U) is measured. The reversibility of binding of tRNAPhe to the complex 30-S-subunit . poly(U) is proved in a direct way. The main reason for the heterogeneity of ternary complexes was found to be due to the ability of high-molecular-weight poly(U) to form complicated aggregates with 30-S subunits. If a fraction of poly(U) of moderate molecular weight (30 000) is used, then the ternary complexes are homogeneous in stability and yield the same affinity constants for deacylated, aminoacylated and peptidyl-tRNAPhe (1 X 10(8) M-1 at 20 mM Mg2+, 200 mM NH+4 and 0 degrees C). Ribosomal protein S1 increases the binding constant of poly(U) with 30-S subunits but does not change the binding constant of tRNAPhe with the 30-S-subunit . poly(U) complex. All 30-S subunits, even partially stripped of S1 protein, are active in the binding of both poly(U) and tRNAPhe.

Isolation and study of some properties of the highly active 30S and 50S Escherichia coli ribosomal subunits.

A method for the isolation of highly active Escherichia coli ribosomal subunits has been described and used to obtain 30S subunits, which are fully active in the cistron-specific binding of tRNA, and reassociated 70S ribosomes, which are at least 35% active in the synthesis of polypeptides. The dissociation constants (Kd) of the 30S-poly(U)-tRNAPhe complex, which proved to be practically identical for tRNAPhe in the deacylated and aminoacylated forms, as well as for the chemically synthesized peptidyl-tRNA, have been measured. Changes in the binding conditions (temperatures from 0 to 30 degrees, Mg2+ concentrations from 20 to 5 mM, and NH4+ concentrations from 200 to 50mM) have a significant effect on the value of Kd without altering the number of active 30S subunits. It has been shown that the codon-specific binding of tRNA to the 30S subunits is completely reversible. The 30S subunits are not only not inactivated after a single act of binding of a tRNA molecule, but are capable of undergoing this process repeatedly without any appreciable loss in activity.