B-Z DNA reversible conformation changes effected by high pressure.

There are numerous data showing that a DNA molecule with alternate pirymidine-purine sequence can adopt a left-handed, double-helical Z-DNA conformation. Such structural changes of DNA occur as a consequence of environmental conditions (e.g. 4 M NaCl) or chemical modification (e.g. methylation or bromination of bases). In this paper, we found for the first time that high pressure (several kilobars) can change the DNA conformation from the B to the Z form. When the pressure is reduced to an atmospheric one, DNA conformation returns back to the B-form. The Z-DNA structure formation was confirmed by circular dichroism (CD) and ultraviolet (UV) measurements. However, we found, that the values of the ratio of absorbance at the wavelengths 295 and 257 nm in the range of 0.3-0.4 is not a fully conclusive proof for the Z-DNA conformation. Although the ratio is typical for Z-DNA form, it is not obvious that the negative band in CD spectrum will be observed. On the other hand, methylated DNA does not undergo B----Z DNA transitions at the high pressure. These conformational changes of DNA molecules could be interpreted as the effect a of different hydration of various DNA forms.

High pressure effects on conformation of homo- and heteroduplexes of nucleic acids.

Four different chemically synthesized single stranded complementary oligonucleotides: DNA I, d(GCGCGCATATAT); RNA I, r(AUAUAUGCGCGC): RNA II, r(GGCCGGUUAAUU); and RNA III, r(AAUUAACCGGCC) were studied in order that the effects of high pressure on heteroduplex and homoduplex structures could be understood. The oligonucleotides were subjected to a high pressure at low and/or high salt buffer and analyzed by circular dichroism spectroscopy. In these conditions, both DNA-RNA and RNA-RNA duplexes with different purine-pyrimidine sequences change their conformation. The heteroduplex DNA I-RNA I with the complementary alternating purine-pyrimidine sequence, does not change its conformation of A type at high salt alone or at high salt and high pressure applied together. The homoduplex RNA II-RNA III with purine-purine-pyrimidine-pyrimidine sequence does not change strongly its. A-RNA conformation either. However, a structure of the homoduplex is affected by high pressure alone or with high salt as concluded from shifting the maximum of the CD spectrum to around 265 nm and inducing higher Cotton effect. These observations clearly suggest some conformational changes of the homoduplex. A single stranded oligonucleotide (RNA I) and oligodeoxynucleotide (DNA I) alone showed up a different conformation. The CD spectrum of RNA I is similar to that of A-RNA structure, out that of DNA I shows a very small Cotton effect and has not an ordered structure.

The non-enzymatic specific amino-acylation of transfer RNA at high pressure.

This paper shows that the phenylalanine-specific tRNA of Escherichia coli as well as the yellow lupin methionine initiator tRNAMet can be charged specifically with phenylalanine and methionine, respectively, in the absence of specific aminoacyl-tRNA synthetases, under high pressure of a maximum of 6 kbar (1 bar = 10(5) Pa; 1 atm = 1.01 x 10(5) Pa). The esterification reaction takes places at the 3' end of the tRNA molecules. The yield of Phe-tRNAPhe or Met-tRNAMet at high pressure is approximately 10 times lower than that of the enzymatic aminoacylation reaction. This reaction seems to be specific, and mis-aminoacylation of tRNAPhe and tRNAMet with serine is negligible. It is well known that tRNA undergoes conformational changes during interaction with an aminoacyl-tRNA synthetase. Similarly, on the basis of circular dichroism spectra, we showed that the conformation of tRNA at high pressure differs slightly from its original A-RNA form. Therefore, it can be speculated that the chargeable conformation of tRNA induced by the aminoacyl-tRNA synthetase during enzymatic aminoacylation and the one created at high pressure are similar and are most probably formed by a dehydration mechanism. We think that the 'unique' tertiary structure of tRNA existing under high pressure creates an active centre which might itself catalyse ester bond formation. Therefore, the structure of the amino acid stem of tRNA may determine (code) the charging of the particular amino acid to specific tRNA. This code is clearly distinct from the rules of the classical genetic code.

A-Z-RNA conformational changes effected by high pressure.

This paper reports evidence obtained by circular dichroism (CD) spectroscopy measurements indicating that two oligoribonucleotide duplexes with the alternating purine-pyrimidine sequences r(GC)6 or r(AU)6 change their A-RNA conformation under high pressure. Under the high-pressure conditions at which B-Z-DNA transition easily occurs, RNA acquires a conformation which only differs slightly from that of A-RNA. However, exposure of r(GC)6 or r(AU)6 to high pressure (6 kbar) in the presence of 5 M NaCl causes a conformation change of both oligoribonucleotide duplexes from their A- to their Z-RNA form. The departure of RNA or DNA duplexes from their original conformations under high pressure depends on the water structure itself and involves displacing an active (structural) water molecule outside the nucleic acid molecules. Experiments carried out until now in many laboratories have shown that B-Z or A-Z transitions of DNA or RNA, respectively, do not depend on the conditions applied, but the common mechanism for these processes seems to be dehydration. This same effect can be observed either at high salt concentrations or in the presence of an alcohol or at high pressure. Our results also support the view that the higher stability of RNA compared with DNA duplexes is due to the strong interaction of the 2'-hydroxyl groups of RNA with water molecules.

tRNA aminoacylated at high pressure is a correct substrate for protein biosynthesis.

tRNA can be aminoacylated specifically with amino acids at high pressure of 6 kbar (1 bar = 1.013 atm = 0.1 MPa = 10(5) Pa) in the absence of the specific aminoacyl-tRNA synthetase and ATP. In this paper we present new evidence obtained by HPLC chromatography and TLC analysis that the esterification reaction under pressure really takes place at the 3' end of the tRNA molecule. If so, tRNA to be aminoacylated undergoes conformational changes similar to those induced with aminoacyl-tRNA synthetase. The most important finding is that aminoacyl-tRNA obtained at high pressure binds to ribosomes and participates in the synthesis of polyphenylalanine in vitro. This is the best proof of proper charging of tRNA at high pressure.