[Isolation of total RNA from baker's yeast].

A simple method of production of total RNA from baker's yeast was developed. Total RNA was isolated from yeast (Saccharomyces cerevisiae) biomass using lysis with sodium dodecyl sulfate at 100 degrees C for 40-60 min and subsequent precipitation of the target product with 3 M NaCl. The preparation obtained was characterized in detail: yield of total RNA from 1 kg of pressed yeast, 9.25 g; optical density at 260 nm of 1 mg of RNA dissolved in 1 ml of water, 20.2 U; content of the acid-soluble fraction, 2.02%; and protein content, 1.8%. Total tRNA was isolated from total RNA by fractional precipitation with ethanol followed by gel filtration.

[Interaction of HIV-1 reverse transcriptase with new minor groove ligands and their conjugates with oligonucleotides].

The influence of new non-natural regular minor groove binders (MGB), containing 2-4 imidazole, pyrrole or thiazole residues, and their conjugates with oligonucleotides, on the polymerization reaction catalyzed by HIV-1 reverse transcriptase was analyzed. Various model template-primer complexes: poly(A)-oligo(U), poly(A)-oligo(dT), poly(dA)-oligo(U), poly(dA)-oligo(dT) and activated DNA were used. The concentration of oligopeptides, giving 50% inhibition (I50) of the RT-dependent polymerization reaction, was shown to depend strongly on the structure of template-primer complexes, number and type of the heterocycle rings in the MGBs analyzed. The range of I50 for the most of the compounds studied is 7.7 x 10(-3)-1.0 x 10(-5) M. The affinity of MGB is minimal for poly(A)-oligo(U). However, some of imidazole and pyrrole-containing MGBs demonstrated unusually high affinity (I50 = 3 x 10(-9)-4 x 10(-8) M) to the above template-primer in complex with RT. The affinity of conjugates of thiazolecarboxamides with oligonucleotides complementary or partially complementary to the template, is 1-4 orders higher compared to free thiazolecarboxamides. The possible reasons of the dependence of I50 values upon the structure of the template-primer complexes, the structure of MGB, and their conjugates with oligonucleotides are discussed.

[Interaction of proteins from general transcription complex RNA polymerase II with oligoribonucleotides].

We have analyzed an interaction of the general transcription complex RNA polymerase II proteins (RNA polymerase II, factors TBP, TFIIB, TFIIF, TFIIE and TFIIH) S. cerevisiae with the oligoribonucleotides. With the help of method EMSA was shown that labeled 32P labeled oligoribonucleotide 5'-ACUCUCUUCCGCAUCGC-3' (r-17) binds with the proteins and generates three species of the complexes with the three major shifts. All the three species of the complexes are RNA specific because a total RNA S. cerevisiae was a competitor for all three species but the TATA-containing oligodeoxyribonucleotide (500-fold molar excess) was not a competitor for its. Complexes 32P-r-17 with the proteins belonging to the middle shift are the sequence specific because unlabeled r-17 was a competitor for its binding (100-fold molar excess) but unlabeled UA-rich oligoribonucleotide (5'-AUAUUAUGUUCAAAA-3) was not a competitor for this shift (500-fold molar excess). Complexes belonging to the upper shift are RNA specific probably. We think 32P-r-17 interaction with the proteins belonging to the under shift is nonspecific corresponding to a sorbtion of 32P-r-17 on a protein. The data presented demonstrate that oligoribonucleotide and oligodeoxyribonucleotide don't compete for the binding sites on a basal transcription complex proteins.

[The role of the 3'-CCA sequence in the interaction of tRNA(Phe) from E. coli and Thermus thermophilus with homologous phenylalanyl-tRNA-synthetases]. / Rol' 3'-CCA-posledovatel'nosti vo vzaimodeistvii tRNA(Phe) iz E. coli i Thermus thermophilus s gomologichnymi fenilalanil-tRNK-sintetazami.

The 3'-CCA end of tRNA(Phe) from E. coli and Thermus thermophilus was modified by stepwise degradation and ligation of the shortened tRNA with different trinucleotides (pUpUpA, (pA)3, (pC)3, (pU)3). Kinetic parameters for the aminoacylation reaction of modified tRNAs have been determined. The role of the 3'-terminal trinucleotide of tRNA(Phe) in tRNA binding and aminoacylation by phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus is postulated.

[Interaction of catalytically active antibodies with oligoribonucleotides]. / Vzaimodeistvie kataliticheski aktivnykh antitel s oligoribonukleotidami.

The interaction of antibodies from blood sera of patients with autoimmune pathology, systemic lupus erythematosus with oligoribonucleotides was studied. The RNA-hydrolyzing activity was shown to be an intrinsic property of autoantibodies. Enzymic activity of antibodies in hydrolysis of poly(U) was estimated at 20-40% of that of RNase A. In contrast to known eukaryotic RNases, the autoantibodies possess a specific RNA-hydrolyzing activity for oligo r(A). The RNA-nicking activity of antibodies in hydrolysis of oligoadenylates was more higher than with hydrolysis of oligo d(A). Optimal conditions of r(pA)13 hydrolysis were selected, including the optimal of pH = 8.7.

