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1

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

Capillary electrophoretic analysis of the stability of RNA-peptide complex in human blood plasma

Mucha P., Szyk A., Rekowski P., Barciszewski J.

Chemia Analityczna

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2004

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Vol. 49, No. 6

845-853

EN

Capillary electrophoresis mobility shift assay (CEMSA) was employed to qualitatively study the stability of RNA and RNA-peptide complex in human blood plasma. RNA-protein interactions play an important role in the replication cycle of the human immunodeficiency virus type 1 (HIV-1). Thus, the CEMSA method was adapted to study the interactions between viral trans-activation response element (TAR RNA) and the arginine-rich fragment (49-57) of viral trans-activation protein Tat HIVn1. The stability of the free and peptide- complexed TAR in human blood plasma was investigated using a polyacrylamide (LPA)-coated capillary and a sieving matrix with the separation buffer. Under the applied conditions the studies on micromolar concentrations of TAR could be performed without labelling in less than 30 min. In the presence of Tat peptide, a significant increase of the migration time of TAR from 18.66 min to 20.12 min was observed. The differences between the behaviour of the free and complexed TAR in human blood plasma were apparent during CEMSA analysis. Both species degraded progressively in time. The first products of degradation were detected immediately after spiking the plasma sample with the free TAR. Migration times of the degradation products were longer than for the free TAR. Free TAR was degraded completely after 60 min. Since TAR was unlabelled, the products of its degradation could not be identified. TAR complexed with the Tat-peptide was much more stable in plasma compared to the free TAR. Even after 180 min of incubation a large amount of the complexed TAR still could be detected. This work is the first to present the application of CE to the stability studies of the free and peptide-complexed RNA in human blood plasma.

PL

Przedstawiono metodę elektroforezy kapilarnej do jakościowej oceny stabilności RNA i kompleksu RNA-peptyd w plazmie ludzkiej krwi. Z powodu dużego znaczenia oddziaływań RNA-białko w cyklu replikacyjnym wirusa HIV-1, metodę zaadaptowano do badań oddziaływań slruktury TAR RNA z bogatym w argininę fragmentem (49-57) wirusowego biafka transaktywowanego Tat HIV-1. Oddziaływanie TAR-Tat jest odpowiedzialne za efektywną clongację wirusowego mRNA. Używając pokrytej poliakryloamidem (LPA) kapiiary i buforu zawierającego czynnik przesiewający badano stabilność wolnego i skomplcksowanego z peptydem TAR. Zastosowane warunki pozwoliły na badanie stabilności kompleksu TAR-Tat bez znakowania RNA, w stężeniu mikromolowym w czasie krótszym niż 30 min. W obecności Tat pcptydu zaobserwowano znaczące przesunięcie czasu migracji TAR od 18.66 do 20.12 min. Zachowanie wolnego i skompleksowancgo TAR było łatwo obser-wowalne przy zastosowaniu CE. Pierwsze produkty degradacji zaobserwowano natychmiast po zmieszaniu wolnego TAR z próbką plazmy. Czasy migracji tych produktów były dłuższe niż wolnego TAR. Całkowita degradacja TAR nastąpiła po l godz. TAR skompleksowany z Tat pcptydem był znacznie stabilniejszy w plazmie w porównaniu do wolnego TAR, Czasy migracji produktów degradacji przypominały te obserwowane w wypadku wolnego TAR. Prezentowana procedura opisuje po raz pierwszy zastosowanie CE do badania zachowania wolnego i skompleksowancgo zpeptydem RNA w ludzkiej plazmie krwi.

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.