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G G Karpova

Publications and source records attributed to G G Karpova.

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↗
PubMed2001

Location of template on the human ribosome as revealed from data on cross-linking with reactive mRNA analogs.

In this review we summarize data on the location of template on the human ribosome that we obtained from cross-linking (affinity labeling) experiments using reactive mRNA analogs. Types of mRNA analogs, model complexes of these analogs with 80S ribosomes, and methods for analysis of the ribosomal components (proteins and rRNA nucleotides) cross-linked with the mRNA analogs are reviewed. From analysis of the cross-linking data, we suggest a scheme for the arrangement of mRNA on the human ribosome and compare the organization of the mRNA binding center on human and Escherichia coli ribosomes.

Affinity Labels↗
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↗
PubMed1998

Isolation, structural analysis and mapping of the functional gene of human ribosomal protein S26.

The nucleotide sequence of the gene of human ribosomal protein S26 has been assembled from cDNA and genomic PCR-amplified DNA fragments, and its transcription start site has been determined by primer extension. The gene is composed of four exons and three introns spanning 2027bp. Like other ribosomal protein genes of vertebrates, this gene contains a short first exon corresponding exactly to the short untranslated 5'- UTR. Its transcription start site is embedded in a polypyrimidine tract. Using PCR on DNAs from hybrid cell lines with a different set of human chromosomes, the intron-containing gene of ribosomal protein S26 was mapped to human chromosome 12.

Base Sequence↗
PubMed1998

Nucleotides of 18S rRNA surrounding mRNA at the decoding site of translating human ribosome as revealed from the cross-linking data.

mRNA analogs, 4-(N-2-chloroethyl-N-methylamino)benzylmethyl-[5'-32P]-phosphamide derivatives of oligoribonucleotides pAUGUn (n=0, 3 or 6), were used for affinity labelling of human 80S ribosomes in complexes with codon-anticodon interaction at the P-site. These complexes were obtained in the presence of fractionated lysate from rabbit reticulocytes deprived of endogenous ribosomes and mRNAs. In all cases, 40S subunits were labelled preferentially. Within the subunits, both ribosomal proteins and 18S rRNA were modified. Ribosomal proteins cross-linked to pAUGUn derivatives were identified earlier. In this paper, nucleotides G-1010, G-1029, G-1033, G-1051, G-1054 and G-1059 of 18S rRNA cross-linked to both pAUG and pAUGU3 derivatives were identified by reverse transcription analysis.

Affinity Labels↗
PubMed1998

[Cloning and structure-function analysis of the human S26 ribosomal protein gene].

Gene HRPS26 encoding the S26 human ribosomal protein has been sequenced. Gene HRPS26 consists of four exons and three introns. Its size is 2027 bp; the size of its mRNA is 438 nucleotides. As most of the genes of ribosomal proteins of vertebrates, the HRPS26 gene has a short first exon, which corresponds to the 5'-untranslated region of mRNA; the origin of transcription is located in the polypyrimidine tract. The functional activity of the cloned HRPS26 promoter region has been confirmed by transcription of hybrid plasmids in HeLa cells.

Base Sequence↗
PubMed1997

Protein environment of mRNA at the decoding site of 80S ribosomes from human placenta as revealed from affinity labeling with mRNA analogs--derivatives of oligoribonucleotides.

Affinity labeling of 80S ribosomes from human placenta has been studied using various mRNA analogs, namely, 2',3'-O-[4-(N-2-chloroethyl)-N-methylamino]benzylidene derivatives of oligoribonucleotides (Up)(n-1)U[32P]pC (n = 3, 6 or 12) and AUGU3[32P]pC as well as ([4-(N-2-chloroethyl)-N-methylamino]benzylmethyl-[5'-32P]-phospham ide derivatives of pAUGUn (n = 3 or 6). Labeling of 80S ribosomes with the derivatives of oligouridylates was carried out in complexes obtained nonenzymatically in the presence of saturating amounts of Phe-tRNA(Phe). Complexes with derivatives bearing AUG codon were obtained using a fractionated lysate from rabbit reticulocytes which contained protein translation factors and was deprived from endogeneous ribosomes and mRNAs. In all cases, 40S subunits were labeled preferentially. Within the subunits, both 18S rRNA and proteins were found to be modified. Sites of cross-linking in 18S rRNA have been identified earlier. Here, it is shown that the main targets of cross-linking among the ribosomal proteins were S3 and S3a (with minor modification of S26) for the 3'-derivatives of (Up)5UpC and (Up)11UpC. For the same derivative of (Up)2UpC, the reverse modification pattern was observed. 5'-derivatives of pAUGUn were cross-linked to proteins S3 and S3a in comparable extent; 3'-derivative of AUGU3pC modified protein S3a preferentially.

Affinity Labels↗
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↗
PubMed1997

Nucleotide G-1207 of 18S rRNA is an essential component of the human 80S ribosomal decoding center.

mRNA analogues-derivatives of oligoribonucleotides consisting of two different codons and bearing an aryl azide group at the 5'-phosphates-were crosslinked to human 80S ribosomes by UV-irradiation of the various model complexes obtained in the presence of the cognate tRNAs. Three sequences, namely pUUUGUU (coding for Phe and Val), pUUCUAAA (first triplet coding for Phe and second being stop-codon), and pGUGUUU (coding for Val and Phe), have been used. Sequences of 18S rRNA containing nucleotides crosslinked to the mRNA analogues were examined by hydrolysis with RNase H in the presence of various cDNA probes. Crosslinked nucleotides were identified by primer extension. In all cases, only nucleotide G-1207 (equivalent to G-926 in Escherichia coli 16S rRNA) has been detected as crosslinked. Crosslinking of the mRNA analogues to the large ribosomal subunit was negligible.

