[The effect of modification of nucleotide-37 on the interaction of aminoacyl-tRNA with the A-site of the 70S ribosome]. / Vliianie modifitsirovannogo nukleotida v polozhenii 37 na vzaimodeistvie aminoatsil-tRNK s A-saitom 70S ribosomy.

To estimate the effect of modified nucleotide-37, the interaction of two yeast aminoacyl-tRNAs (Phe-tRNAK+YPhe and Phe-tRNAK-YPhe) with the A site of complex [70S.poly(U).deacylated tRNA(Phe) in the P site] was assayed at 0-20 degrees C. As comparisons with native Phe-tRNAK+YPhe showed, removal of the Y base decreased the association constant of Phe-tRNAK-YPhe and the complex by an order of magnitude at any temperature, and increased the enthalpy of their interaction by 23 kJ/mol. When the Y base was present in the anticodon loop of deacylated tRNA(Phe) bound to the P site of the 70S ribosome, twice higher affinity for the A site was observed for Phe-tRNAK-YPhe but not for Phe-tRNAK+YPhe. Thus, the modified nucleotide 3' of the Phe-tRNA(Phe) anticodon stabilized the codon-anticodon interaction both in the A and in the P sites of the 70S ribosome.

[Mechanism of tRNA translocation on the ribosome]. / Mekhanizm translokatsii tRNK na ribosome.

During the translocation step of the elongation cycle of peptide synthesis two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. Translocation is catalyzed by the elongation factor G (EF-G) and requires GTP hydrolysis. The fundamental biochemical features of the process were worked out in the 1970-80s, to a large part by A.S. Spirin and his colleagues. Recent results from pre-steady-state kinetic analysis and cryoelectron microscopy suggest that translocation is a multistep dynamic process that entails large-scale structural rearrangements of both ribosome and EF-G. Kinetic and thermodynamic data, together with the structural information on the conformational changes of the ribosome and of EF-G, provide a detailed mechanistic model of translocation and suggest a mechanism of translocation catalysis by EF-G.

[A modified method of isolation of "tight" 70S ribosomes from Escherichia coli highly active at different stages of the elongation cycle]. / Modifitsirovannyi sposob polucheniia "tight" 70S ribosom iz Escherichia coli, vysokoaktivnykh v otdel'nykh stadiiakh tsikla élongatsii.

The functional activity of the wide-spread "tight" 70S ribosomes is usually equal to 55-80%. We show here that the inactive fraction of this type of ribosomes is virtually blocked by residual endogenous RNA's. These RNA's are shown to be removable by introducing an additional stage in the isolation procedure including: 1. short heating (15 min, 37 degrees C) of "tight" 70S under dissociation conditions, i. e. in a buffer containing 3 mM MgCl2 and 200 mM NH4Cl; 2. washing off endogenous RNA's on a sucrose density gradient in the same buffer; 3. final selection of purified "tight" 70S on the sucrose gradient containing 5 mM MgCl2 and 50 mM NH4Cl. "Tight" 70S ribosomes isolated by such a procedure are 90-100% active with respect to tRNA binding (including the factor-dependent one), peptide bond synthesis and translocation.

[Effect of the concentration ratio of bi- and monovalent ions on the functional activity of 30S ribosome subunits of Escherichia coli]. / Vliianie sootnosheniia kontsentratsii dvukh- i odnovalentnykh kationov na funktsional'nuiu aktivnost' 30S subchastits ribosom Escherichia coli.

Experiments on poly(U)-dependent binding of Phe-tRNAPhe to 30S subunits revealed the existence of a critical [Mg2+]/[NH4+] ratio in a medium (approximately 0.05-0.1) with respect to the binding capacity of subunits. If the ratio is greater than the critical one, 30S subunits undergo reversible inactivation even at the highest Mg2+ concentrations (up to 20 mM). The stronger is the deviation from the [Mg2+]/[NH4+] value = 0.05-0.1, the greater are both the rate and extent of such an inactivation. Two sites for tRNA in initially active 30S subunits have been shown to be inactivated in an interdependent way. On the other hand, a progressive decrease of [Mg2+]/[NH4+] ratio in a medium (from the value of 0.05 and lower) does not produce inactivation, but rather results in reduced affinity constants of Phe-tRNAPhe for active sites of 30S subunits.

[The effect of the antibiotic edeine on tRNA interaction with A, P and E sites of Escherichia coli 70S ribosomes]. / Osobennosti vliianiia antibiotika édeina na vzaimodeistvie tRNK s uchastkami A, P i E 70S ribosom Escherichia coli.

Edeine inhibits poly(U)-dependent binding of tRNAPhe to the P and A sites simultaneously, both on 30S subunits and 70S ribosomes. Hence, edeine cannot be considered as antibiotic, "complementary" to tetracycline for selective adsorption of tRNA only to the P or to the A site. Further, edeine decreases the affinity constant of tRNAPhe for the P-site by more than two orders of magnitude, no matter poly(U) is present or not. Neither edeine nor tetracycline affect interaction of deacylated tRNAPhe with the E-site of E. coli 70S ribosomes.

