[Interaction between RNA molecules of a two-componet trans-analog of antigenomic HDV-ribozyme].

Association of RNA molecules forming a two-component B:LS trans-analog of antigenomic HDV ribozyme was studied. From previously synthesized trans-ribozymes the B:LS ribozyme differs by length and sequence of its RNA molecules (33 and 34 bp, respectively), topology of functional parts and the absence of very short reaction product. The ribozyme displays a biphasic kinetics of self-cleavage similar to that of cis-ribozyme. Our original kinetic scheme for the B:LS trans-ribozyme self-cleavage (www.cardio.ru\labgen\RZ_e.html)describes a possible cause of biphasic nature of the reaction curve, namely, variation of the rate-limiting stage in the series of successive conformational transformations which coincide with the ribozyme self-cleavage. Interactions between the molecules involved in the reaction, i.e., "multimerization" of entire ribozyme and its components can be regarded as another cause of the biphasic kinetics. B:LS trans-ribozyme is a convenient model for the investigation of this process, since the binding of LS and B allows the formation of complexes with 1B:2LS or 2B:1LS stoichiometry and complexes with the cleavage products. We examined the factors determining dissociation-association of the ribozyme components using a series of electrophoreses under nondenaturing conditions. The possibility of interaction between cis- and transribozyme components was confirmed experimentally. In the presence of LS excess over B the ribozyme can form multimeres. These findings suggest the involvement of intermolecular interactions in native cis-ribozyme self-cleavage.

Properties of antigenomic hepatitis delta virus ribozyme that consists of three RNA oligomer strands.

A three-strand ribozyme, a derivative of antigenomic hepatitis delta virus (HDV) ribozyme, which consists of subfragments of 16 (L), 17 (S), and 33 nucleotides (B), has been constructed. The ternary B-L-S complex formed by the subfragments in stoichiometric ratio was able to catalyze a self-cleavage reaction. Kinetics of this reaction exhibited biphasic behavior and the same parameters as in the case of natural cis-ribozyme. Study of kinetics of reaction initiated by adding various reaction components and the study of binary complex formation between subfragments B and L, B and S, and also ternary B-L-S complex formation revealed that: 1) in the presence of Mg2+, B and S form a stoichiometric complex, L and S do not form complex at all, while B and L form 2 types of complexes, probably B-L and 2B-L; and addition of S subfragment prevented the formation of the latter complex; 2) the reaction initiated by S subfragment proceeds much slower than that initiated by other components pointing to the possibility that in the absence of S L may form a nonproductive complex with B, which is slowly displaced by S followed by productive ternary complex formation. Dissociation constants for binary B-L, B-S and ternary B-L-S complexes have been estimated.

[A kinetic model of the cleavage of permutational variants of the antigenomic HDV-ribozyme]. / Kineticheskie modeli rasshchepleniia permutatsionnykh variantov antigenomnogo HDV-ribozima.

The kinetic characteristics have been studied for noncircularly permuted variants of the human hepatitis delta virus (HDV) antigenomic ribozyme to find out the cause of the two-phase kinetics of the self-cleavage reaction. Different ways of reaction initiation, suboptimal conditions, and jumpwise changes of reaction conditions have been used, and the temperature dependences have been studied. A correlation has been shown between the apparent kinetic constant of the first reaction phase and the portion of the ribozyme molecules that self-cleaved during the first phase. Partial restoration of the initial reaction characteristics has been shown by the reinitiation of reaction being stopped after completing the first phase. On the basis of all the data obtained, a scheme of the self-cleavage reaction has been proposed including: (i) activation of the ribozyme with energy of 40-50 kcal/mol and a characteristic time of several deciminutes under optimal reaction conditions; (ii) fast and reversible reaction of the phosphodiester bond cleavage; (iii) reaction leading to isomerization of the 3',5'-phosphodiester bond to the 2',5' bond in the self-cleavage site with a characteristic activation time of tens of minutes; and (iv) practically irreversible conformational change leading to fixation of the cleavage by immobilization of the 5'-terminal nucleotide of the product in the center of the formed structure and displacement of the 3'-terminal nucleotide to the periphery. The latter process has a characteristic time of tens of minutes and a low activation energy.

