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Metody otrzymywania nienaturalnych aminoacylo-tRNA

Plitta B., Giel-Pietraszuk M., Barciszewski J.

Wiadomości Chemiczne

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2007

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[Z] 61, 1-2

61-77

EN

Despite an enormous progress in the development of biophysical methods to establish protein structure and function, there is still a lack of precise ways of detection and mapping the specific fragments of molecules, in terms of their structure and properties. This is due to the fact that only a few amino acids show specific properties - like fluorescence of tryptophan - which enable analysis of the interaction and formation mechanism of protein - nucleic acids complexes. These problems one can easily overcome using proteins containing non-natural amino acid. However, all methods used in in vitro protein synthesis require up till now aminoacyl-tRNA as a substrate. Hence, the acylation of tRNA is a key and limiting step of every method. In the present article, we show all known non-enzymatical methods of tRNA acylation. One of them is based on ribozymes obtained by in vitro evolution (SELEX). These ribozymes that transfer amino acid bound to its 5'-end to 3'-end of tRNA can specifically recognize amino acid or tRNA. Other attempts aimed at chemical synthesis of aminoacyl-nucleotides, which were further ligated to appropriately prepared tRNA. As an amino acid donor, the peptidylnucleic acid (PNA) was used as well. An alternative method is an acylation under high hydrostatic pressure which allows to attach any amino acid to tRNA in a one-step procedure. All mentioned methods can be used in protein translation in vitro. For in vivo synthesis of protein containing non-natural amino acid, orthogonal pairs of tRNA-AARS are used. In orthogonal pairs mutated aminoacyl-tRNA synthetase recognizes specific amino acid and acylates suppressor amber tRNA.

2

Interaction of a Transcription Factor IIIA Peptide Containing Two Zinc Fingers with 5S rRNA

Giel-Pietraszuk M., Mucha P., Rekowski P., Kupryszewski G., Barciszewska M. Z.

Polish Journal of Chemistry

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2002

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Vol. 76 / nr 6

815-822

EN

The interaction of lupin ribosomal 5S RNA with a chemically synthesized peptide containing 60 amino acid, derived from Xenopus laevis transcription factor IIIA, is analyzed. The results show that such short fragment retains the ability of binding to 5S rRNA molecule, as shown by electrophoretic gel shift and RNase footprint assay. The peptide protects from hydrolysis with specific nucleases helix II and V of 5S rRNA.

3

The Ribosome and the Spliceosome Are the True Ribozymes

Giel-Pietraszuk M., Barciszewski J.

Polish Journal of Chemistry

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2001

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Vol. 75, nr 12

1807-1816

EN

Recent advances in ribosome crystallography revealed an atomic resolution structure of the peptidyl-transferase active site. Similarly big progress in biochemical studies of spliceosomes provided a good basis to modify our view concerning functions of these particles. In this review the problem if the ribosomes and the spliceosomes are the ribozymes is discussed.