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Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method
We have used the in vitro selection method to search for catalytically active variants of the antigenomic delta ribozyme with mutations in the regions that constitute the ribozyme active site: L3, J1/4 and J4/2. In the initial combinatorial library 16 nt positions were randomized and the library con...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2006
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1388270/ https://www.ncbi.nlm.nih.gov/pubmed/16513845 http://dx.doi.org/10.1093/nar/gkl018 |
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author | Łęgiewicz, Michał Wichłacz, Agnieszka Brzezicha, Bartosz Ciesiołka, Jerzy |
author_facet | Łęgiewicz, Michał Wichłacz, Agnieszka Brzezicha, Bartosz Ciesiołka, Jerzy |
author_sort | Łęgiewicz, Michał |
collection | PubMed |
description | We have used the in vitro selection method to search for catalytically active variants of the antigenomic delta ribozyme with mutations in the regions that constitute the ribozyme active site: L3, J1/4 and J4/2. In the initial combinatorial library 16 nt positions were randomized and the library contained a full representation of all possible sequences. Following ten cycles of selection-amplification several catalytically active ribozyme variants were identified. It turned out that one-third of the variants contained only single mutation G80U and their activity was similar to that of the wild-type ribozyme. Unexpectedly, in the next one-third of the variants the C76 residue, which was proposed to play a crucial role in the ribozyme cleavage mechanism, was mutated. In these variants, however, a cytosine residue was present in a neighboring position to the polynucleotide chain. It shows that the ribozyme catalytic core possesses substantial ‘structural plasticity’ and the capacity of functional adaptation. Four selected ribozyme variants were subjected to more detailed analysis. It turned out that the variants differed in their relative preferences towards Mg(2+), Ca(2+) and Mn(2+) ions. Thus, the functional properties of the variants were dependent on both the structure of their catalytic sites and divalent metal ions performing catalysis. |
format | Text |
id | pubmed-1388270 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2006 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-13882702006-03-07 Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method Łęgiewicz, Michał Wichłacz, Agnieszka Brzezicha, Bartosz Ciesiołka, Jerzy Nucleic Acids Res Article We have used the in vitro selection method to search for catalytically active variants of the antigenomic delta ribozyme with mutations in the regions that constitute the ribozyme active site: L3, J1/4 and J4/2. In the initial combinatorial library 16 nt positions were randomized and the library contained a full representation of all possible sequences. Following ten cycles of selection-amplification several catalytically active ribozyme variants were identified. It turned out that one-third of the variants contained only single mutation G80U and their activity was similar to that of the wild-type ribozyme. Unexpectedly, in the next one-third of the variants the C76 residue, which was proposed to play a crucial role in the ribozyme cleavage mechanism, was mutated. In these variants, however, a cytosine residue was present in a neighboring position to the polynucleotide chain. It shows that the ribozyme catalytic core possesses substantial ‘structural plasticity’ and the capacity of functional adaptation. Four selected ribozyme variants were subjected to more detailed analysis. It turned out that the variants differed in their relative preferences towards Mg(2+), Ca(2+) and Mn(2+) ions. Thus, the functional properties of the variants were dependent on both the structure of their catalytic sites and divalent metal ions performing catalysis. Oxford University Press 2006 2006-03-02 /pmc/articles/PMC1388270/ /pubmed/16513845 http://dx.doi.org/10.1093/nar/gkl018 Text en © The Author 2006. Published by Oxford University Press. All rights reserved |
spellingShingle | Article Łęgiewicz, Michał Wichłacz, Agnieszka Brzezicha, Bartosz Ciesiołka, Jerzy Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method |
title | Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method |
title_full | Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method |
title_fullStr | Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method |
title_full_unstemmed | Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method |
title_short | Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method |
title_sort | antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1388270/ https://www.ncbi.nlm.nih.gov/pubmed/16513845 http://dx.doi.org/10.1093/nar/gkl018 |
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