Size distribution of the urokinase mRNA decay intermediates in different tissues and cell lines.

Many genes, particularly those encoding the products participating in the regulation of transcription, replication and tissue remodeling, produce short-lived mRNA. It has been commonly accepted that once mRNA is disintegrated, the degradation process is so rapid that the decay intermediates cannot be detected. In the present study we verified this postulate and focused our attention on the quantification of the decay products of the urokinase-type plasminogen activator (uPA) mRNA that belongs to short-lived mRNAs. Using a previously described modified quantitative RT-PCR method, we have shown that intact uPA mRNA coexists in normal human tissues, Jurkat and 5637 cells with a great abundance of its degradation products. The uPA mRNA decay products were not detected in T24P cells. The content of intact uPA mRNA in normal tissues was as low as 5% of the total amount of its poly(A)(+) fraction. The size distribution of the mRNA decay products suggests that the mRNA is digested by exonucleases or/and non-specific endonuclease with cut sites evenly distributed along the mRNA chain. Different decay degrees were demonstrated for subpopulation of the uPA mRNA molecules with intact 3' and 5' ends.

[Analogs of nucleotides, modified by a sugar residue and pyrimidine base, in a DNA synthesis reaction, catalyzed by Thermus aquaticus DNA polymerase]. / Analogi nukleotidov, modifitsirovannye po sakharnomu ostatku i pyrimidinovomu osnovaniiu, v reaktsii sinteza DNK, kataliziruemogo DNK-polimerazoi Thermus aquaticus.

Substrate properties of dNTP analogues in the DNA synthesis reaction catalyzed by Thermus aquaticus DNA polymerase were studied. It was shown that most of dNTP analogues which were known as terminators of DNA synthesis of E. coli DNA polymerase I were able to terminate DNA synthesis catalyzed by Thermus aquaticus DNA polymerase. An interesting feature of Thermus aquaticus DNA polymerase was the ability to utilize 3'-azido-2',3'-dideoxythymidine triphosphate as terminating substrate. Relative efficiency of tested dNTP analogues incorporation into the DNA growing chain was estimated.

[Substrate properties of C'-methylnucleoside triphosphates in a reaction of RNA synthesis catalyzed by Escherichia coli RNA-polymerase]. / Substratnye svoistva C'-metilnukleozidtrifosfatov v reaktsii sinteza RNK, kataliziruemoi RNK-polimerazoi Escherichia coli.

2 theta-C-methyl substituted and phosphonate analogs of UTP were prepared and together with the synthesized earlier 3'-C-methyl-UTP were investigated in the RNA synthesis reaction catalysed by Escherichia coli RNA-polymerase. Substrate properties of UTP analogs were studied in the presence of all natural triphosphates, in the absence of UTP and under conditions of soil substrate reaction. It was shown that UTP(3'CH3) is incorporated into the RNA chain and terminates further RNA elongation. Another analog UTP (2'CH3) may substitute natural UTP in RNA synthesis, but the effectivity of its incorporation is diminished. Phosphonate analog UTP(5'CH2) is a pseudoterminator of RNA synthesis. The conformational analysis of 2'- and 3'C-methylnucleosides by force-field method of calculation was carried out in order to find energetically forbidden conformations of these analogs due to the collision of bulky methyl group and a heterocyclic base. An attempt was made to fix the conformation of the substrate during its enzymatic transformation.

3'-Fluoro-3'-deoxyribonucleoside 5'-triphosphates: synthesis and use as terminators of RNA biosynthesis.

3'-Fluoro-3'-deoxy-uridine, -cytidine, -adenosine and -guanosine have been synthesized by glycosylation of the corresponding silylated bases with 1-O-acetyl-2,5-di-O-benzoyl-3-fluoro-3-deoxy-D-ribofuranose in the presence of Friedel-Crafts catalysts and were converted to the 5'-triphosphates, NTP(3'-F). It was shown that NTP(3'-F) are terminators of RNA synthesis catalyzed by DNA-dependent RNA polymerase from E. coli and may thus serve as tools for DNA sequencing.