[Structural elements of 80S ribosomes located near the 5'-region of the mRNA-binding center]. / Strukturnye élementy 80S ribosomy, raspolozhennye vblizi 5'-oblasti mRNK-sviazyvaiushchego tsentra.

Affinity labeling of 80S ribosomes with 4-(N-2-chloroethyl-N-methylamino)benzylmethylphosphoramides of oligoribonucleotides [32P]AUGUn--mRNA analogs--was studied in three model complexes: 80S.ClRCH2N(CH3)-pAUGU6.Met(Phe)2-trRNA(Phe), 80S.ClRCH2N(CH3)pAUGU3.MetPhe-tRNA(Phe), and 80S.ClRCH2N(CH3)-pAUG.Met- tRNA(Met). Two of these complexes imitate the posttranslocational state of 80S ribosomes. Small subunits were labeled preferentially; both 18S rRNA and ribosomal proteins were modified by the mRNA analogs. The relative modification extents of proteins and rRNA depended on the length of the reagent oligoribonucleotide moiety. Extension of the latter resulted in decrease in the relative extent of 18S rRNA modification from 95 a to 16% (for proteins, increase from 5 to 84%, respectively). Fragments of 18S rRNA containing cross-linking sites were identified using blot hybridization. In all cases, fragment 976-1164 was found to be modified. In the case of ClRCH2N(CH3)pAUGU6, labeling occurred also within fragments 593-673 and 1748-1869. Analysis of the modified proteins revealed that proteins S14/S15 were labeled with all three reagents and were the single target of modification with ClRCH2N(CH3)pAUGU6. Proteins S3/S3a, S6, and S16/S18 were modified only with ClRCH2N(CH3)pAUGU3; protein S20 only with ClRCH2N(CH3)pAUG; and proteins S5 and S17 were labeled with both reagents (n = 0, 3).

[Interaction of mRNA analogs--oligoribonucleotides AUGU3, AUGU6 and (pU)6 with 80S ribosomes from human placenta in the presence of related tRNA and a protein-synthesizing system]. / Vzaimodeistvie analogov mRNK--oligoribonukleotidov AUGU2, AUGU6, and (pU)6 s 80S ribosomami iz platsenty cheloveka v prisutstvii rodstvennykh tRNK i beloksinteziruiushchei sistemy.

Binding of oligoribonucleotides AUGUn (n-3 and 6) and (pU) to 80S ribosomes from human placenta in the presence of cognate tRNAs and a "ribosome-free" protein-synthesizing system from rabbit reticulocytes has been studied. The binding of the mRNA analogues resulted in formation of stable post-translocational complexes (which may be easily isolated by centrifugation in sucrose density gradient): 80S.AUGU3.MetPhe-tRNA(Phe); 80S.AUGU6.Met(Phe)2.tRNA(Phe); 80S.(pU)6.(Phe)2-tRNA(Phe). In these complexes the ratios of the bound ligands are close to the theoretically expected values. Comparison of the results obtained with the previously reported data on nonenzymatic binding of oligouridylates and Phe-tRNA(Phe) to 80S ribosomes lead one to the conclusion that translation factors significantly stabilize the complexes of tRNA with 80S ribosomes and oligoribonucleotide templates.

[Affinity modification of 40S ribosomal subparticles from human placenta with mRNA analogs--AUGUnC oligoribonucleotide analogs with an alkylating group at the 3'-end]. / Affinnaia modifikatsiia 40S ribosomnykh subchastits iz platsenty cheloveka analogami mRNK--proizvodnymi oligoribonukleotidov AUGUnC s alkiliruiushchei gruppoi na 3'-kontse.

Using 2',3'-O-[4-N-(2-chloroethyl)-N-methylamino]benzylidene derivatives of AUGUn[32P]pC (mRNA analogues), affinity labelling of human placenta 40S ribosomal subunits has been investigated in model initiation complexes obtained in the presence of the ternary complex eIF-2.GTP.Met-tRNA(fMet). The regions of 18S rRNA labelled with these mRNA analogues were identified. The main targets of 18S rRNA alkylation by the derivative of AUG[32P]pC were located within positions 1610-1747 and 1748-1869. The site of covalent attachment of AUGU3[32P]pC derivative to 18S rRNA was found within positions 593-673. Taking into account the data on labelling of human placenta ribosomes with the same derivatives of oligourydilates obtained previously, the conclusion was made that the arrangement of the codon U3 in the mRNA-binding centre of the initiation complex 40S.AUGU3[32P]pC derivative.eIF-2.GTP.Met-TPHK(fMet) differs from the arrangement of the same codon at the A-site of the complex imitating the pretranslocation state of ribosomes.

