کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
8340219 1541220 2016 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
In vitro evolution of coenzyme-independent variants from the glmS ribozyme structural scaffold
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
In vitro evolution of coenzyme-independent variants from the glmS ribozyme structural scaffold
چکیده انگلیسی
Uniquely among known natural ribozymes that cleave RNA sequence-specifically, the glmS ribozyme-riboswitch employs a small molecule, glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. In vitro selection was employed to search for coenzyme-independent variants of this ribozyme. In addition to shedding light on the catalytic mechanism of the ribozyme, such variants could resemble the evolutionary ancestors of the modern, GlcN6P-regulated ribozyme-riboswitch. A mutant pool was constructed such that the secondary structure elements, which define the triply-pseudoknotted global fold of the ribozyme, was preserved. A stringent selection scheme that relies on thiol-mercury affinity chromatography for separating active and inactive sequences ultimately yielded a triple mutant with a cleavage rate exceeding 3 min−1 that only requires divalent cations for activity. Mutational analysis demonstrated that a point reversion of the variant toward the wild-type sequence was sufficient to partially restore GlcN6P-dependence, suggesting that coenzyme dependence can be readily be acquired by RNAs that adopt the glmS ribozyme fold. The methods employed to perform this selection experiment are described in detail in this review.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Methods - Volume 106, 15 August 2016, Pages 76-81
نویسندگان
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