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M N Repkova

Publications and source records attributed to M N Repkova.

50 records · Page 3Linked to original sources
PubMedDate not supplied

[Protein environment of the sense codon of the template in the A site of the human ribosome as inferred from crosslinking to oligoribonucleotide derivatives].

The protein environment of each nucleotide of the template codon located in the A site of the human ribosome was studied with UUCUCAA and UUUGUU derivatives containing a Phe codon (UUC and UUU, respectively) and a perfluoroarylazido group at U4, U5, or U6. The analogs were positioned in the ribosome with the use of tRNA(Phe), which is cognate to the UUC or UUU codon and directs it to the P site, bringing a modified codon in the A site with a modified nucleotide occupying position +4, +5, or +6 relative to the first nucleotide of the P-site codon. On irradiation of ribosome complexes with tRNA(Phe) and mRNA analogs with mild UV light, the analogs crosslinked predominantly to the 40S subunit, modifying the proteins to a greater extent than the rRNA. The 18S rRNA nucleotides crosslinking to the analogs were identified previously. Of the small-subunit proteins, S3 and S15 were the major targets of modification in all cases. The former was modified both in ternary complexes and in the absence of tRNA, and the latter, only in ternary complexes. The extent of crosslinking of mRNA analogs to S15 decreased when the modified nucleotide was shifted from position +4 to position +6. The results were collated with the data on ribosomal proteins located at the decoding site of the 70S ribosome, and conclusion was made that the protein environment of the A-site codon strikingly differs between bacterial and eukaryotic ribosomes.

Codon↗
PubMedDate not supplied

[The first nucleotide of the codon located in the P site of the 80S ribosome is close to G1702 of the 18S rRNA as revealed by crosslinking to a pUUUGUU derivative containing a perfluorophenylazido group at the guanine].

Modification of the 18S rRNA with a pUUUGUU derivative carrying a perfluorophenylazido group at N7 of G was studied in the complex with the human 80S ribosome and Val-tRNA(Val), which directs modified GUU to the P site. Reverse transcription reported modification of invariant G1702 of the 18S rRNA. On evidence of the results and the earlier data on affinity modification of the human ribosome with tetra- or heptaribonucleotide derivatives carrying an alkylating group at the 3' end, the template was assumed to make a bend between the A- and P-site codons, which brings both codons closer to G1702 of the 18S rRNA.

Base Sequence↗
PubMedDate not supplied

[Part of the matrix on the 5' side from codon in the E-segment is close to protein S26 on the human 80S ribosome].

Positioning of mRNA on the 80S ribosome upstream the E site bound codon was studied using derivatives of nona- and dodecaribonucleotides containing the triplet UUU coding for Phe at the 3'-terminus, and a perfluorophenylazide cross-linker on either the first or the third nucleotide. Two sets of the mRNA analogues were used, with the photoactivatable groups on either the C5 atom of the uridine or the N7 atom of the guanosine. The modified nucleotides were directed to positions from -4 to -9 with respect to the first nucleotide of the P site bound codon by tRNA(Phe) cognate to the triplet UUU targeted to the P site. Mild UV-irradiation of ribosomecomplexes with tRNA(Phe) and mRNA analogues resulted in the cross-linking to the 40S subunits preferentially, mainly to the proteins. The principal target for the cross-linking was protein S26 in all cases. Location of the photoactivatable group on the nucleotide at position -4 lead also to the minor cross-linking to protein S3, and at position -6 to protein S14. In the absence of tRNA, all mRNA analogues cross-linked to protein S3.

Codon↗
PubMedDate not supplied

[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.

Electrophoretic Mobility Shift Assay↗
PubMedDate not supplied

[Positioning of mRNA 3' of the a site bound codon on the human 80S ribosome].

