Biomedical subjects
Publications and source records attributed to J Barciszewski.
Evidence is presented for a new role for elongation factor EF-Tu. This involves conformational restraint or conformational selection of any aminoacyl-tRNA for channeling it to the ribosomal decoding site.
Recently we have proposed a new three-dimensional model of plant 5S rRNAs structure. To verify this proposal we present here new data on RNase T1 digestion and hydroxyl radical hydrolysis of lupin and wheat germ 5S rRNAs at various buffer and temperature conditions. Interestingly, the guanosine residues 85-87 in the loop D of these RNAs, are resistant to RNase T1 at native but not at denaturating conditions. On the other hand, the reaction of 5S rRNA with the hydroxyl radicals showed different reactivity of many nucleotides in various parts of the molecule and suggest conformational changes, which occur mostly in the loops. The experimental data clearly support involvement of the nucleotides occupying conserved positions in the loops in the tertiary interactions in plant 5S rRNA structure.
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.
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.
RNA phosphodiester bonds can be cleaved by metal ions, of which Pb2+ is one of the most effective. It can cleave both generally and site-specifically, depending on the substrate and the conditions. In addition, metal ions are also known to cleave ester bonds between amino acid and the 3'-end of transfer RNA. Here we report that in aminoacylated transfer RNA, Pb2+ ions cleave internucleotide bonds in the 3'-end of tRNA and also cleaves the bond between tRNA and its amino-acid, attached at the 3'-end via an ester bond to the terminal ribose in aminoacyl tRNA. The two reactions proceed at different rates. The rate of deacylation is significantly faster than the rate of cleavage of phosphodiester bonds, with a pH-optimum of 7. This dual hydrolytic role is not seen for other metal ions examined, namely Zn(II), Cd(II) and Mn(II). The rate of the two kinds of hydrolyses by Pb2+ ions is compared with that of other metal-ions. The mechanism of cleavage is investigated further by modification of the 3'-end of tRNA.
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The primary structure of the 5S rRNA from Arabidopsis thaliana was determined. A comparison of this nucleotide sequence with that of 5S rRNA gene showed two differences. Furthermore, we compared all plant 5S rRNA and 5S rDNA sequences known to date and found that lack of colinearity is widespread among higher plant 5S rRNAs and occurred mainly in the double stranded regions. To explain this, we suggest a mechanism which converts putative products of pseudogene ("cryptogene") to mature 5S rRNA molecule. This kind of editing mechanism functions primarily in stems of 5S rRNA in order, to correct mispairing and thereby restoring the Watson-Crick base pairs. This idea could explain why so many different 5S rRNA genes or gene like sequences and only one 5S rRNA species exist in the plant cell. The editing can serve as a new mechanism of regulation of 5S rRNA synthesis in addition to transcription of 5S rRNA gene.
A series of short oligo-DNA probes (8-10-mers) complementary to various regions of the plant ribosomal 5S ribonucleic acid (5S rRNA) have been synthesized. The results of their hybridization to free 5S rRNA and to ribosomes pointed to the availability of nucleotides in loop "C" for complexation. We found a correlation between hybridization of selected oligonucleotides and their inhibitory effect on enzymatic binding of Phe-tRNA and poly(Phe) synthesis on wheat germ 80S ribosomes. Evidence was obtained for involvement of 5S rRNA in the elongation of polypeptide chain during protein biosynthesis. 5S rRNA seems to play a critical role in protein biosynthesis, probably through causing conformational changes of loop C.
DNA damage due to oxidative free radicals is considered to be a major cause of ageing and age-related diseases including cancer. Of more than 20 modifications formed in DNA by the action of hydroxyl radicals, 8-hydroxy-2'-deoxyguanosine (oh8dG) is potentially highly mutagenic and is known to occur most frequently. Using HPLC combined with electrochemical (HPLC/EC) detection of oh8dG, fivefold higher levels of oh8dG are detected in the DNA of cultured normal human skin fibroblasts as compared with SV40-transformed human fibroblasts MRC-5V2. In comparison, the levels of oh8dG were similar in the growth medium of both types of cells. Applications of this method range from studies on the genomic stability and instability of normal and cancerous cells to the clinical and laboratory testing of toxic substances and drugs.
