Preparation of cyclic 2',3'-monophosphates of oligoadenylates (A2'p)nA > p and A3'p(A2'p)n-1A > p.

The action of the guanylyl-preferring RNase from Bacillus intermedius (binase) on a mixture of oligoadenylates with randomly distributed 2'-5' and 3'-5' internucleotide bonds [(A2'/3'p)n] under conditions sufficient for complete hydrolysis of poly(A) results in a mixture containing a single circular oligoadenylate and two series of linear oligoadenylates ending in cyclic 2',3'-phosphate. Individual compounds formed upon digestion of (A2'/3'p)n with binase have been isolated. Their structure was determined on the basis of their chemical and enzymatic conversions and confirmed by 1H-, 13C- and 31P-NMR spectra. According to these data, the circular triadenylate contains one 2'-5' and two 3'-5' internucleotide bonds, linear oligoadenylates of one series contain exclusively 2'-5' internucleotide bonds [(A2'p)nA > p], while each compound of the other series contains a single 3'-5' internucleotide bond connecting the 5'-ultimate nucleotide residue with the penultimate one [A3'p(A2'p)n-1A > p]. The incubation of compounds of the former series A3' p(A2'p)n > p at pH 1.0 and the subsequent action of phosphatase results in successive formation of compounds of two other new series: A3'p(A2'p)nA2'(3')p and A3'p(A2'p)nA.

Laser photofootprinting of d(C).d(G).d(G) intramolecular triplex.

Supercoiling-induced structural transition of the d(C24GC21).d(G21CG24) sequence in plasmid DNA in the presence of Mg2+ at neutral pH results in alterations of efficiencies of not only single-quantum (pyrimidine[6-4]pyrimidone adducts) but also two-quantum (alkali-sensitive lesions of dG residues) photomodifications of nucleoside residues within this sequence. The generation of both types of photoreactions was achieved by the application of high-intensity laser UV radiation (intensity approximately 10(11) W/m2, pulse duration approximately 10(-8) s, lambda = 266 nm) for irradiation of a plasmid DNA. The modification extent sufficient for analysis of photoreaction efficiency distributions along both strands of the insert (photofootprinting) was obtained by the action of a single nanosecond pulse of laser UV radiation. The pattern of a laser photofootprinting is consistent with the d(C).d(G).d(G) triplex formation in the presence of Mg2+ within the insert and shows some details of this triplex structure.

Principles of selective inactivation of viral genome. VIII. The influence of beta-propiolactone on immunogenic and protective activities of influenza virus.

The influence of beta-propiolactone action on the immunogenic and protective activity of the influenza virus A/WSN/33 (H1N1) has been studied. The production of antibodies against virion surface antigens in mice immunized intramuscularly by the modified virus was enhanced with the increase of inoculating dose from 6 x 10(7) to 1.5 x 10(8) viral particles per animal. The immunizing dose of 6 x 10(7) produced complete protection of immunized animals against a lethal challenge of A/WSN/33 virus. The inhibition of virus reproduction in animal lungs was increased with the increase of the virus immunizing dose up to 6 x 10(8). At a constant dose the inhibition of virus reproduction decreases with the increase of the virus modification extent.

Principles of selective inactivation of viral genome. VII. Some peculiarities in determination of viral suspension infectivity during inactivation by chemical agents.

When the infectivity of the influenza virus is determined by means of titration on chicken embryos, calculating the infection titre according to Reed and Muench, the course of inactivation with beta-propiolactone shows an anomaly - the fraction of infected embryos in a batch initially increases and then decreases as the degree of dilution of the virus-containing sample is increased. This anomaly occurs because a slight dilution lowers the agent concentration insufficiently so that inactivation goes on after the dilution of the sample before and/or after the inoculation of the solution into the embryo. The anomaly can be avoided either by neutralizing or removing the agent prior to titration or by starting titration from high dilutions.

Principles of selective inactivation of viral genome. VI. Inactivation of the infectivity of the influenza virus by the action of beta-propiolactone.