[Affinity modification of 40S ribosomal subparticles from human placenta by derivatives of oligoribonucleotides, containing an AUG codon. Ribosomal proteins, forming a codon-anticodon interaction region]. / Affinnaia modifikatsiia 40S ribosomnykh subchastits iz platsenty cheloveka proizvodnymi oligoribonukleotidov, soderzhashchikh kodon AUG. Ribosomnye belki, formiruiushchie oblast' kodon-antikodonovykh vzaimodeistvii.

Using 2',3'-O-[4-N-(2-chloroethyl)-N-methylamino]benzylydene derivatives of AUGUn [32P]pC and 4-[N-(2-chloroethyl)-N-methylamino]benzylmethylphosphoamide derivatives of [32P]pAUGUn, the affinity labelling of human placental 40S ribosomal subunits was studied within 40S initiation complexes obtained in the presence of a ternary complex eIF-2.GTP.Met-tRNA(fMet). Analysis of the ribosomal proteins labelled by these mRNA analogues revealed that proteins S3/S3a, S6, S7, and S14/S15 play a key role in the interaction of the template with the 40S subunit in the presence of the ternary complex eIF-2.GTP.Met-tRNA(fMet). Proteins S2, S4, S5, S8, S9, and S17 are also involved in this interaction.

[Affinity modification of 80S ribosomes from human placenta with mRNA analogs--derivatives of oligouridylates with alkylating groups at the 3'-end]. / Affinnaia modifikatsiia 80S ribosom iz platsenty cheloveka analogami mRNK--proizvodnymi oligouridilatov s alkiliruiushchei gruppoi na 3'-kontse.

2',3'-O-[4-(N-2-chloroethyl-N-methylamino)]benzylidene derivatives of oligoribonucleotides (Up)5U[32P]pC and (Up)11U[32P]pC were used for affinity labeling of 80S ribosomes from human placenta in the presence of cognate Phe-tRNA(Phe). The derivatives retained the ability of tRNA(Phe)-dependent binding with 80S ribosomes and within the corresponding complexes were cross-linked to 40S subunits (preferentially to 18S rRNA). Sites of the mRNA analogs attachment were identified by blot-hybridization of the modified rRNA with restriction fragments of corresponding rDNA. They are located within positions 1610-1747 and 1748-1865 for both analogs. These fragments are situated at the 3'-end region of 18S rRNA in a highly conserved part of eukaryotic small subunit rRNA secondary structure.

[Affinity modification of the 40S subparticle from human placenta with derivatives of pAUG and pAUGU3]. / Affinnaia modifikatsiia 40S subchastits iz platsenty cheloveka proizvodnymi pAUG i pAUGU3.

Affinity labeling of 40S subunits from human placenta with 4-(N-2-chloroethyl-N-methylamino)benzylmethyl-[32P]phosphoamide s of oligoribonucleotides pAUG and pAUGU3 was studied. Covalent attachment of these derivatives to 40S subunits within the complexes with 40S subunits, formed in the presence of Met-tRNAf.eIF-2.GTP, was detected. Both rRNA and ribosomal proteins were modified. Fragments of 18S rRNA, containing sites of the reagent attachment were identified: 1058-1164 for pAUG derivative and 976-1057--for pAUG and pAUGU3 ones. The data obtained allowed to conclude that the presence of the neighbouring codon at the A-site, regardless of the presence of the tRNA in it, affects significantly the arrangement of the trinucleotide template in the codon-anticodon interaction region. The large subunit does not cause significant alterations in the structural organization of the codon-anticodon interaction region.

[RNA ligase from bacteriophage T4. VIII. Solid phase enzymatic synthesis of oligoribonucleotides]. / RNK-ligaza bakteriofaga T4. VIII. Tverdofaznyi fermantativnyi sintez oligoribonukleotidov.

A method of the solid-phase enzymic synthesis of oligoribonucleotides has been suggested. The donor is fixed through its 3'-end on a water-insoluble matrix followed by the stepwise RNA ligase- and T4 polynucleotide kinase-assisted coupling of trinucleoside diphosphates in the 5'-direction. As an example, (pA)6pAox was immobilised on Biogel P-300 hydrazine and the RNA ligase-catalyzed addition of acceptor ApApA to the donor gave (Ap)9 with the 50% yield.

[RNA-ligase of bacteriophage T4. IV. Synthesis of pentadecaribonucleotide ApUpG(pU)6(pA)5pAp]. / RNK-ligaza bakteriofaga T4. IV. Sintez pentadekaribonukleotida ApUpG(pU)6(pA)5pAp.

Oligoribonucleotide ApUpG(pU)6(pA)5pAp (I) has been prepared by means of RNA ligase. ApUpG, synthesised by the phosphotriester approach, was elongated in the 3'-direction by adding (pU)6 and then (pA)6, which was 3'-blocked with the phosphate or with the periodate-oxidized AMP residue, the latter giving considerably lower level of by-products. Condensation of ApUpG(pU)6 with blocked (pA)6 was carried out under conditions optimal for poor acceptors (20 degrees C, 48 h, pH 8,7) to afford (I) with the yield of 20% (105 OE260); ApUpG(pU)6(pA)10pAp was identified as a byproduct.