[Dimerization of short RNA fragments from avian leukosis virus as revealed with 2-aminopurine fluorescence].

The dimerization of genomic retroviral RNA is well studied for several groups of viruses, the dimerization of human immunodeficiency (HIV) RNA being investigated in more detail. Regions of dimerization apparently involve the short sequences RNA which are directly responsible for the formation of two type dimers: kissing loop-loop (KD) and linear (LD). The 5'-end sequences from RNA avian viruses, where the dimers are basically formed, considerably differ from those of HIV. However, as it was described earlier, the mechanism of dimerization of RNA from human immunodeficiency and from avian leukosis viruses are identical. The fluorescence of adenine analogue 2-aminopurine (2-AP) incorporated into loop sequence of short fragments RNA ALV was used for analysis of dimers formation. Using the temperature dependence of fluorescence intensity 2-AP we have determined RNA melting temperature under various conditions for KD RNA ALV formed by two strands. Effects of magnesium and aminoglycoside antibiotic paromomycin on stabilization of kissing loop-loop dimer RNA have been studied. Under the experimental conditions KD RNA ALV was found to have the stability at the magnesium concentration higher than 1 mM and at paromomycin concentration higher than 2.5 mkM.

[Characteristic of the two-step RNA dimerization in avian sarcoma and leukosis viruses].

Dimerization of two copies of genomic RNA is a necessary step of retroviral replication. In the case of human immunodeficiency virus type 1 (HIV-1) the process is explored in many details. It is proved that conserved stem-loop structure is an essential element in RNA dimerization. Similar model of two-step dimerization mechanism can be considered for avian sarcoma and leukosis virus group (ASLV) in spite of the absence of homology between dimer initiation site (DIS) of ASLV and that of HIV-1. In this paper, short RNA fragments of two viruses: avian sarcoma virus CT-10 and avian leukosis virus HPRS-103 have been chosen in order to investigate the structural requirements of dimerization process and compare them to that of HIV-1. The rate of spontaneous transition from loose to tight dimer was studied as a function of stem length and temperature. Although both types of dimers were observed for both avian retroviruses chosen, fragments of CT-10 requires much higher RNA concentration to form loose dimer. In spite of identical sequence of the loops (5'-A-CUGCAG-3') avian sarcoma virus CT-10 RNA fragments dimerization was greatly impaired. The differences can be explained by deletion of adenine 271 in avian sarcoma virus CT-10 in the stem and by resulting shortening of the self-complementary loop.

[The role of structural elements in dimerization of RNA fragments in avian retroviruses]. / Rol' strukturnykh élementov v dimerizatsii fragmentov RNK retrovirusov ptits.

The slipped loop structure, earlier identified as an unusual DNA structure, was found to be a possible element of the RNA folding. In order to experimentally test this suggestion, model oligoribonucleotides capable of forming the SLS were synthesized. Treatment of the oligoribonucleotides with nuclease S1 and RNases specific for single- and double-stranded RNA demonstrated the steric possibility of SLS formation. To determine the possible functional role of SLS-RNA, various naturally occurring RNAs were screened in silico. Among the most interesting findings were dimerization initiation sites of avian retroviral genomic RNAs. Analysis of RNA from 31 viruses showed that formation of the intermolecular SLS during RNA dimerization is theoretically possible, competing with the formation of an alternative hairpin structure. Identification of the secondary structure of selected RNA dimers employing nuclease digestion techniques as well as covariance analysis of the retroviral RNA dimerization initiation site sequences were used to show that the alternative conformation (loop-loop interaction of two hairpins) is the most preferred. Alternative structures and conformational transitions in RNA dimerization mechanisms in avian retroviruses are discussed.

[Why termination tRNAs have not been found? Because they are hidden in the large ribosomal RNA]. / Pochemu ne naideny terminatornye tRNK? Potomy chto oni spriatany v bol'shoi ribosomnoi RNK.

It is well known that protein synthesis in ribosomes on mRNA requires two kinds of tRNAs: initiation and elongation. The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes). The latter participates in the synthesis proper, recognizing the sense codons. The synthesis is assisted by special proteins: initiation, elongation, and termination factors. The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon. No termination tRNA capable of recognizing stop codons by its anticodon is known. The termination factors are thought to do this. We discovered in the large ribosomal RNA two regions that, like tRNAs, contain the anticodon hairpin, but with triplets complementary to stop codons. By analogy, we called them termination tRNAs (Ter-tRNA1 and Ter-tRNA2), though they transport no amino acids, and suggested them to directly recognize stop codons. The termination factors only condition such a recognition, making it specific and reliable (of course, they fulfill the hydrolysis of the ester bond between the polypeptide and tRNA). A strong argument in favor of our hypothesis came from vertebrate mitochondria. They acquired two new stop codons, AGA and AGG (in the standard code, they are two out of six arginine codons). We revealed that the corresponding anticodons appear in Ter-tRNA1.

