Andrzej Krzyżaniak

Studies on the Synthesis of O-Ribosyl-Adenosine – a new Minor Nucleoside Of tRNA

Abstract A synthesis of new minor nucleoside of yeast tRNA, O-β-D-ribofuranosyl- (l′–>2′)-adenosine ([A∗], 1) and a dinucleosidemonophosphate [A∗]pG (10) is presented.

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 = 105 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.

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.

Structure and Function of Nucleic Acids Under High Pressure

Abstract We have studied effects of high pressure on structure and function of synthetic and natural DNA and RNA. An analysis of circular dichroism (CD) spectra identified that at pressure of 6kbar, poly d(CG) changes its B conformation to Z-DNA form. Synthetic oligoribonucleotides (CG) 6 and (AU) 6 change their conformation from A to Z-RNA at 6 kbar and in the presence of high salt (5M NaCl) concentration, but not at high pressure only. Heterodimer of DNA-RNA responds to high pressure slightly changing its original A-like conformation. We suggested that high pressure effects the nucleic acids structure through their dehydration, of which the first step is a change of water structure itself. Detailed inspection of the CD spectra of tRNA before and after pressure treatment suggests some changes in its conformation. What is more interesting, high pressure catalyses charging specific amino acid to its cognate tRNA. Such obtained aminoacylated tRNA molecule, is fully active active in ribosomal poly(U) directed polyphenylalanine synthesis.