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Biomedical subjects

A M Kopylov

Publications and source records attributed to A M Kopylov.

At least 19 recordsLinked to original sources
PubMed2003

Rate of tetracycline photolysis during irradiation at 365 nm.

Kinetics of photolysis of the antibiotic tetracycline (TC) during irradiation at 365 nm was studied in three buffer solutions usually used for studies on TC binding to its main cell targets--a transcriptional repressor protein TetR and to the ribosome. These buffer solutions contain magnesium ions and an antioxidant--mercaptoethanol or dithiothreitol. The rate of TC photolysis was maximal in medium which contained 14 mM mercaptoethanol and 5 mM magnesium ions. In the absence of mercaptoethanol the photolysis rate was more than twofold decreased. The rate constants and quantum yields of the photolysis were determined under various conditions.

Buffers↗
PubMed2002

X-ray analysis of ribosomes: the static of the dynamic.

This review considers a brief history, comments, and consequences of recent remarkable achievements: X-ray analysis on the level of atomic resolution of structures of bacterial ribosomes, their subunits, and functional complexes.

Bacterial Proteins↗
PubMed2001

Neuronal BC1 RNA structure: evolutionary conversion of a tRNA(Ala) domain into an extended stem-loop structure.

By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brain-specific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 5' domain of BC1 RNA, derived from tRNA(Ala), does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNA(Ala) domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 3' end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA.

Aldehydes↗
PubMed2000

A direct photo-activated affinity modification of tetracycline transcription repressor protein TetR(D) with tetracycline(1).

Results of a first successful application of a direct photo-induced affinity modification of Tet repressor (TetR(D)) protein with tetracycline within a complex of known three-dimensional structure are described. The conditions of the modification have provided suitable yields of the modified complex and allowed characterization of the modified segments of the protein. The potential of tetracycline as a fine modifying reagent was established. In the complex of TetR(D) protein with tetracycline, the antibiotic modifies at least two segments, Ile59-Glu73 and Ala173-Glu183, which form a binding tunnel for the drug according to the X-ray analysis. These data open possibilities for the use of different tetracycline targets for structural studies in solution.

Amino Acid Sequence↗
PubMed1999

A study of the thermophilic ribosomal protein S7 binding to the truncated S12-S7 intercistronic region provides more insight into the mechanism of regulation of the str operon of E. coli(1).

A study of the ability of His6-tagged ribosomal protein S7 of Thermus thermophilus to interact with the truncated S12-S7 intercistronic region of str mRNA of Escherichia coli has been described. A minimal S7 binding mRNA fragment is a part of the composite hairpin, with the termination codon of the S12 cistron on one side and the initiation codon of the next S7 cistron on the other. It has a length in the range of 63-103 nucleotides. The 63 nucleotide mRNA fragment, which corresponds to a putative S7 binding site, binds very poorly with S7. Tight RNA structure models, which behave as integral systems and link the S7 binding site with the translational regulation region of the hairpin, are suggested. This observation provides more insight into the mechanism of S7-directed autogenous control of translational coupling of str mRNA.

Ammonium Chloride↗
PubMed1998

An extremely high conservation of RNA-protein S7 interactions during prokaryotic ribosomal biogenesis.

Direct determination of RNA-protein complex structures is often facilitated by the use of thermophilic proteins; however E. coli is the most investigated system so far. A hybrid approach is to form heterologous complexes of E. coli RNA with thermophilic proteins. The rationale for this approach to RNA-protein interactions in ribosomes is based on the ability of the thermophilic protein S7 to replace a homologous counterpart in vivo. In vitro, the protein S7 of Thermus thermophilus is able to form complexes with both the minimal 16S rRNA fragment and the intercistronic region of the str operon mRNA from E. coli (Kd = 1.4 x 10(7) M and 1.1 x 10(-7) M respectively). The interaction of Thermus S7 with the E. coli intercistronic mRNA is surprising, because this region does not exist in the thermophilic str operon. It suggests a high degree of conservation of an RNA-binding site on S7.

Bacterial Proteins↗
PubMed1995

In vivo assembly of plasmid-expressed ribosomal protein S7 of Thermus thermophilus into Escherichia coli ribosomes and conditions of its overexpression.

Researchers still have great difficulty in isolating individual ribosomal proteins from the ribosome in quantities high enough for structural research. To this end, when studying protein S7, we created an E. coli overproducer of the recombinant protein S7 of Thermus thermophilus. The vector for expression was pQE-32 having a strong promoter of E. coli phage T5 and six triplets of His at the 5'-end. This N-terminal six His tag of the fusion protein is responsible for binding to Ni-NTA-resin and allows purifying the protein in one step. The yield of the recombinant protein was 20% and more of the total cellular proteins. In addition we have shown that the recombinant thermophilic protein is incorporated in vivo into the ribosome of E. coli despite the fact that these proteins (thermophilic and mesophilic) have a rather low homology, only 52%. This fact provides a base for the system to study functions of individual proteins.

Amino Acid Sequence↗
PubMed1995

Segment selection during 'phase variation'-type regulation of gene replacement mediated by FLP recombinase in the yeast Saccharomyces cerevisiae.

FLP recombinase has recently been used as a tool to direct the exchange between invertible DNA segments, called 'Phase variation'-type regulation of gene replacement in eukaryotic cells. Using an appropriate selective medium, positive segment selection was shown to be efficient during the regulation of gene replacement. The efficiency was determined from the copy number ratio of invertible segments with the use of the neomycinphosphotransferase II (NPTII) gene bearing invertible segments located on the episomal yeast plasmid, and the resident 2-microns circle. Without the selection the segments copy number ratio was retained in growing cells. The results obtained are an evidence for the efficiency of positive segment selection during the 'Phase variation'-type regulation of gene replacement in eukaryotic cells.

