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A A Malygin

Publications and source records attributed to A A Malygin.

At least 19 recordsLinked to original sources
PubMed2005

Recombinant human ribosomal protein S16: expression, purification, refolding, and structural stability.

The cDNA of human ribosomal protein S16 was cloned into the expression vector pET-15b. Large-scale production of the recombinant protein was carried out in E. coli cells and highly purified protein was isolated. A method for refolding the protein from inclusion bodies was optimized. The secondary structure content of the refolded protein was analyzed by CD spectroscopy. It was found that 21 +/- 4% of the amino acid sequence of the protein forms alpha-helices and 24 +/- 3% is in beta-strands. The protein structure stability was studied at various pH values and urea concentrations. The protein is quickly denatured at pH above 8.0, whereas increasing of urea concentration causes slow unfolding of the protein.

Amino Acid Sequence↗
PubMed2000

Proteins S7, S10, S16 and S19 of the human 40S ribosomal subunit are most resistant to dissociation by salt.

The protein components of human 40S ribosomal subunits were dissociated by centrifugation in gradients of sucrose and LiCl in the presence of 0.5 M KCl. The proteins that split off were analyzed by SDS-PAGE and 2D-PAGE. The order of dissociation of the proteins, depending on the salt concentration (from 0.8 M to 1.55 M), was established. The majority of the proteins started to split off simultaneously at a monovalent cation concentration of 0.8 M. Ten proteins were found to be more resistant; of these proteins S7, S10, S16, and S19 were retained most strongly and thereby may be considered to be core proteins.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1999

Proteins neighboring 18S rRNA conserved sequences 609-618 and 1047-1061 within the 40S human ribosomal subunit.

The structure of human 40S ribosomal subunits has been probed by a cross-linking strategy based on the use of oligonucleotide derivatives that modify proteins in the vicinity of specific 18S rRNA sequences. The oligonucleotide derivatives carried a p-azidoperfluorobenzamide group at the 5' ends and were complementary to 18S rRNA sequences 609-618 and 1047-1061, homologous to the highly conserved regions designated as the "530 stem-loop" and "790 stem-loop", respectively, in Escherichia coli 16S rRNA. Ribosomal proteins surrounding these sequences were the main targets of the cross-linking. Proteins S3 and S5 were cross-linked to the derivative complementary to the sequence 609-618, and proteins S2 and S3 were modified by the derivative complementary to the sequence 1047-1061. Cross-linking was not affected by binding of 40S subunits to either poly(U) or poly(U) and Phe-tRNA(Phe).

Base Sequence↗
PubMed1997

Studying functional significance of the sequence 980-1061 in the central domain of human 18S rRNA using complementary DNA probes.

Region 980-1061 in human 18S rRNA has been chosen on the basis of our previous results, indicating that cross-linking sites of the alkylating mRNA analogs are located within this region. In the present study, we have used 10 DNA 15-mers complementary to various overlapping sequences within the 18S rRNA positions 980-1061. Their abilities to bind selectively to the target rRNA sequences were proved by hydrolysis of 18S rRNA within heteroduplexes with the corresponding probes by RNase H. Four sequences (980-994, 987-1001, 1025-1039 and 1032-1046) were found to be well accessible for binding of the respective cDNA probes within 40S subunits. None of the oligomers inhibited tRNA(Phe)-dependent binding of oligo(U) messenger to 40S subunits and binding of Met-tRNA(imet) to 40S subunits in the presence of eIF-2 and nonhydrolysable GTP analog. Nevertheless, two probes (complementary to the 18S rRNA sequences 987-1001 and 1025-1039) being covalently attached to 40S subunits, inhibited translation of poly(U) by human 80S ribosomes in a cell-free system. The same oligomers revealed the most pronounced inhibitory action on the binding of messenger trinucleotide in the complex pAUG.40S.Met-tRNA(imet).eIF-2.GTP. Results of these functional assays demonstrate the importance of the 18S rRNA sequences 987-1001 and 1025-1039 for translation process on human ribosomes, most probably at the initiation step.

Alkylation↗
PubMed1994

Arrangement of mRNA at the decoding site of human ribosomes. 18S rRNA nucleotides and ribosomal proteins cross-linked to oligouridylate derivatives with alkylating groups at either the 3' or the 5' termini.