The kinetics of inactivation of the infectivity of the influenza virus by beta-propiolactone have been studied. Rate constants have been determined for inactivation of the A/Leningrad/385 (H3N2) and B/Leningrad/489/80 influenza virus under the action of beta-propiolactone on a virus-containing allantoic fluid and on a purified viral suspension. The data obtained allow calculation of the time required for inactivation of the influenza virus infectivity to a given extent in virus-containing solutions at any initial concentration of beta-propiolactone.

Principles of selective inactivation of viral genome. V. Rational selection of conditions for inactivation of the viral suspension infectivity to a given extent by the action of beta-propiolactone.

The influence of the initial concentration of beta-propiolactone, the composition of the solution, temperature, and pH on the bacteriophage MS2 infectivity inactivation kinetics has been studied. Rate constants have been determined for the infectivity inactivation and for the change in the concentration (the consumption) of the reactant under inactivation conditions. These constants have been shown to permit a sufficiently precise description of the phage MS2 survival curves under the action of beta-propiolactone. These data have been used to put forward a kinetic approach for the rational determination of conditions for inactivation of the viral infectivity to a required extent with agents whose concentration decreases during inactivation as a result of hydrolysis and reactions involving the medium components.

A transition from a twice-folded structure to a hairpin in oligo(dG) in the presence of magnesium ions.

A long (dG)n stretch can fold twice forming an intramolecular tetrahelix stabilized by guanine tetrads and stacking interactions between them (G-structure). In this paper, we show that magnesium ions induce a transition of the (dG)n stretch from the G-structure to the hairpin stabilized by Hoogsteen-like G.G base pairs (G-hairpin). This transition between the G-structure and the G-hairpin is detected by chemical probing. The characteristic time of the transition for a (dG)28 at 4 degrees C exceeds five hours, which allowed us to separate these two forms by electrophoresis at low temperature. We believe that the slowness of the transition is due to the fact that half of the deoxyguanosines in the insert must change their conformation from syn to anti or vice versa.

Direct tRNA-protein interactions in ribosomal complexes.

Nucleotide residues in E. coli tRNA(Phe) interacting directly with proteins in pre- and posttranslocated ribosomal complexes have been identified by UV-induced cross-linking. In the tRNA(Phe) molecule located in the Ab-site (pretranslocated complex) residues A9, G18, A26 and U59 are cross-linked with proteins S10, L27, S7 and L2, respectively. In tRNA(Phe) located in the Pt-site (posttranslocated complex) residues C17, G44, C56 and U60 are cross-linked with proteins L2, L5, L27 and S9, respectively. The same cross-links (except for G44-L5) have been found for tRNA in the Pb-site of the pretranslocated ribosomal complex. None of the tRNA(Phe) residues cross-linked with proteins in the complexes examined by us are involved in the stabilization of the secondary structure, but residues A9, G18, A26, G44 and C56 participate in stabilization of tRNA tertiary structure. Since translocation of tRNA(Phe) from Ab- to P-site is accompanied by changes of tRNA contacts with proteins L2 and L27, we postulate that this translocation is coupled with tRNA turn around the axis joining the anticodon loop with the CCA-end of the molecule. This is in agreement with the idea about the presence of a kink in mRNA between codons located in the ribosomal A- and P-sites. In all E. coli tRNAs with known primary structure positions 18 and 56, interacting with L27 protein, when tRNA is located either in A- or P-site, are invariant, whereas positions 17 and 60, interacting with proteins only when tRNA is in the P-site, are strongly conserved. In positions 9, 26 and 59 purines are the preferred residues. In most E. coli tRNAs deviations from the consensus in these three positions is strongly correlated.

Determination of tRNA nucleotide residues directly interacting with proteins in the post- and pretranslocated ribosomal complexes.

Nucleotide residues of E. coli tRNA interacting directly with proteins in pre- and posttranslocated ribosomal complexes have been identified by analysis of photo-induced tRNA-protein cross-links. A9, G18, A26 and U59 residues of NAcPhePhe-tRNA, located in the Ab-site (pretranslocated complex) have been cross-linked with proteins S10, L27, S7 and L2 respectively. In deacylated tRNA, located in the Pb-site, residues C17, G44, C56 and U60 have been cross-linked with proteins L2, L5, L27 and S9 respectively. The G44-L5 cross-link disappeared after translocation (NAc-PhePhe-tRNA located in the Pt-site).

