Группа регуляции биосинтеза белка


Заведующий – Л.П. Овчинников
The protein composition of mammalian mRNPs (informosomes) has been determined (Fig. 1).

It was shown that free non-translated mRNPs contain one 50 kDa major protein (p50), while polysomal mRNPs have two major proteins, p50 and 70 kDa poly(A)-binding protein (PABP). The amount of p50 in polysomal mRNPs is twice as low as in free mRNPs. Thus,
i) p50 appears the universal major mRNP protein, and
ii) mRNA transition from its non-translated to translated state is accompanied by attachment of PABP and dissociation of half of p50.
Fig. 2. The amino acid sequence of the universal major mRNP protein p50 has been determined.

It appeared that
i) p50 is identical to Y-box binding transcription factor YB-1 as to its amino acid sequence;
ii) p50/YB-1 consists of three domains. Its central domain is extremely evolutionarily conserved and widely distributed from bacteria to human beings. At present, this domain is termed the cold chock domain (CSD).
iii) both CSD and the C-terminal domain display affinity for nucleic acids. Besides, in the absence of mRNA, the C-domain is capable of multimerization.
iv) YB-1 is a member of the large cold-shock domain protein family (Fig. 3).

It was proposed and experimentally supported that when interacting with mRNA, YB-1 alone, without other mRNP proteins, gives RNA-protein complexes displaying all unique physico-chemical properties of natural mRNPs. Thus, YB-1-mRNA complexes may serve as the simplest model for studies of the structural organization of natural mRNPs. It was shown that depending on the YB-1/mRNA ratio, YB-1 changes its quaternary structure, mode of its interaction with mRNA, and the overall mRNP packaging (Fig.4).

At comparatively low YB-1/mRNA ratios typical of polysomal translated mRNPs, YB-1 binds to mRNA as a monomer through its both RNA-binding domains (CSD and C-domain) and form an unfolded translationally active mRNP structure. Upon mRNA saturation with YB-1, which is characteristic of free non-translated mRNPs, YB-1 interacts with mRNA predominantly through the cold shock domain. The displaced C-domains undergo multimerization. Most probably, mRNA is located on the surface of these YB-1 multimers, since it is extremely sensitive to endoribonucleases. One YB-1 multimer (~600 kDa) packs an mRNA segment of 600-700 nucleotide residues. More extended mRNA molecules and YB-1 form polyparticles. In such complexes, mRNA is translationally inactive and stabilized both in lysates and in vivo, probably due to inaccessibility of mRNA termini for exoribonucleases that usually trigger mRNA degradation.


Группа молекулярной генетики


Заведующий – Н.И. Матвиенко
1. In eightieths the genes of 16s RNA, g-factor, and several genes of proteins of small ribosomal subunit of Thermus thermophilus HB8 had been cloned and sequenced.
2. In ninetieths more than 30 site-specific endonucleases and DNA-methylases had been isolated and characterized from natural bacterial strains. 4 of them have not prototypes.
3. Site-specific DNA-nickase had been found in 2000. With this enzyme high sensitive method of hybridization analysis of DNA have been developed.


Группа генной инженерии


Заведующий – В.Н. Ксёнзенко
1. Complete genomes of two relative bacteriophages, T5 and BF23 were sequenced, and their annotation was carried out. T5-like bacteriophages have a number of unique features. That is why they represent an attractive model in molecular biological researches. Complete genomes of bacteriophage T5 (121916 bp) and its close relative, bacteriophage BF23 (118822 bp), were sequenced. Computer analysis of the phage T5 nucleotide sequence revealed 162 open reading frames (ORFs), and for 54 of their gene products functional role can be assigned. Besides, the genes for 25 tRNAs, and for 9 RNAs with unknown functions were found in T5 genome (slide 1).

In general, genomes of T5 and BF23 bacteriophages are very similar (slide 2). 143 out of 161 BF23 ORFs have their homologs in T5 genome. In the most cases, ORFs which are different in T5 BF23 genomes encode proteins with unassigned functions. An exception concerns the genes encoding so called HNH homing endonucleases (hegs, marked with red in slide 2). Thus, only one out of nine phage T5 hegs is present in BF23 genome. At the same time, additional gene for HNH endonuclease was found in BF23. Substantial differences were observes between tRNA gene regions of both phages as well as between genes encoding some virion proteins. Probably, identification of two ORFs encoding putative NAD+-dependent DNA ligase was the most surprising finding in course of analysis of bacteriophages T5 and BF23 genomes. We have cloned these ORFs and demonstrated that they really encode two-subunit DNA ligase. In the subsequent experiments we have characterized enzymatic and DNA binding properties of this ligase. It should be noted that NAD+-dependent DNA ligase of T5-like bacteriophages represents the first example of two-subunit DNA ligases.

2. Complex study of site-specific endonucleases encoded by T5-like bacteriophages. Ten nicking and one DNA double strand cleaving endonucleases encoded by three T5-like bacteriophages were identified and their cleavage points in the phage genomes were mapped. Two of them, F-TflI and F-TflII, were expressed in Escherichia coli cells and purified nearly to homogeneity. Activity of both enzymes is stimulated by a wide range of divalent metal ions, and reaches optimal levels at extreme conditions (pH 10.0; 50oC – 55oC). F-TflI, F-TflII as well as homologous to them F-TflIV was shown to be able initiate homing. Recognition properties of F-TflII endonuclease were studied in detail. DNA footprinting, modification-interference assays and mutational analysis allowed us to determine sequence requirements for substrate recognition by this endonuclease. Modeling of F-TflII/DNA complex also permitted us to predict amino acid residues which participate in complex formation (slide 3).


Группа физики нуклеопротеидов


Заведующий – И.Н. Сердюк
1969-1985 гг. Сформулированы и экспериментально обоснованы принципы структурной организации рибосом.

1985-1986 гг. Получены кристаллы 70S рибосом Thermus thermophilus, пригодные для рентгено-структурных исследований.

1987-1995 гг. Предложен теоретически и обоснован экспериментально новый метод вариации контраста в рассеянии нейтронов - метод тройного изотопического замещения.

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