[The effect of modification of tRNA nucleotide-37 on the tRNA interaction with the P- and A-site of the 70S ribosome Escherichia coli].

A modified nucleotide on the 3'-side of the anticodon loop of tRNA is one of the most important structure element regulating codon-anticodone interaction on the ribosome owing to the stacking interaction with the stack of codon-anticodon bases. The presence and identity (pyrimidine, purine or modified purine) of this nucleotide has an essential influence on the energy of the stacking interaction on A- and P-sites of the ribosome. There is a significant influence of the 37-modification by itself on the P-site, whereas there is no such one on the A-site of the ribosome. Comparison of binding enthalpies of tRNA interactions on the P- or A-site of the ribosome with the binding enthalpies of the complex of two tRNAs with the complementary anticodones suggests that the ribosome by itself significantly endows in the thermodynamics of codon-anticodon complex formation. It happens by additional ribosomal interactions with the molecule of tRNA or indirectly by the stabilization of codon-anticodon conformation. In addition to the stacking, tRNA binding in the A and P sites is futher stabilized by the interactions involving some magnesium ions. The number of them involved in those interactions strongly depends on the nucleotide identity in the 37-position of tRNA anticodon loop.

[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.

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.

Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome.

Elongation factor G (EF-G) is a GTPase that is involved in the translocation of bacterial ribosomes along messenger RNA during protein biosynthesis. In contrast to current models, EF-G-dependent GTP hydrolysis is shown to precede, and greatly accelerate, the rearrangement of the ribosome that leads to translocation. Domain IV of the EF-G structure is crucial for both rapid translocation and subsequent release of the factor from the ribosome. By coupling the free energy of GTP hydrolysis to translocation, EF-G serves as a motor protein to drive the directional movement of transfer and messenger RNAs on the ribosome.

[Interaction of deacylated phenylalanyl tRNA from yeasts with Escherichia coli ribosomes. The role of the modified nucleotide in codon-anticodon interaction]. / Vzaimodeistvie deatsilirovannoi fenilalaninovoi tRNK iz drozhzhei s ribosomami Escherichia coli. Rol' modifitsirovannogo nukleotida v kodon-antikodonovom vazimodeistvii.

The method of anticodon loop replacement has been used to make derivatives of yeast tRNA(Phe)GmAAY with the substitution at the 37 position (tRNA(Phe)GAAA), and at both the anticodon (tRNA(Phe)GCAG) and the 37 position. A quantitative study of the interaction of various types of yeast deacylated tRNA: tRNA(Phe)GmAAY, tRNA(Phe)GAAA, tRNA(Phe)GCAG, and tRNA(Phe)-Y with the P site of the 70S ribosome.poly(U) complex was carried out at different Mg2+ concentrations and temperatures. The replacement of the Y base on the nonmodified adenosine decreases the interaction enthalpy from 39 to 24 kcal/mole, whereas the complete removal of the Y base reduces the interaction enthalpy to 16 kcal/mole. The replacement of the second letter of the anticodon (A) with cytosine leads to a drop in the enthalpy to 6 kcal/mole, which is typical of tRNA interaction with the P site in the absence of poly(U). In the absence of poly(U) the affinity of tRNA(Phe)-Y for the P site of the 70S ribosome is 5 times lower than the affinity of tRNA(Phe)GmAAY and tRNA(Phe)GCAG. Thus, in the ribosome the modified nucleotide not only stabilizes the codon-anticodon interaction owing to the stacking interaction with the stack of codon-anticodon bases, but also lowers the free energy of binding as a result of the interaction of the modified nucleotide itself with the hydrophobic center of the P site on the ribosome.

[Binding of the yeast phenylalanine tRNA with Escherichia coli ribosomes. Effect of the removal of a modified base from the 3'-end of the anticodon on codon-anticodon interaction]. / Sviazyvanie fenilalaninovoi tRNK iz drozhzhei s ribosomami Escherichia coli. Vliianie udaleniia modifitsirovannogo osnovaniia s 3'-storony antikodona na kodon-antikodonovoe vzaimodeistvie.

Phe-tRNAPhe+Y and N-acetyl-Phe-tRNAPhe+Y from yeast interact with prokaryotic 30S subunits and 70S ribosomes with slightly lower affinity than respective tRNA's of E. coli (decrease of standard free energy change of interaction less than 10%). The removal of Y-base from Phe-tRNAPhe+Y results in two orders of magnitude decrease of association constant of Phe-tRNAPh-Ye with P site of the 30S X poly(U) complex and one ordef of magnitude or more of that with A site. The same modification decreases the association constants of Phe-tRNAPhe-Y and N-acetyl-Phe-tRNAPhe-Y 60 and 15 times respectively with P site of the 70S X poly(U) complex. In the absence of poly(U) the affinity of N-acetyl-Phe-tRNAPhe-Y to P-site of 70S ribosome was 20-fold lower than that of native N-acetyl-Phe-tRNAPhe+Y. The sign of interaction enthalpy of N-acetyl-Phe-tRNAPhe+/-Y and Phe-tRNAPhe-Y changes below 6-7 degrees C exposing the hydrophobic part of P-site interactions. Similar removal of Y-base does not change both the enthalpy of interaction with P-site and magnesium concentration dependence.

[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.

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.

[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.