Short mRNA analogues carrying a UUU triplet at the 5'-termini and a perfluorophenylazide group at either the N7 atom of the guanosine or the C5 atom of the uridine 3' of the triplet were applied to study positioning of mRNA 3' of the A site codon. Complexes of 80S ribosomes with the mRNA analogues were obtained in the presence of tRNAPhe that directed UUU codon to the P site and consequently provided placement of the nucleotide with cross-linker in positions +9 or +12 with respect to the first nucleotide of the P site bound codon. Both types mRNA analogues cross-linked to the 18S rRNA and 40S proteins under mild UV-irradiation. Cross-linking patterns in the complexes where modified nucleotides of the mRNA analogues were in position +7 were analyzed for comparison (cross-linking to the 18S rRNA in such complexes has been studied previously). The efficiency of cross-linking to the ribosomal components depended on the nature of the modified nucleotide in the mRNA analogue and its position on the ribosome, extent of cross-linking to the 18S rRNA being decreased drastically when the modified nucleotide was moved from position +7 to position +12. The nucleotides of 18S rRNA cross-linked to mRNA analogues were determined. Modified nucleotides in positions +9 and +12 cross-linked to the invariant dinucleotide A1824/A1825 and to variable A1823 in the 3'-minidomain of 18S rRNA as well as to protein S15. The same ribosomal components have been found earlier to cross-link to modified mRNA nucleotides in positions from +4 to +7. Besides, all mRNA analogues cross-linked to the invariant nucleotide c1698 in the 3'-minidomain and to and the conserved region 605-620 closing helix 18 in the 5'-domain.

Base Sequence↗
PubMedDate not supplied

[Silencing of c-myc gene expression by enzymatically and chemically synthesized siRNAs].

Small interfering RNAs (siRNA) provide a powerful approach for sequence-specific silencing of gene expression. In the present study we investigated inhibition of c-myc gene expression by siRNAs targeted to the sequence 1452-1470 b. in third exon of c-myc mRNA and to homologous regions in second exons of c-myc (697-715 b.) and N-myc (302-320 b.) mRNAs. siRNAs were prepared enzymatically according to the scheme, including dsDNA-templates preparation using Klenow fragment, separate in vitro transcription of each RNA strand with subsequent hybridization and removal of leader sequences by T1 RNase. Investigation of c-myc gene silencing by siRNAs revealed that enzymatically prepared siRNAs induce stronger inhibition of c-myc expression, than siRNA with the same sequence prepared by chemical synthesis. It was found that down-regulation of c-myc gene expression by investigated siRNAs results in efficient inhibition and even complete arrest of carcinoma cell proliferation, moreover, the extend of growth inhibition correlates with the level of siRNA-mediated reduction of c-myc mRNA.

Base Sequence↗
PubMedDate not supplied

[Synthesis of leucine codons and their use in the study of tRNALeu codon correspondence].

The leucine codon--UUA, UUG, CUU, CUC, CUA, CUG--was prepared via the phosphotriester approach. The purity of codon was confirmed by reverse phase chromatography in micro scale. The absence of migration of internucleotidic linkages was confirmed by hydrolysis with pancreatic RNase. Synthetic codon were used to study the codon response of six isoacceptor tRNALeu from the cow mammary gland.

Chemical Phenomena↗
PubMedDate not supplied

[Interaction of Escherichia coli RNA polymerase with oligoribonucleotides, homologous to "10"- and "35"- segments of the SPC promotor of bacterial genes].

It was shown previously that E. coli RNA polymerase in a highly selective manner recognizes and binds 11-14-mere oligodeoxyribonucleotides related to the "-10" region of the nontranscribed DNA strand of bacterial gene promoters. The oligodeoxyribonucleotides cover the Pribnow box with flanking nucleotides up to the transcription start. These affinity oligodeoxyribonucleotides inhibit competitively the transcription of bacterial DNA carried out by E. coli RNA polymerase. The present work has demonstrated that E. coli RNA polymerase is not capable of binding the oligoribonucleotides homologous to the affinity oligodeoxyribonucleotides related to the "-10" area of the spc promoter, but binds the oligoribonucleotides which are complementary to the latter. The oligoribonucleotides with a high affinity for the E. coli RNA polymerase strongly inhibit transcription of the bacterial DNA. Attachment of alkylating groups to the 5'-ends of the affinity oligodeoxy- and oligoribonucleotides provides their covalent binding to the E. coli RNA polymerase subunits. It was shown that the modified affinity 32P-labelled oligodeoxyribonucleotide is covalently bound to the sigma-subunit while the modified affinity 32P-labelled oligoribonucleotide is covalently bound to the beta'beta-subunits of the E. coli RNA polymerase. It is suggested that the affinity oligoribonucleotides can be transcribed from the non-transcribed DNA strand in the region of the open complex and functions presumably as a primer which is splitted later from the nascent RNA or as a regulator of transcription.

Base Sequence↗
PubMedDate not supplied

[Structural elements of 80S ribosomes located near the 5'-region of the mRNA-binding center].

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).

Affinity Labels↗
PubMedDate not supplied

[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].

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 Labels↗
PubMedDate not supplied

[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].

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.

Binding Sites↗