Extended conformation of mammalian translation elongation factor 1A in solution.

The conformation of mammalian elongation factor eEF1A in solution was examined by the small angle neutron scattering and scanning microcalorimetry. We have found that in contrast to the bacterial analogue the eEF1A molecule has no fixed rigid structure in solution. The radius of gyration of the eEF1A molecule (5.2 nm) is much greater than that of prokaryotic EF1A. The specific heat of denaturation is considerably lower for eEF1A than for EF1A, suggesting that the eEF1A conformation is significantly more disordered. Despite its flexible conformation, eEF1A is found to be highly active in different functional tests. According to the neutron scattering data, eEF1A becomes much more compact in the complex with uncharged tRNA. The absence of a rigid structure and the possibility of large conformational change upon interaction with a partner molecule could be important for eEF1A functioning in channeled protein synthesis and/or for the well-known capability of the protein to interact with different ligands besides the translational components.

[Comparative study of the reactability of phosphoric acid residues in tRNA(Ser) and tRNA(Leu) from Thermus thermophilus]. / Sravnitel'noe izuchenie reaktsionnoi sposobnosti ostatkov fosfornoi kisloty, vkhodiashchikh v sostav tRNK(Ser) i tRNK(Leu) iz Thermus thermophilus.

The reactivity of phosphates in the Thermus thermophilus tRNA(Ser) (GCU) and tRNA(Leu) (CAG) was studied using the ethylnitrosourea modification. It was shown that phosphates of nucleotides 58-60 (T loop), 20-22 (D loop), and 48 (at the junction of the variable and T stems) were poorly modified in both tRNAs. The most pronounced differences in the reactivity were observed for phosphates at the junctions of the variable stem with T-stem (47q, 49) and anticodon stem (45). This indicates differences in orientations of the long variable arm relative to the backbone in the tRNAs studied.

Three tRNA binding sites in rabbit liver ribosomes and role of the intrinsic ATPase in 80S ribosomes from higher eukaryotes.

Three tRNA binding sites have been found in organisms of all domains (former kingdoms) with only one exception: Four binding sites have been reported for cytoplasmic 80S ribosomes from rabbit liver. Therefore, the issue was reconsidered, and the data revealed that rabbit liver ribosomes contain three tRNA binding sites, underlining the universal character of this ribosomal feature. Furthermore, a first analysis of the role of the ribosome intrinsic ATPase was performed. This ATPase is found in ribosomes of higher eukarya but not in lower eukarya such as yeast or ribosomes of the domains archea and bacteria. The results suggest that the intrinsic ATPase fulfills the same function as the essential third elongation factor EF-3, an ATPase in higher fungi (yeast etc.), that facilitates the release of the deacylated tRNA from the E site.

Evidence for the formation of an unusual ternary complex of rabbit liver EF-1alpha with GDP and deacylated tRNA.

Eukaryotic translation elongation factor 1alpha is known to interact in GTP-bound form with aminoacyl-tRNA promoting its binding to the ribosome. In this paper another ternary complex [EF-1alpha*GDP*deacylated tRNA], never considered in widely accepted elongation schemes, is reported for the first time. The formation of this unusual complex, postulated earlier (FEBS Lett. (1996) 382, 18-20), has been detected by four independent methods. [EF-1alpha*GDP]-interacting sites are located in the acceptor stem, TpsiC stem and TpsiC loop of tRNA(Phe) and tRNA(Leu) molecules. Both tRNA and EF-1alpha are found to undergo certain conformational changes during their interaction. The ability of EF-1alpha to form a complex with deacylated tRNA indicates that the factor may perform an important role in tRNA and aminoacyl-tRNA channeling in higher eukaryotic cells.

[Determination of interacting segments of tRNA(Leu) from cow mammary glands with homologous aminoacyl-tRNA-synthetase by a chemical modification method]. / Opredelenie uchastkov vzaimodeistviia tRNK(Leu) iz molochnoi zhelezy korov s gomologichnoi aminoatsil-tRNK-sintetazoi metodom khimicheskoi modifikatsii.

The interaction of the cow mammary gland tRNA(IAGLeu), having a long variable loop, with the cognate aminoacyl-tRNA synthetase has been studied by the alkylation with ethylnitrosourea. It was shown that leucyl-tRNA synthetase protects from alkylation 3'-phosphates of the nucleotides 12-13 in D-loop, 23-24 in D-stem and 37-43 in the anticodon arm of tRNA(IAGLeu). All regions of interaction with the aminoacyl-tRNA synthetase are located in the same plane of tRNA whereas the long variable loop is in another plane.

[A study of the conformation of tRNA(IAGLeu) from the cow mammary gland using chemical modification methods]. / Issledovanie élementov prostranstvennoi struktury tRNK(IAGLeu) molochnoi zhelezy korov metodami khimicheskoi modifikatsii.

The nucleosides of tRNA(IAGLeu) (with a long variable loop) from the cow mammary gland included in formation of the three-dimensional structure have been analysed by the chemical modification methods. Exposed guanosine and cytidine residues were detected by means of dimethylsulfate, whereas diethylpyrocarbonate was used to detect exposed adenosine residues. The low level of the modification was characteristic of guanosine residues in positions 10 (m2G), 13, 15, 23, 24, 29, 30, 47 H, 51, 52, 53, 57; of cytidine residues in positions 48 (m5C), 56 and those involved in Watson--Crick pairing; of adenosine residues in positions 14, 22, 31, 42, 59, 64. Most bases of tRNA(IAGLeu) thus detected are similarly located in the yeast tRNA(Phe) molecule, which suggests a common role of these bases in the formation of the spacial structure of both tRNAs.

[Determination of phosphate residues participating in the formation of the spatial structure of tRNA- Leu IAG from cow mammary glands]. / Opredelenie ostatkov fosfornoi kisloty, uchastvuiushchikh v obrazovanii prostranstvennoi struktury tRNK- Leu IAG molochnoi zhele korov.

The phosphates of the tRNA-Leu IAG from cow mammary gland (tRNA which has a long variable loop) participating in the formation of three-dimensional structure were studied by alkylation with ethylnitrosourea and methylnitrosourea. A low degree of modification was observed for the phosphates of the following nucleotides: 7, 8, 9, 10 (at the bend site between the acceptor and D-stem); 18, 19, 20A and 21 in the D-loop; 47H and 49 at the joint of variable and T-stem; 57, 58 and 59 in the T-loop.