DNA Nucleotidyltransferases↗
PubMed1992

Identification of the Escherichia coli 30S ribosomal subunit protein neighboring mRNA during initiation of translation.

To identify the proteins of the 30S ribosomal subunit of E coli that neighbor mRNA in the ternary initiation complex (mRNA*30S subunit*tRNA(fMet), we used an affinity cross-linking approach in which photoactivated groups were attached to different positions along the mRNA chain. A series of mini-genes originating from the 5'-end region of the cro gene of lambda bacteriophage were constructed as templates for mini-mRNA synthesis. Two strategies were used to introduce photo-reactive agents into the message. According to the first, two transcripts were isolated from E coli and chemically derivatized at their 5'-ends with a photoinducible diaziril group. One of these messages allowed for localization of the 5'-end of the Shine-Dalgarno sequence while the other one allowed for labeling of the ribosome at the 5'-end side of the initiation AUG codon in the P site. According to the second approach, 5-azidouridine (5N3U) was randomly incorporated into mRNA transcripts during a T7 RNA polymerase catalyzed reaction by using a mixture of 5N3UTP and UTP. A message that had U residues at either -4, -3, -1, +2 and +14, +19, +20 positions was used (A from cro AUG is +1). Whereas cross-links with the 5N3U transcripts were essentially 'zero-length', the 5'-derivatized transcripts were covalently attached to ribosomal components about 14 A from the 5'-end. We found that proteins S1, S7, S5, S3 and S4 compose, or were close to, the ribosomal mRNA-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1992

Photochemical cross-linking of the anticodon loop of yeast tRNA(Phe) to 30S-subunit protein S7 at the ribosomal A and P sites.

Yeast tRNA(Phe), containing the photoreactive nucleoside 2-azidoadenosine at position 37 within the anticodon loop, has been cross-linked to the aminoacyl-tRNA (A) and peptidyl-tRNA (P) binding sites of the Escherichia coli ribosome. The 30S subunit was exclusively labeled in each case, and cross-linking occurred to both protein and 16S rRNA. Electrophoretic and immunological analyses demonstrated that S7 was the only 30S-subunit protein covalently attached to the tRNA. However, digestion of the A and P site-labeled S7 with trypsin revealed a unique pattern of cross-linked peptide(s) at each site. Thus, while the anticodon loop of tRNA is in close proximity to protein S7 at both the A and P sites, it neighbors a different portion of the protein molecule in each. The placement of the aminoacyl- and peptidyl-tRNA binding sites is discussed in relationship to recent models of the 30S ribosomal subunit.

Anticodon↗
PubMed1990

[The 5S rRNA-protein complex of Escherichia coli studied by carbodiimide modification].

5S rRNA-protein complex has been reconstituted from 5S rRNA and total protein of large (L) ribosomal subunit of Escherichia coli. The complex consists of 5S rRNA and 3 proteins only: L5, L18, L25. A water-soluble carbodiimide [N-cyclohexyl-N'-(2-morpholinoethyl)-carbodiimide-methyl-p-toluolsulp honate] cross-links L18 to 5S rRNA at pH 7.2 and L25 to 5S rRNA at pH 7.7. This pH-dependence of cross-linked proteins is a consequence of the difference in stability of the initial complex: the complex has all three proteins at pH 7.7 but L18 mainly at pH 7.2. It has been shown that L18 stimulates the chemical modification of U87 and U89 residues of 5S rRNA by carbodiimide. A model of L18-5S rRNA complex has been proposed.

Bacterial Proteins↗
PubMed1989

[Synthesis in E. coli cells of short RNA encoded in plasmids].

The synthesis of 5S rRNA and 4.5S RNA in E. coli HB 101 cells harbouring plasmids pKK 5-1 and pKK 247-2 was studied. The plasmids were derived from pBK 322 and contained genes coding for 5S rRNA and 4.5S RNA with regulatory elements of an rRNA transcription operon rrn B. When the cells were grown on enriched or minimal media (2 and 0.3 duplications per hour), the synthesis of both 5S rRNA and 4.5S RNA was proportional to the gene dosage and was greater in the plasmid than in the host strain. Such RNA accumulation did not change the cell growth parameters and was thus not toxic for the cells. At high growth rates, the RNA synthesis in the cells became excessive, and the processing system was upset with the accumulation of RNA precursors. The fact confirms the hypothesis, according to which the whole rRNA operon is essential for its own feedback regulation.

Escherichia coli↗
PubMed1985

The nucleotide sequence of the gene coding for the 16S rRNA from the archaebacterium Halobacterium halobium.

The complete 1473-bp sequence of the 16S rRNA gene from the archaebacterium Halobacterium halobium has been determined. Alignment with the sequences of the 16S rRNA gene from the archaebacteria Halobacterium volcanii and Halococcus morrhua reveals similar degrees of homology, about 88%. Differences in the primary structures of H. halobium and eubacterial (Escherichia coli) 16S rRNA or eukaryotic (Dictyostelium discoideum) 18S rRNA are much higher, corresponding to 63% and 56% homology, respectively. A comparison of the nucleotide sequence of the H. halobium 16S rRNA with those of its archaebacterial counterparts generally confirms a secondary structure model of the RNA contained in the small subunit of the archaebacterial ribosome.

Archaea↗