We studied the interaction of wheat germ 5S rRNA with synthetic polypeptides whose amino acid sequences were similar to that of the second zinc finger of Xenopus laevis transcriptional factor IIIA (TFIIIA). The results clearly show that in addition to weak 5S rRNA binding activity (data not shown), these two 30 amino acid long polypeptides hydrolyse some phosphodiester bonds of wheat germ 5S rRNA. The cleavage pattern of plant 5S rRNA is very specific and the cuts occur only after the pyrimidine residues. The same properties of these peptides were furthermore observed for E. coli tRNA(Phe). We found that the digestion specificity of both the zinc finger peptides is very similar to that of a pancreatic ribonuclease (RNase A).
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Pb2+ ions in sub-millimolar concentrations are known to cleave internucleotide bonds of phenylalanine-specific transfer RNA (tRNAPhe) from Saccharomyces. cerevisiae specifically between nucleotides D17 and G18 in the D-loop, with additional minor cleavages after D16 and G15. This makes lead(II) a sensitive structural probe for correct folding of tRNAPhe. In the present paper we use Pb2+ ions as a functional probe to determine whether this part of tRNA is protected by the Escherichia coli elongation factor EF-Tu in the ternary complex formed between Phe-tRNAPhe and EF-Tu.GTP. Our results show that for tRNA in complex with EF-Tu:GTP, the phosphodiester bond after D17 is cleaved, yet the phosphodiester bonds after D16 and G15 are not. To our knowledge, this is the first time that Pb2+ ions, bound at a specific site in tRNA, have been used both to investigate the correct folding of tRNA in complex, and to footprint a functional complex with components whose individual structures are known.
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The new model for the tertiary structure of ribosomal 5 S rRNA from plants recently proposed by some of us has been already supported by RNase H digestions in the presence of complementary oligodeoxynucleotides. These results are confirmed now by the new biochemical and NMR spectroscopy data. Diethylpyrocarbonate (DEP) and monoperphthalic acid (MPA) are the reagents with the high specificity toward single-stranded adenosine residues. Our experiments clearly show that under native conditions adenosine 100 (A100) of lupin 5 S rRNA is not available for reaction toward these reagents. However under denaturing conditions this residue reacts with DEP and MPA. The detailed analysis of the lupin 5 S rRNA by NMR spectra provide the data on the specific interaction of A100-U53. Thus, we have seen for the first time the NMR signal due to the A100-U53 tertiary base pair, which as we believe, stabilizes interactions between loops B and E.
Ribosomal 5S RNA is present in all eubacterial and eukaryotic ribosomes. Despite a large amount of experimental data on the primary and secondary structures of these types of molecules, details of their tertiary structure and their precise function in protein biosynthesis are still not known. Recently we have proposed a new model for the tertiary structure of plant 5S rRNA. In this study we applied the Fe(II)-mediated cleavage reaction to test the model. The data presented here provide experimental evidence that in the 5S rRNA molecule only a few nucleotides are buried in the tertiary structure. Similar experiments performed with methionine initiator tRNA gave results which imply the difference in its structure when compared with the X-ray structure of yeast tRNAPhe.
We show that kinetin, a non-natural product with strong cytokinin activity, is incorporated into prokaryotic and eukaryotic tRNAs in the exchange reaction catalysed by a putative tRNA-kinetin transglycosylase. We also show that kinetin is specifically incorporated into E. coli tRNA(Tyr) and most probably at position 37. To our knowledge, this is the first report of a nucleic acid base exchange reaction occurring at this position.
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The dynamic structure of initiator and elongator tRNAs was analyzed using a very sensitive reaction with hydroxyl radicals. The main target for this reagent is the ribose moieties not buried (accessible) in the tertiary structure of the RNA molecule. At variable time, temperature and magnesium concentrations, some nucleoside residues of lupin initiator tRNA or yeast tRNA(Phe) become accessible or not depending on the actual tRNA conformation. The nucleotides in the thymidine stem of yeast tRNA(Phe) and amino acid stem in both tRNAs do not change their reactivity and conformation. Also the reactivities of the nucleosides of the anticodons are not changing. Our data clearly suggest that hydroxyl radicals can be very useful for the analysis of the tertiary structure of tRNA.