Affinity labeling of human placental 80S ribosomes with mRNA analogs of up to 12 uridyl residues, i.e. alkylating derivatives of oligouridylates bearing either 4-(N-2-chloroethyl-N-methylamino)benzylmethylphosphamide group at the 5'-termini or 2',3'-O-[4-(N-2-chloroethyl-N-methylamino)]benzylidene residue attached to the 3'-termini, in the presence of cognate Phe-tRNA(Phe) has been investigated. All the mRNA analogs modified only the 40S subunit. The fraction of 18S rRNA modified by the mRNA analogs with the alkylating group at the 5'-end decreased dramatically with extension of the reagent oligouridylate moiety. Nucleotides of 18S rRNA alkylated with the mRNA analogs were determined using a reverse transcription technique. For the mRNA analogs with the alkylating groups at the 3'-termini, G1702 and G1763/G1764 were identified as the cross-linking sites. The intensities of the bands corresponding to reverse transcriptase stops depended on the length of the reagent oligouridylate moieties. Cross-linking sites of the mRNA analogs with the alkylating group at the 5'-termini on 18S rRNA were A1023, C1026, C1057 and A1058 for the (pU)3 and (pU)4 derivatives and a single nucleotide C1057 for the (pU)6 one. Ribosomal protein S26 was found as the main target of modification with the same derivatives of (pU)6 and (pU)12.

Affinity Labels↗
PubMed1993

Structural arrangement of the codon-anticodon interaction area in human placenta ribosomes. Affinity labelling of the 40S subunits by derivatives of oligoribonucleotides containing the AUG codon.

Using the derivatives of the oligoribonucleotides pAUGUn and AUGUnC (n = 0; 3) bearing an alkylating group at either the 5' or 3' end, respectively (mRNA analogues), the affinity labelling of the 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 and ribosomal proteins labelled with these mRNA analogues were identified. The sites of covalent attachment of the pAUGUn derivatives with a reactive group at the 5' end were located between 18S rRNA positions 976 and 1164. The derivative of AUGU3C with an alkylating group at the 3' end modified 18S rRNA mainly at the 593-673 region. The main targets of the 3' end derivative of AUGC were located between positions 1610 and 1869. The proteins S3/S3a, S6, S7 and S14/S15 were modified by both types of the oligoribonucleotide derivatives regardless of the point of the reactive group attachment to the oligonucleotide moiety. The proteins S2 and S4 were modified by both the 3' end derivative of AUGC and 5' end derivative of pAUGU3; and the protein S8 was modified by the 3' end derivative of AUGC. The proteins S5 and S9 were labelled by the 5' end derivative of pAUGU3, and the protein S17 was modified by the 5' end derivative of pAUG.

Affinity Labels↗
PubMed1993

[Affinity modification of 80S ribosomes from human placenta with mRNA analogs--derivatives of oligouridylates with and alkylating group at the 5'-end].

Affinity labelling of 80S ribosomes from human placenta with 4-(N-methylamino-N-2-chloroethyl)benzylmethylphosphoramide derivatives of oligouridylates pUn (n = 3, 4, 6, 12) bearing 5'-32P-label was studied. Complexes of these derivatives with 80S ribosomes where codon-anticodon interaction took place either in P-site (in the case of pU3-and pU4-derivatives), or in P- and A-site simultaneously (in the case of pU6- and pU12-derivatives) were obtained in the presence of Phe-tRNA(Phe). All the reagents modified only the 40S subunit. The extent of 18S rRNA modification by pU3-, pU4-, pU6- and pU12-derivatives as a fraction of the total modification extent of 18S rRNA and proteins in the 40S subunit equaled 96, 93, 24 and 4%, respectively. The pU4-derivative was covalently attached at positions 976-1061 and 1058-1164 and pU12-derivative was covalently attached within regions 976-1061, 1058-1164, 593-673 and 1748-1869 of the 18S rRNA. By means of the primer extension technique, modified bases in 18S rRNA were determined to be: A-1023, C-1026, A-1027, A-1058, G-1059 for pU3- and pU4-derivatives and A-1058 for pU6-derivative.

Affinity Labels↗
PubMed1993

[Affinity modification of 40S ribosomal subparticles from human placenta with mRNA analogs--AUGUnC oligoribonucleotide analogs with an alkylating group at the 3'-end].

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 Labels↗
PubMed1992

Functional topography of human ribosomes as studied by affinity labeling with reactive mRNA analogs.