[Synthesis of 2',3',-dideoxy-3'-fluoradenosine and -guanosine, their 5'-triphosphates and a study of 2',3'-dideoxy-3'-fluoronucleoside- 5'-triphosphates as substrates for DNA-polymerases]. / Sintez 2',3'-didezoksi-3'-ftoradenozina i -guanozina, ikh 5'- trifosfatov i izuchenie 2',3'-didezoksi-3'-ftornukleozid-5'-trifosfato v kachestve substratov DNK-polimeraz.

Potential antiviral and antitumour nucleosides, 3'-fluoro-2', 3'-dideoxy-adenosine and -guanosine, have been synthesized by the chemical transglycosylation reaction using 5'-O-acetyl-3'-fluoro-2', 3'-dideoxy-thymidine and -uridine as donors of the carbohydrate fragment and persilylated 6-N-benzoyladenine and 2-N-palmitoylguanine as acceptors, respectively. 5'-Triphosphates of 3'-fluoro-2', 3'-dideoxy-thymidine, -cytidine, -adenosine, and -guanosine (dNTP(3'F] were synthesized and tested as terminators in cell-free system of DNA synthesis catalyzed by RNA-directed DNA polymerase (reverse transcriptase, RT) from the avian myeloblastosis virus (AMV) and E. coli DNA polymerase I (Klenow fragment). A method of estimating relative effectiveness of dNTP(3'F) incorporation into DNA growing chain in comparison with the natural substrates was developed. It is shown that, in case of AMV-RT, dATP(3'F), dCTP(3'F) incorporate 14 times less efficiently than dATP and dCTP respectively, and dTTP(3'F) 3 times less effectively than the corresponding natural substrates, whereas dGTP (3'F) is as efficient as dGTP. With E. coli DNA polymerase I (Klenow fragment) dATP (3'F) and dCTP(3'F) are ca. 100 times less efficient, and dTTP(3'F) and dGTP(3'F) are ca. 50 times less efficient than the respective natural substrates.

[A method of determining the primary structure of oligodeoxyribonucleotides]. / Metod opredeleniia pervichnoi struktury oligodezoksiribonukleotidov.

Sanger method was modified to fulfill the requirements of sequencing of oligodeoxyribonucleotides. E. coli DNA polymerase I Klenow fragment was used for all the reactions. The method consists of three steps made in succession in one tube: 1. Optional hydrolysis of a 5'-labeled oligodeoxyribonucleotide primer in order to get a set of primers of different lengths. 2. Elongation of the produced set of primers in the presence of a template, natural dNTPs and chain terminating dNTP analogs. 3. Hydrolysis of the products of the previous step in order to remove the unterminated molecules. Change of steps in achieved just by varying the reaction conditions without any product purification. The method in insensitive to the presence of admixture of oligonucleotides which is not complementary to the primer or to the template.

[Properties of cloned promoters]. / Izuchenie svoistv klonirovannykh promotorov.

Strong early bacteriophage T7 promoters A2 and A3 and also A2 and lac UV5 promoters with altered segments downstream the initiation of RNA start point were cloned using specially constructed plasmid vectors pBRS188 and PBRS240. The relative signal strengths of these promoters in vivo and in vitro were evaluated and the kinetic parameters of their interaction with RNA polymerase were determined. It has been shown that the nucleotide sequence of the transcribed region plays a significant role in specific promoter-RNA polymerase interaction and that the rate-limiting step of RNA synthesis initiation is different for various promoters.

Stability of cloned promoter-containing fragments.