Sequence-specificity of the alkali-sensitive lesions induced in DNA by high-intensity ultraviolet laser radiation.

The action of high-intensity (10(9)-10(12) W/m2) UV (266 nm) laser radiation pulses (duration ca 10 ns or ca 40 ps) on liquid aqueous solutions of DNA is known to cause not only single- but also two-quantum modification of nucleic bases. The action of hot piperidine on the laser-irradiated DNA results in non-random splitting of polynucleotide chain. Hence, at least some of the modified nucleoside residues are alkali-sensitive lesions (ASLs). The distribution of ASLs along the DNA chain shows that the position of these lesions corresponds with pyrimidines in the PyPy sequences (similar to those formed via single-quantum conversions) as well as with deoxyguanosine residues. The last ASLs result from two-quantum reactions and occur much more efficiently than the direct photo-induced cleavage of the internucleotide (phosphodiester) bond. It has been shown with fragments of plasmids pUC18, pUC19 and pBR322 (total length over 600 base pairs) that the relative efficiency of ASLs at deoxyguanosine sites depends on the primary structure context and can differ by an order of magnitude. The highest efficiency of modification is observed when a purine is 3' neighbour to the 2'-deoxyguanosine, i.e. at 5'-GPu-3' sites. However, considerable variations in the modification efficiency were also found in these sequences.

Laser (two-quantum) photolysis of polynucleotides and nucleoproteins: quantitative processing of results.

The paper offers and substantiates a technique for quantitative processing of the photochemical reactions in polynucleotides, nucleoproteins and their components caused by the action of powerful ultraviolet laser pulse radiation.

G-DNA: a twice-folded DNA structure adopted by single-stranded oligo(dG) and its implications for telomeres.

Our dimethyl sulfate modification experiments suggest that (dG)n stretches within single-stranded DNA fragments, which represent the simplest model for telomeric sequences, adopt a complex intrastrand structure other than a simple hairpin. We present a molecular model for the DNA structure that conforms to dimethyl sulfate methylation data. The principal element of this G-DNA structure is a quadruple helix formed by pairwise antiparallel segments of the twice-folded (dG)n stretch. This quadruple core has two wide and two narrow grooves connected by three loop-shaped segments. The strong stacking interactions of the neighboring guanine tetrads and the large number of hydrogen bonds formed can be the primary reasons that such structures are favored over a common hairpin for long (dG)n stretches. Such compact structures may be formed from (dG)n stretches of telomeric sequences.

Irradiation of the template with high-intensity (pulse-laser) ultraviolet light results in DNA-polymerase termination events at deoxyguanosine residues.

During primer elongation by Escherichia coli DNA-polymerase I large fragments on the template were irradiated with UV laser pulses at an intensity greater than or equal to 10(10) W/m2. In addition to the termination events at photoproducts typical of low-intensity UV irradiation, termination is observed before deoxyguanosine residues. The effect of the UV light intensity on the ratio of termination efficiencies before dPy and dG suggests that the termination of polymerization before deoxyguanosine residues results from the formation of photoproducts yielded by two-quantum reactions. The results obtained herein, together with data published previously, imply that photomodification of dG residues is the major two-quantum reaction under the action of high-intensity UV radiation on DNA.

Magnesium-dependent supercoiling-induced transition in (dG)n.(dC)n stretches and formation of a new G-structure by (dG)n strand.

