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O A Dontsova

Publications and source records attributed to O A Dontsova.

36 records · Page 2Linked to original sources
PubMed1996

[Phosphate sites of RNA-ligands interacting with ribosome at different stages of translation. Thiophosphate method of analysis].

A novel footprinting method was recently developed which identifies phosphate groups of RNA involved in strong RNA-RNA and RNA-protein interactions. The method is based on iodine-dependent RNA cleavage at phosphothioate groups as long as these groups are not protected from iodine. Our recent studies of mRNA and tRNA regions protected in active ribosomes are summarized; initiation state of ribosomes as well as two elongation states in pre- and post-translocational states were analyzed. Only one phosphate group of mRNA, which was two positions upstream of the decoding codons, was weakly protected in longation complexes, whereas this group and the phosphate groups in the Shine-Dalgarno sequence were protected in the initiation complex. No protection was observed downstream of the decoding codons. On the contrary, numerous phosphate residues of tRNA were protected by the ribosome. The tRNA protection patterns significantly varied between two tRNAs simultaneously bound to the ribosome. The protection pattern of an individual tRNA was not significantly affected by translocation. The data indicate that both tRNA molecules are tightly bound to the ribosome, whereas mRNA is fixed predominantly by two tRNAs via codon-anticodon interaction. A possible translocation mechanism is suggested.

Base Sequence↗
PubMed1995

[Directed cleavage of the 16S rRNA molecule at a single internucleotide bond].

Cleavage of 16S ribosomal RNA (rRNA) from E. coli "hammerhead" type ribozymes as well as by RNAase iI in the presence of "hymeric" (2'-deoxy-F-thymidine containing) oligonucleotides has been studied. The conditions for the cleavage of a desired single internucleotide bond have been found for a large molecule with a very complicated secondary and three-dimensional structure.

Base Sequence↗
PubMed1993

mRNA-ribosome interactions.

Synthetic mRNA analogues were constructed with sequences related to the Cro-protein mRNA from lambda-phage and prepared by T7 transcription. Each mRNA contained several thiouridine (thio-U) residues. The regions upstream from the AUG initiator codon of the mRNA were the same in all the messages, whereas in the downstream part the thio-U residues were placed in selected positions. These positions covered the region from +4 to +16 (A in the initiator AUG codon being defined as +1). After binding to the ribosome in the presence of initiator tRNA the thio-U residues were activated by u.v. irradiation and the resulting sites of cross-linking to 16 S rRNA and ribosomal proteins were analysed. Cross-links to several ribosomal proteins were identified in different types of complex. Changes in the conformation of the small ribosomal subunit in different initiation and elongation complexes are discussed.

Bacteriophage lambda↗
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↗
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↗
PubMedDate not supplied

Structure and function of 5S rRNA in the ribosome.

5S rRNA is a small RNA molecule that is a component of a ribosome from almost all living organisms. In this review, we discuss the biogenesis of 5S rRNA and its properties as an independent structural domain of a ribosome as well as the current concepts concerning the higher order structure of 5S rRNA in free state and in its complexes with ribosomal proteins and its folding in the ribosome. Special attention is paid to recent experimental approaches that have been useful in 5S rRNA studies. Our own data on topography of 5S rRNA in the ribosomes are discussed in detail. The hypothesis describing the possible functional role of 5S rRNA for ribosome functioning is discussed.

Base Sequence↗
PubMedDate not supplied

[Study of ribosome structure using the biochemical methods: judgment day].

The data on RNA-RNA interactions between the components of the E. coli translation machinery obtained by X-ray crystallography and chemical methods are compared. The approaches to the study of RNA secondary and tertiary structure are assessed. The following conclusions are made: comparative sequence analysis and compensatory mutations approach both give reliable data on the RNA secondary structure. The chemical modification technique provides good results. Local cleavage of internucleotide bonds by hydroxyl radicals is reliable in the frame of its 40 A resolution, in contrast to the application of copper-phenanthroline complex as a cleavage reagent, which is unreliable. Direct UV irradiation and nitrogen mustard treatment are the best methods of crosslink generation. In vitro transcription is the only good method for the incorporation of nucleotide analogs in RNA. RNase H hydrolysis and/or nucleotide-specific RNases fingerprints must be applied for the crosslink site determination in parallel with reverse transcription.

Base Sequence↗
PubMedDate not supplied

[Telomerase: structure and properties of the enzyme, characteristics of the yeast telomerase].

Telomerase is a ribonucleoprotein that extends the telomeric ends of the chromosomes to counterbalance the natural shortening due to incomplete DNA replication in eukaryotic cells. The core enzyme consists of catalytic reverse transcriptase subunit TERT (Telomerase Reverse Transcriptase) and RNA subunit TER (Telomerase RNA), a short specific region of which serves as a template for synthesis of the telomeric repeats. In this review we focus on the telomerase from yeast Saccharomyces cerevisiae. Despite the intensive research of telomerase in different organisms, the enzyme mechanism remains unclear. The observed peculiarities of the yeast telomerase is of great interest too. Unlike ciliate and human telomerases, yeast enzyme can add only one telomeric repeat to a DNA oligonucleotide (primer) imitating the single-stranded telomeric end of the chromosome and remains stably bound to it after elongation. This review is an attempt to summarise results of numerous studies of the structure and functions of the core enzyme components, their interactions between each other and with a primer, telomerase activity on different substrates in vitro. Also the peculiarities of the telomerase functioning in a cell and accessory proteins of the telomerase complex are discussed.

5' Flanking Region↗
PubMedDate not supplied

[The structural changes in the ribosome during the elongation cycle].

The plenty of data about structural changes in the ribosome during its functioning has been accumulated. The most interesting information on such changes was obtained by cryo-EM of various ribosomal complexes with the ligands and by combination of rRNA site-directed mutagenesis with the analysis of structural changes in ribosome by chemical modification technique (chemical probing). The most studied structural transformations of the ribosome interacting with tRNAs and elongation factors are considered in this review. The structural rearrangements are discussed in the context of interactions between the functional centers of the ribosome. We also describe the system of tertiary contacts between the rRNA helices and proteins which forms the universal structure in the ribosome. We pay attention that by means of such system the allosteric conformational signal can be transmitted between the functional centers. Besides the discussion of different biochemical data in the scope of structural data we also consider the hypothesis that the position of GTPase associated center (GAC) in the ribosome regulates the binding of elongation factors.

Cryoelectron Microscopy↗