[Interaction of tRNA with ribosomes]. / Vzaimodeistvie tRNK s ribosomami.

Definition of the site of tRNA-binding to ribosomes is suggested on the basis of a free energy of tRNA-ribosome interaction. From this point of view disagreements that have arisen in recent years concerning the numbers of tRNA binding sites on the ribosome, their distribution between subunits, the properties of the third site E in ribosomes and the compatibility of new experimental data with different models of elongation cycle are discussed. The observation of the third site in the ribosome (messenger independent and with a presumably exit function) is not a refutation but an extension of Watson's model of translating ribosome.

[How do ribosome binding sites recognize the functional state of tRNA?]. / Kak uchastki sviazyvaniia ribosom razlichaiut funktsional'nye formy tRNK?

The order of relative affinity of three different functional form of tRNA (aminoacyl-tRNA, peptidyl-tRNA and deacylated tRNAOH) was established for three sites of the ribosome. The affinity increases for A-site in consecutive order: tRNAOH less than AcPhe-tRNA less than aa-tRNA; for P-site with messenger RNA: AcPhe-tRNA less than aa-tRNA less than tRNAOH; without messenger RNA: aa-tRNA less than AcPhe-tRNA less than tRNAOH; for E-site: (AcPhe-tRNA, aa-tRNA) much less than much less than tRNAOH. The dependence of association constants versus magnesium concentration for all forms of tRNA conforms the equation: delta lg Ka/delta lg[Mg2+] = n. Number "n" varies in the range 1 to 8 depending on the site of adsorption the form of tRNA and the presence of mRNA. Such magnesium dependence of affinity of tRNAs shows that the electrostatic interactions play an important role in the recognition of functional forms of tRNA by ribosomal sites.

[Mechanism of codon-anticodon interaction in ribosomes. Interaction of aminoacyl-tRNA with 70S ribosomes in the absence of elongation factor EF-Tu and GTP]. / Mekhanizm kodon-antikodonnogo vsaimodeistviia v ribosomakh. Vzaimodeistvie aminoatsil-tRNA s 70S ribosomami Escherichia coli v otsutstvie faktora élongatsii EF-Tu i GTP.

Purified Phe-tRNAPhe revealed higher affinity to the donor (D) site of vacant 70S . poly(U) complex than to the acceptor (A) site, independent on Mg2+ concentration. As a result, in excess of ribosomes Phe-tRNAPhe binds exclusively to the D site. This was proved using the tests in the presence of tetracycline and puromycin. Preferential binding of Phe-tRNAPhe to the D site was used to measure equilibrium association constants of this interaction at different temperatures and Mg2+ concentrations. A large value of reaction enthalpy (ca. -26 Kcal/mole) was found.

[Quantitative studies of interaction of polyuridylic acid with 30S subunits of ribosomes of Escherichia coli]. / Kolichestvennoe izuchenie vzaimodeistviia poliuridilivoi kisloty s 30S subchastitsami ribosom Escherichia coli.

Polyuridilic acid of average molecular weight 18 000 binds to the 30S subunits with stoichiometry 1 : 1 but two kinds of 30S.poly(U) complexes with different stability are formed. The main reason for such heterogeneity was found to be due to the presence or absence of ribosomal protein Sl in 30S subunits. In its presence the association constant of 30S.poly(U) complex is equal 2.7.10(8) M-1, and in the opposite case it is much less 1.5.10(6) M-1. In the same conditions (20 mM MgCl2, 200 mM HN4Cl, 0 degrees) the association constant of binary complex Sl.poly(U) is equal 5.10(7) M-1.

[Nature of the heterogeneity of 30S ribosomal subparticles in vitro. I. Effect of large centrifugal fields during 30S subparticle isolation on their capacity for codon-dependent tRNA binding]. / Priroda geterogennosti 30S ribosomnykh subchastits in vitro. I. Vliianie bol'shikh tsentrobezhnykh polei pri vydelenii 30S subchastits na ikh sposobnost' k kodonzavisimomu sviazyvaniiu tRNK.

Measurements of association constants (Ka) of specific [14C]Phe-tRNAPhe with a 30S..poly(U) complex revealed that values of these constants vary from 0.5.10(7) up to 1.5.10(8) M--1 when different 30S subunit preparations were used at the same medium conditions (20 mM Mg2+, 200 mM NH4, 0 degrees C). Analysis of these data showed that the higher the rotor speeds were used during separation of 70S ribosomes into subunits, the less Ka values were measured. In special experiments on sedimentation of pure 30S subunits at different rotor speeds it was found that the decrease of Ka values was caused due to the additional reversible dissocation of ribosomal proteins from 30S subunits at high (the order of 100 000.g) centrifugal fields. As a possible mechanism of such dissociation we suggest the influence of high hydrostatic pressure on the association constants of S-proteins with 30S subunits. Data presented in this paper demonstrate that at least one of the reasons for the physical and functional heterogeneity of 30S subunits in vitro derives from the application of high centrifugal fields during isolation of ribosomal subunits.