[The Z-form of DNA. Nonmonotonous change in stability with increase in ionic strength]. / Z-forma DNK. Nemonotonnoe izmenenie stabil'nosti s rostom ionnoi sily.

To specify the stability area of the Z-DNA conformation as a function of ambient conditions (ionic strength, temperature, water activity) a 3-D phase diagram for the (B,Z) equilibrium as exemplified by poly(dG-5ethyldC) has been constructed in experiment. Its main peculiarity proves to be the nonmonotonic change in stability of the Z form with ionic strength, with a minimum at 0.1 M NaCl, i.e. the increase or decrease in salt concentration from the above value leads to stabilization of the Z form relatively to the B form. The shape of the phase diagram section (temperature, ionic strength) is indicative of the existence of two B-type conformations, equilibrium between them being unaffected by ionic strength though being sensitive to temperature. The B1 form is stable at t < 30 degrees C while the B2 one--at t > 30 degrees C. Using the general 3-D phase diagram together with a polyelectrolyte theory of the B-Z transition, thermodynamic parameters were calculated for the B1-Z, B2-Z, and B1-B2 conformational shifts as well as the B/Z junction energy. The totality of the obtained results with poly(dG-5ethyldC) and those earlier obtained by us with poly(dG-5methyldC) is in full qualitative and quantitative agreement with the polyelectrolyte model, and at the same time it makes unlikely the explanation suggested by others for the low-ionic Z to B transition as the one totally stipulated by a metal ion contamination.

[SLS--a new type of polynucleotide chain folding]. / SLS--novyi tip ukladki polinukleotidnoi tsepi.

Short tandem repeats (5-8 base pairs) are not uncommon in the prokaryotic and eukaryotic DNA. Regions with such sequence motifs, when under superhelical stress, manifest unusual sensitivity to single-strand specific nuclease. To explain this, it has been suggested that one DNA thread should be shifted relatively to another, so that they could form two single-stranded loops protruding from the opposite chains and separated on the DNA helix by the length of a direct repeat. The structure was proposed to play a role in the regulation of transcription, organization of chromatin and in the recombination. Such type of folding could have been extra-stabilized by base pairing between the loops. This attractive possibility of the interloop minihelix formation requires a delicate stereochemical analysis and direct experimental support. Formation of the interloop minihelix in the Slipped Loop Structure (SLS) was tested by a chemical modification method at one nucleotide level resolution. The results show that bases located within the proposed interloop helix are well protected from the probes used. This fact encourages us to publish a 3-D model for the SLS-form DNA (and RNA). The SLS is characterized by a remarkable symmetry having three mutually perpendicular dyad axes. Scanning the bank of nucleotide sequences has revealed more than 500 sites, the transcripts of which are capable of folding into the SLS form, which allow us to regard the SLS form as a novel universal structural form. Remarkably, the abundance of SLS in intrones three times exceeds that of the coding sequences. This may reflect a functional role (or roles) of the SLS conformation.

[Study of cooperative transitions in DNA using phase diagrams]. / Izuchenie kooperativnykh perekhodov v DNK pri pomoshchi fazovykh diagramm.

The study of the triple equilibria (A, B, Coil) and (A, B, Z) makes it possible to obtain interconnection between thermodynamic parameters of different co-operative transitions in DNA. Melting of DNA of poly[d(A--T)] was performed in a broad interval of trifluoroethanol (TFE) concentrations including those of the B to A transition range. A branch, which characterizes the helix-coil equilibrium, has a universal shape for different DNAs and the polynucleotide: melting temperature is minimum near 30% TFE and increases sharply within the region of the A form stability (greater than 65% TFE). The B-A equilibrium depends on temperature only slightly. The slope angles of the branches in the vicinity of the triple point (A, B, Coil) allow to estimate the co-operativity length of the B-A transition: it proves to be of the order of 20 base pairs for DNA and the polynucleotides. Due to the finite transition widths a peculiar situation, not studied up to now, is realized in our cases, at which three different conformations may be present in one polymeric molecule. This results in some new effects, predicted by a theory, such as stabilization by the B/A junctions of the helical state and the increase in width of the melting curves within the B-A transition interval. It thus appears that the phase diagram method may become a very informative approach to the studies of DNA metamorphosis in solution.