Plasmids containing (dG)27.(dC)27 inserts (pPG27), (dG)37.(dC)37 inserts (pPG37), and (dG)24C(dG)21.(dC)24G(dC)21 inserts (pPG46C) were constructed for the study of structural transitions within (dG)n.(dC)n stretches. Two-dimensional gel electrophoresis has shown that a Mg2+-dependent supercoiling-induced structural transition takes place at pH 8 in plasmid pPG46C. The transition occurs at -0=0.06 and involves a supercoiling release corresponding to 5 superhelical turns. After denaturation of the restriction fragments containing (dG)n.(dC)n inserts, the strands do not renature completely and (dG)n-containing strand migrates in PAGE much faster than the (dC)n-containing one. Chemical modification experiments with the (dG)n-strand have revealed the periodic nature of the protection of guanines against dimethyl sulfate methylation. The (dG)n strand in the presence of Mg2+ forms complexes with the complementary (dC)n strand, which differ from the native duplex in mobility. We believe these effects to be due to the formation of an intrastrand structure within the (dG)n strand stabilized by G.G interactions (we called it G-structure), which in the presence of Mg2+ forms an interstrand complex. with the (dC)n strand.

Nucleotide residues of tRNA, directly interacting with proteins within the complex of the 30 S subunit of E. coli ribosome with poly(U) and NAcPhe-tRNA(Phe).

With the aid of photoinduced tRNA-protein cross-linking, nucleotide residues A21, U45 and U60 were shown to interact directly with proteins S5, S7 and S9 respectively, in the complex of the 30 S subunit of E. coli ribosome with poly(U) and NAcPhe-tRNA(Phe). These nucleotide residues are located in the central part of the L-form in the tertiary structure of RNA.

Structural characteristics and classification of some tRNA-binding sites of elongating Escherichia coli ribosome.

Ultraviolet(254 nm)-irradiation-induced cross-linkages in ribosomal complexes allowed identification of proteins in contact with tRNA at different elongation steps. Both the set and the ratio of cross-linked proteins, i.e. the structural characteristics of the tRNA-binding sites of the ribosome, were shown to depend strongly not only on the position of the mRNA codon with which tRNA interacts as a component of a ribosomal complex, but also on its functional state, i.e. on the elongation step. A new classification of tRNA-binding sites of ribosome is suggested.

Induction of polynucleotide-protein cross-linkages by ultraviolet irradiation. Peculiarities of the high-intensity laser pulse irradiation.

The efficiency and specificity of RNA-protein cross-linking in the 30S subunit of Escherichia coli ribosomes, induced by low-intensity (10(15) photons cm-2 s-1, 254 nm) and high-intensity [(1.6-6.8) X 10(24) photons cm-2 s-1, 266 nm, pulse duration 10(-8) s] ultraviolet radiation, are studied. Under the former conditions proteins S4, S7 and S9/S11, and under the latter conditions these proteins together with S3, S18 and S20, are cross-linked to 16S RNA. Biphotonic processes operate in the latter case. In the presence of 2-mercaptoethanol cross-linking occurs either directly, via a higher excited state or via activated intermediates with life-times less than 25 ns. Cross-links thus formed are specific, i.e. they are formed between regions of macromolecules which are in contact in the native (non-disturbed) complex prior to excitation. The efficiency of cross-linking (per photon absorbed) is 20-100 times higher upon two-step excitation as compared with single-step excitation and an analysable number of cross-links are produced in a single pulse. Only base U-1239 of 16S RNA is cross-linked to protein S7 by low-intensity radiation, whereas the adjacent base, G-1240 is also involved in laser-induced cross-linking. A transition from the former to the latter conditions allows one to reduce the duration of irradiation from several minutes to several nanoseconds.

Footprinting of DNA secondary structure by high-intensity (laser) ultraviolet irradiation.

The action of high-intensity ultraviolet pulse laser radiation on a 161 bp fragment of pBR 322 DNA (EcoRI-MspI fragment) was studied. At doses up to 5 X 10(18) photons/cm2 the N-glycosidic bond splitting is negligible. The action of hot piperidine on irradiated DNA leads to chain splitting at the residues, modified via biphotonic processes. The modification and, hence, splitting efficiencies depend on the type of base (G greater than T greater than A greater than C) and on its position in the sequence. Preferentially modified bases in the opposite strands of double-stranded DNA belong, mainly, to the same or adjacent base pairs. Residues in the Pribnow box are modified considerably less, than in the sequences, immediately upstream and downstream. This approach seems to be useful in footprinting of DNA secondary structure peculiarities and alterations, conjugated with the functional role and state of the respective fragment.