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A spermidine-induced conformational change of long-armed hammerhead ribozymes: ionic requirements for fast cleavage kinetics.
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MedLine Citation:
PMID:  9365249     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The catalytic activity of the trans cleaving hammerhead ribozyme 2as-Rz12, with long antisense flanks of 128 and 278 nt, was tested under a wide range of different reaction conditions for in vitro cleavage of a 422 nt RNA transcript derived from human immunodeficiency virus type 1 (HIV-1). Depending on the reaction conditions, in vitro cleavage rates varied by a factor of approximately 100. Increasing concentrations of magnesium up to 1 M were found to enhance the reaction. Sodium when added simultaneously with magnesium showed an inhibitory effect on the cleavage reaction. Addition of sodium during pre-annealing, however, produced a stimulating effect. It was found that the additional inclusion of spermidine during pre-annealing further increased the reaction rate markedly. In accordance with accelerated cleavage, it was possible to identify a distinct, spermidine-induced conformer of the ribozyme-substrate complex. Under the most favourable conditions cleavage rates of 1/min were obtained, which are in the range of rates obtained for conventional hammerhead ribozymes with short antisense flanks. A comparison of thermodynamic data for short- and long-armed hammerhead ribozymes suggested that the activation entropy became unfavourable when helices I and III formed a long chain ribozyme-substrate complex. We conclude that in the absence of spermidine folding into the active conformation is impaired by increased friction of long helices, resulting in relatively low cleavage rates in vitro.
Authors:
C Hammann; R Hormes; G Sczakiel; M Tabler
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Nucleic acids research     Volume:  25     ISSN:  0305-1048     ISO Abbreviation:  Nucleic Acids Res.     Publication Date:  1997 Dec 
Date Detail:
Created Date:  1998-01-20     Completed Date:  1998-01-20     Revised Date:  2010-09-10    
Medline Journal Info:
Nlm Unique ID:  0411011     Medline TA:  Nucleic Acids Res     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  4715-22     Citation Subset:  IM; X    
Affiliation:
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, PO Box 1527, GR-71110 Heraklion, Crete, Greece.
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MeSH Terms
Descriptor/Qualifier:
Catalysis
Crystallography, X-Ray
Entropy
Humans
Kinetics
Magnesium / metabolism
Models, Chemical
Protein Conformation / drug effects
RNA, Catalytic / chemistry,  drug effects*
Sodium / metabolism
Spermidine / pharmacology*
Chemical
Reg. No./Substance:
0/RNA, Catalytic; 124-20-9/Spermidine; 7439-95-4/Magnesium; 7440-23-5/Sodium
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): Nucleic Acids Res
ISSN: 0305-1048
ISSN: 1362-4962
Article Information
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Print publication date: Day: 1 Month: 12 Year: 1997
Volume: 25 Issue: 23
First Page: 4715 Last Page: 4722
ID: 147112
PubMed Id: 9365249
Publisher Item Identifier: gka752

A spermidine-induced conformational change of long-armed hammerhead ribozymes: ionic requirements for fast cleavage kinetics.
C Hammann
R Hormes
G Sczakiel
M Tabler
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, PO Box 1527, GR-71110 Heraklion, Crete, Greece.


Article Categories:
  • Research Article


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