Strong bacteriophages lambda and T7 promoters for Escherichia coli RNA polymerase were cloned in a multicopy plasmid. To achieve this result, two variants of the promoter-probe vectors were constructed. It was found that (i) modifications of the nucleotide sequence, apart from the commonly accepted promoter region, both upstream and downstream of the RNA initiation point greatly influenced the efficiency of promoters in vivo, (ii) a recombinant DNA composed of one of the promoter-probe plasmids and a tandem of A1, A2, and A3 promoters of T7 bacteriophage DNA induced a reproducible secondary change in plasmid DNA upon cloning. This change was substitution of the part of the recombinant that originated as T7 by a large portion of the host DNA.

[Construction of promoter-probe plasmid vector]. / Konstruirovanie plazmidnogo vektora dlia klonirovaniia promotorov.

A new plasmid vector, designated pBRS188 has been constructed for cloning of promoter-containing DNA fragments. This plasmid is a derivative of the E. coli drug-resistance plasmid pBR322 in which a small region (13 base pairs long) within the Tc promoter is eliminated. As a result of the alteration pBRS188 has lost the ability to confer Tc resistance to the host strain. Cloning of foreign DNA fragments, carrying promoters for E. coli RNA polymerase, into the unique EcoRI site of pBRS188 allows to isolate the recombinant TcR transformants. Our construction required the use of new techniques, involving partial hydrolysis of DNA fragments by E. coli DNA polymerase I in the presence of one deoxyribonucleosidetriphosphate and by nuclease S1. An important feature of this method is the ability to regenerate restriction endonuclease recognition sites at junctions of DNA fragments.

[Influence of ionic strength on RNA-polymerase structure]. / Vliianie ionnoi sily na strukturu RNK-polimerazy.

Chromatography of RNA polymerase holoenzyme preincubated under different ionic strength conditions on the DNA agarose column was studied. Ratio of two peaks identified to be core and holoenzyme was analysed. In the range of 0.15 to 0.05 M KCl the relative content of the holoenzyme peak gradually decreased from 100 to 50%. At the same time a peak of free sigma-subunit appeared as detected by the chromatography on DNA agarose gel A-1.5 m. The dissociation of half of the sigma-subunit amount occured within the enzyme dimer-monomer transition range. The results suggest that the dimerization follows the equation: E sigma + E sigma in equilibrium with E2 sigma. Reconstitution of the RNA polymerase holoenzyme from purified core enzyme and sigma-subunit was also studied by the same method. Reconstitution did not occur at a low ionic strength (0--0.1 M KCl), but takes place at ionic strength of 0.2 M or higher. Possible function of the dimerisation of the enzyme in search of promoter site and regulation of RNA synthesis is discussed.

The study of DNA-RNA-polymerase complexes by kinetic formaldehyde method.

A modification of the kinetic formaldehyde method has been proposed providing a possibility for locally denatured regions (defects) formed in DNA preincubated with RNA polymerase (in the absence of nucleoside triphosphates) to be detected. This modification consists in a previous fixation of DNA-enzyme complex with small concentrations of formaldehyde, which do not induce formation of defects in DNA alone. The method has been calibrated under the conditions favourable to RNA synthesis. Studies of the effect of the fixation conditions on the number of defects in DNA interacting with RNA polymerase have shown that the number of defects is constant with formaldehyde fixation concentration between 0.05% and 0.3-0.5% and with fixation time between 2 min and 100 min. The dependence of the number of defects in DNA on RNA polymerase concentration at low ionic strength (0.05 M KCl) is presented by a curve with a plateau. From the initial linear part of the curve it has been found that the enzyme bound to DNA as a monomer. At the excess of the enzyme the mean number of nucleotide pairs between defects is 400-500. Increase of ionic strength results in decrease of the number of defects in DNA. The number of defects depends on temperature of preincubation of the complex. There were no defects in DNA at temperatures below 20 degrees C. At temperatures above 30 degrees C the number of defects reaches saturation. A sharp transition occurs in the range of temperatures between 20 degrees C and 30 degrees C. Analysis of the experimental and literature data, concerning the interaction of formaldehyde and amino acid methylol derivatives with DNA bases, leads to the conclusion that the mechanism of the formation of defects in helical DNA most likely consists in its unwinding or sharp weakening upon binding of RNA polymerase, prior to addition of formaldehyde.