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M N Repkova

Publications and source records attributed to M N Repkova.

At least 37 records · Page 2Linked to original sources
PubMed1990

[Highly selective labeling of the E. coli RNA-polymerase promoter complex with reactive derivatives of oligonucleotide primers of various specificity].

A technique of highly selective affinity labelling, which includes covalent modification of the enzyme-T7A2 promoter complex with reactive oligonucleotide derivatives and subsequent elongation of the attached oligonucleotide residue with a radioactive substrate was used to study the product-binding site of E. coli RNA polymerase. Different oligonucleotides complementary to the T7A2 promoter (with lengths ranging from 2 to 8 residues) containing 5'-terminal phosphorylating, alkylating or aldehyde groups were used for the labelling. The procedure resulted in labelling DNA and beta-, beta'- or sigma-subunits of the enzyme, which are therefore believed to contact with growing RNA in the course of initiation. Consideration of the labelling patterns as a functions of the oligonucleotide's length as well as of the structure and chemical specificity of the reactive groups led to a tentative topographic scheme of the RNA polymerase product-binding region.

Affinity Labels↗
PubMed1990

[Oligo(2'-methylribonucleotides) and their derivatives. I. Automated synthesis of oligo(2'-methylribonucleotides) via H-phosphonates].

New representatives of the [ H-phosphonate's class, N-acyl-2'-O-methyl-5'-O-dimethoxytritylribonucleoside 3'-H-phosphonates, were synthesized via salicylchlorophosphine and used for the automatic synthesis of oligo(2'-O-methylribonucleotides). The efficiency of the method was demonstrated by the synthesis of a number of pyrimidine oligomers with chain length from 6 to 15 monomers.

Chemical Phenomena↗
PubMed1987

[Synthesis of oligoribonucleotides via ribonucleoside-H-phosphonates].

The synthesis of oligoribonucleotides via 5'-O-dimethoxytrityl-2'-E-tetrahydropyranyl-N-acylribonucleoside 3'-H-phosphonates is described. With these synthones and pivaloyl chloride as condensing agent, the synthesis of CpC and GpA in solution and the polymer support synthesis of (Up)4U and (Up)5C in manual variant and at the automatic synthesizer "Victoria-4M" was performed. The obtained oligonucleotide-H-phosphonates were oxidized to phosphates both with usually employed aqueous iodine solution and with of CCl4-Et3N-H2O (the Todd-Atherton reaction).

Chemical Phenomena↗
PubMed1987

[RNA-ligase of bacteriophage T4. IV. Synthesis of pentadecaribonucleotide 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.

Catalysis↗
PubMedDate not supplied

[Template location on the human ribosome: environment of the mRNA nucleotide adjacent to the A-site codon on the 3'-side].

The 18S rRNA nucleotides close to the 80S ribosome template nucleotide adjacent to the A-site codon on the 3-end (i.e., the nucleotide in position +7 relative to the first nucleotide of the P-site codon) were identified using template-controlled chemical affinity ligation. For this purpose, used the photoreactive mRNA analogues with a perfluorophenylazido group attached through various linkers to the uridine C5,3'-terminal phosphate, or guanosine N7 were used. The position of the mRNA analogues on the ribosome was preset using tRNAPhe, which recognized the phenylalanine codon directed to the P-site. An analysis of the rRNAs isolated from the irradiated complexes of 80S ribosomes showed that all the analogues are almost equally ligated to the 18S rRNA nucleotides we attributed to the A-site codon environment: namely, to nucleotides A1823, A1824, and A1825 of the 3'-minidomain and to the 620-630 fragment of the 18S rRNA 5'-domain. In addition, we identified a new component of the mRNA binding site of human ribosomes, nucleotide C1698 belonging to the 18S rRNA 3-minidomain, using analogues bearing a perfluorophenylazido group on uridine and guanine residues. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

Codon↗
PubMedDate not supplied

[Interaction of tick-borne encephalitis virus RNA with proteins].

