التفاصيل البيبلوغرافية
| العنوان: |
Identification of Separate Structural Features That Affect Rate and Cation Concentration Dependence of Self-Cleavage by the Neurospora VS Ribozyme. |
| المؤلفون: |
Poon, Alan H. L.1, Olive, Joan E.1, McLaren, Meredith1, Collins, Richard A.1 rick.collins@utoronto.ca |
| المصدر: |
Biochemistry. 11/7/2006, Vol. 45 Issue 44, p13394-13400. 7p. 1 Diagram, 6 Graphs. |
| مصطلحات موضوعية: |
*CATIONS, *NEUROSPORA, *CATALYTIC RNA, *RNA, *RADIOGENETICS |
| الشركة/الكيان: |
RIBOZYME Pharmaceuticals Inc. 794531392 RZYM |
| مستخلص: |
The cleavage site of the Neurospora VS ribozyme is located in an internal loop in a hairpin called stem-loop I. Stem-loop I undergoes a cation-dependent structural change to adopt a conformation, termed shifted, that is required for activity. Using site-directed mutagenesis and kinetic analyses, we show here that the insertion of a single-stranded linker between stem-loop I and the rest of the ribozyme increases the observed self-cleavage rate constant by 2 orders of magnitude without affecting the Mg2+ requirement of the reaction. A distinct set of mutations that favors the formation of the shifted conformation of stem-loop I decreases the Mg2+ requirement by an order of magnitude with little or no effect on the observed cleavage rate under standard reaction conditions. Similar trends were seen in reactions that contained Li+ instead of Mg2+. Mutants with lower ionic requirements also exhibited increased thermostability, providing evidence that the shifted conformation of stem-loop I favors the formation of the active conformation of the RNA. In natural, multimeric VS RNA, where a given ribozyme core is flanked by one copy of stem-loop I immediately upstream and another copy 0.7 kb downstream, cleavage at the downstream site is strongly preferred, providing evidence that separation of stem-loop I from the ribozyme core reflects the naturally evolved organization of the RNA. [ABSTRACT FROM AUTHOR] |
| قاعدة البيانات: |
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