Benzoates↗
PubMed1996

Characterization of the human small-ribosomal-subunit proteins by N-terminal and internal sequencing, and mass spectrometry.

Reverse-phase HPLC was used to fractionate 40S ribosomal proteins from human placenta. Application of a C4 reverse-phase column allowed us to obtain 27 well-resolved peaks. The protein composition of each chromatographic fraction was established by two-dimensional polyacrylamide gel electrophoresis and N-terminal sequencing. N-terminally blocked proteins were cleaved with endoproteinase Lys-C, and suitable peptides were sequenced. All sequences were compared with those of ribosomal proteins available from data bases. This allowed us to identify all proteins from the 40S human ribosomal subunit in the HPLC elution profile. By matrix-assisted laser-desorption ionization mass spectrometry the masses of the 40S proteins were determined and checked for the presence of post-translational modifications. For several proteins differences to the deduced sequences and the calculated masses were found to be due to post-translational modifications.

Amino Acid Sequence↗
PubMed1995

[Mapping the genes for ribosomal proteins S14 and S17 on human chromosomes using cDNA from a panel of hybrid cells].

A new method of mapping transcriptionally active genes of ribosomal proteins onto human chromosomes is proposed. The method is based on the detection of the expression of human ribosomal protein mRNA in rodent-human hybrid cells carrying different human chromosomes. Using this method, the functional gene of the human ribosomal protein S17 was mapped and the location of the S14 ribosomal protein gene on chromosome 5 was confirmed.

Animals↗
PubMed1995

[Cloning and determination of the primary structure of DNA complementary to the mRNA of human ribosomal protein L11].

A polymerase chain reaction strategy was employed to isolate cDNA encoding L11 human ribosomal protein. Based on the known nucleotide sequence of 5'-region of the ribosomal protein L11 mRNA, we have designed primers and used them in amplification of corresponding sequence of human cDNA from total placenta cDNA. The fragment of RPS26 cDNA was cloned in plasmid vector and sequenced. Sequence analysis showed that there is high homology (88%) between coding regions of RPS26 mRNAs in rat liver and human placenta. The amino acid exchanges were observed at positions: 91 (Asp-->Glu), 217 (Thr-->Ala), 352 (Lys-->Glu).

Amino Acid Sequence↗
PubMed1995

[Mapping of the genes for ribosomal proteins S26, L19, and L32 on human chromosomes].

A fragment of cDNA and an intron-containing fragment of the human L19 ribosomal protein (RPL19) gene, and introns of the human ribosomal proteins S26 (RPS26) and L32 (RPL32) genes were cloned and sequenced. The intron-containing genes of these ribosomal proteins were mapped to human chromosomes by means of polymerase chain reaction (PCR) using a human/rodent hybrid DNA panel.

Animals↗
PubMed1995

[The 5'-region of mink ribosomal protein S26 cDNA: sequencing and comparative analysis].

The 5'-terminal region of the mink S26 ribosomal protein cDNA was cloned using polymerase chain reaction. The nucleotide sequences of the 5'-UTR (24 bp) and a 120-bp fragment of the coding region of RPS26 mRNA were determined. The homology between the coding regions of the human and mink RPS26 mRNAs proved to be 90.8%. The nucleotide sequences of the 5'-UTRs of mink, human, and rat RPS26 mRNAs, as well as mRNAs of the Drosophila S31 protein and Neurospora crp-5 protein, which are homologous to mammalian RPS26, were compared. A highly conserved 9-bp sequence located immediately upstream of the AUG codon was revealed in the 5'-UTR of the RPS26 mRNAs from different species.

Animals↗
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↗
PubMed1994

mRNA binding track in the human 80S ribosome for mRNA analogues randomly substituted with 4-thiouridine residues.

The interaction between mRNA and 18S rRNA in human 80S ribosomes has been studied using synthetic mRNA analogues randomly substituted with 4-thiouridine, which can be photoactivated for cross-linking. Two mRNA analogues with different sequences have been used for complex formation with ribosomes without or with the presence of a cognate tRNA. Cross-linked 18S rRNA nucleotides were identified by reverse transcription analysis. The base U630 in 18S rRNA was the main target of cross-linking for both of the mRNA analogues studied, and three minor sites of cross-linking, A1060, U1046, and U966, were also identified. Thus, in the case of human 80S ribosomes, the set of nucleotide residues cross-linked to the mRNA analogues is significantly smaller than the twelve sites seen for Escherichia coli with these same two mRNA analogues [Bhangu, R., & Wollenzien, P. (1992) Biochemistry 31, 5937-5944]. The residue U630 is within a highly conserved region corresponding to the 530 loop region of eubacterial 16S rRNA; the cross-link to this site indicates that it plays a key role in interacting with mRNA on 80S ribosomes independently of the presence of a cognate tRNA at the P site.

Base Sequence↗