Binding of tick-borne encephalitis (TBE) virus RNA to proteins was studied by 3 methods: gel retardation, RNA-protein cross-linking under UV light, and RNA-protein blotting. Two cellular proteins with molecular weights about 30 and 22 kDa were detected in the nuclear fraction of two cell strains (PS and RH) and in blood mononuclear cells of patients with TBE. Weak interaction between the studied RNA and viral proteins can result from trace amounts of TBE virus proteins in infected cells and by lack of additional binding factors. Bacterial super-products and affinity chromatography for isolation of native glycoproteins were used to increase the amount of viral proteins. Purified E and NS1 proteins did not react with viral RNA, while incubation with recombinant nonstructural TBE virus NS3 protein led to a shift in the mobility of TBE virus RNA in gel in the absence of cross-linking under conditions of UV exposure after addition of heparin. Poor binding of nonstructural TBE virus NS3 protein is based on electrostatic binding. Oligoribonucleotide 14 nucleotide residues long corresponding to 5'-terminal of TBE virus genome did not react with NS3 protein, probably because of its small size or absence of certain sequences.

Animals↗
PubMedDate not supplied

[Crosslinking of mRNA analog, pUUAGUAUUUAUU derivative with a photoactivated group at the guanosine residue, with human 80S ribosomes].

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg2+, or 4 mM Mg2+ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840-1849 of 18S rRNA, but in the complexes formed with participation of Phe-TPHKPhe (where the G residue carrying the arylazido group occupied position-3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position-3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg2+ concentration and the presence of polyamines.

Azides↗
PubMedDate not supplied

[The mRNA codon environment at the P and E sites of human ribosomes deduced from photo crosslinking with pUUUGUU].

Three mRNA analogs--derivatives of hexaribonucleotide pUUUGUU comprising phenylalanine and valine codons with a perfluoroarylazido group attached to the C5 atom of the uridine residue at the first, second, or third position--were used for photocrosslinking with 80S ribosomes from human placenta. The mRNA analogs were positioned on the ribosome with tRNA recognizing these codons: UUU was at the P site if tRNA(Phe) was used, while tRNA(Val) was used to put there the GUU codon (UUU at the E site). Thus, the crosslinking group of mRNA analog might occupy positions -3 to +3 with respect to the first nucleotide of the codon at the P site. Irradiation of the complexes with soft UV light (lambda > 280 nm) resulted in the crosslinking of pUUUGUU derivatives with 18S RNA and proteins in the ribosome small subunit. The crosslinking with rRNA was observed only in the presence of tRNA. The photoactivatable group in positions -1 to +3 binds to G1207, while that in positions -2 or -3 binds to G961 of 18S RNA. In all cases, we observed crosslinking with S2 and S3 proteins irrespective of the presence of tRNA in the complex. Crosslinking with S23 and S26 proteins was observed mainly in the presence of tRNA when modified nucleotide occupied the +1 position (for both proteins) or the -3 position (for S26 protein). The crosslinking with S5/S7 proteins was substantial when modified nucleotide was in the -3 position, this crosslinking was not observed in the absence of tRNA.

Codon↗
PubMedDate not supplied

[Affinity modification of the 40S subparticle from human placenta with derivatives of pAUG and 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.

Affinity Labels↗
PubMedDate not supplied

[Arrangement of the sense and terminating codons of the template in the A-segment of human ribosomes from photocrosslinking data withe oligonucleotide derivatives].

Oligoribonucleotide derivatives containing Phe codon UUC along with a 3'-flanking sense codon or stop codon carrying a perfluoroarylazido group at G or U were used to study the position of each nucleotide of the latter codon relative to the 18S rRNA in the A site of the 80S ribosome. To place the modified sense or stop codon in the A site, UCC-recognizing tRNA(Phe) was bound in the P site. Regardless of the position in the sense or stop codon, the modified nucleotide crosslinked with invariant dinucleotide A1823/A1824 or nucleotide A1825 in helix 44 close to the 3' end of the 18S rRNA. Located in the second or third position of either codon, the modified G bound with invariant nucleotide G626, which is in the evolutionarily conserved 530 stem-loop segment. The results were collated with the X-ray structure of the bacterial ribosome, and the template codon was assumed to be similarly arranged relative to the small-subunit rRNA in various organisms.

Base Sequence↗