Derivatives of 5'-32P labeled (pU)3 an (pU)6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residue attached to 5'-phosphate via phosphamide bond and (Up)5U[32P]pC and (Up)11U[32P]pC bearing 4-(N-2-chloroethyl-N-methylamino)benzyl residue attached to 3'-end via benzylidene bond were applied for the affinity labeling of 80S ribosomes from human placenta in the presence of a cognate tRNA. The derivatives of 32P-labeled pAUG and pAUGU3 analogous to the 5'-phosphamides of (pU)n were used for affinity labeling of 40S subunits in the presence of ternary complex eIF-2.GTP.Met-tRNA(f). The sites of the reagents' attachment to 18S ribosomal RNA were identified by blot-hybridization of the modified 18S rRNA with restriction fragments of the corresponding rDNA. They were found to be located within positions 976-1057 for (pU)6 and pAUGU3 derivatives and within 976-1164 for (pU)3 and pAUG ones. The sites of 18S rRNA modification with the derivatives of (Up)5UpC and (Up)11UpC were found within positions 1610-1869 at 3'-end of the molecule. All the sites identified here are located presumably within highly conserved parts of the eukaryotic small subunit rRNA secondary structure.

Affinity Labels↗
PubMed1991

[Segments of 18S rRNA, located in the area of the mRNA-binding center of ribosomes from human placenta].

The affinity labelling of human placenta 80S ribosomes by 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphoramide of hexauridylate has been studied. This mRNA analogue has normal coding properties because its binding to placenta ribosomes significantly increases in the presence of the cognate tRNA(Phe). Incubation of the mRNA analogue in the complex with ribosomes and Phe-tRNAPhe) leads to its covalent attachment exclusively to the small subunit (mainly to 18S rRNA). The reaction site has been shown by hybridisation experiments to be located within positions 975-1055 of 18S rRNA. The identified fragment is located in a highly conserved part of the small subunit rRNA domain II.

Affinity Labels↗
PubMed1990

Identification of a site on 18 S rRNA of human placenta ribosomes in the region of the mRNA binding center.

The affinity labeling of human placenta 80 S ribosomes by 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphamide of hexauridylate was studied. This mRNA analog has normal coding properties because its binding to placenta ribosomes significantly increases in the presence of cognate tRNAPhe. Incubation of the mRNA analog in the complex with the ribosomes and Phe-tRNAPhe leads to its covalent attachment exclusively to the small subunit (mainly to 18 S rRNA). The site of the reaction has been identified by hybridization experiments to be located within positions 975 to 1055 of 18 S rRNA. The identified fragment is located in a highly conserved part of the small subunit rRNA domain II.

Affinity Labels↗
PubMedDate not supplied

[Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S ribosomal subunit].

The molecular environment of the internal ribosome entry site (IRES) element of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 was crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group in nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the outer subunit surface between the head and the body next to the "beak" near the entrance into the mRNA-binding channel. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.

Hepacivirus↗
PubMedDate not supplied

[Ribosomal protein binding with the first intron of the human ribosomal protein S26 pre-mRNA stimulates its interaction with proteins extracted from Hela cells].

As shown by nitrocellulose filtration assays with RNA fragments transcribed from various regions of the human ribosomal protein (rp) S26 gene, proteins of the 40S ribosome subunit bind to the first intron of the rpS26 pre-mRNA. The binding involved mostly S23, S26 and, to a lesser extent, S13/16. Negligible binding was observed for S2/3a, S6, S8, S10, S11, and S20. Small-subunit proteins did not affect the efficiency of in vitro splicing of a pre-mRNA fragment corresponding to the first intron, second exon, second intron, and a part of the third exon of the rpS26 gene. However, ribosomal proteins substantially increased UV-induced adduction of the pre-mRNA fragments with nuclear extract proteins of HeLa cells. The same set of HeLa proteins was observed with each pre-mRNA fragment. Ribosomal proteins formed adducts only in the absence of HeLa proteins.

Cell Extracts↗
PubMedDate not supplied

[Affinity modification of 80S ribosomes from human placenta by derivatives of tri- and hexauridylates as mRNA analogs].

Derivatives of 5'-32P]labeled (pU)3 and (pU)6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residues attached to 5'-phosphates via phosphamide bond were applied to the affinity labeling of 80S ribosomes from human placenta. The reagents had normal coding properties and were fixed in the ribosomal mRNA-binding region by codon-anticodon interaction with cognate Phe-tRNA(Rhe) at P site (in the case of (pU)3 derivative) or at both A and P sites (in the case of (pU)6 one). Both reagents were found to modify only the 40S subunit. The sites of the reagents attachment to 18S ribosomal RNA were identified by blot-hybridization of the modified 18S rRNA with restriction fragments of the corresponding rDNA. They were found to be located within positions 976-1057 for (pU)6 derivative and within 976-1164 for (pU)3 one. These sites are located presumably within highly conserved parts of the eukaryotic small subunit rRNA secondary structure.

Alkylating Agents↗
PubMedDate not supplied

[Affinity modification of 80S ribosomes from human placenta with mRNA analogs--derivatives of oligouridylates with alkylating groups at the 3'-end].

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 Labels↗