Mutational analysis of loops 1 and 5 of the hairpin ribozyme.
Mutational analysis of loops 1 and 5 of the hairpin ribozyme.
复制标题
发夹核酶环 1 和 5 的突变分析。
DOI：
10.1021/bi9721288
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发表时间：
1998
期刊：
影响因子：
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通讯作者：
Hampel,A
中科院分区：
文献类型：
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作者：
Shippy,R;Siwkowski,A;Hampel,A
用五嘧啶环将Acis-核酶拴在底物上，对发夹状核酶−底物复合体环1和环5中所有位置的碱基偏好进行了全面的分析。核酶−底物分子被突变，以包含这些环内八个位置上的三个非天然碱基变异中的每一个。测定了每个突变体的催化活性，并与原始天然序列的活性进行了比较。这是这些环中的所有碱基位置第一次被突变为所有变体并具有运动学特征。发现了各种影响，从不变的碱基位置到对任何碱基变化几乎完全耐受的影响。两个位置导致所有非野生型碱基替换的卵裂率低于准确量化的下限。这些位置是核酶中的G8和底物中的Gs6。当A10被嘧啶取代时，自切割活性下降到检测下限以下，而其余位置显示出不同的碱基偏好。这里报告的1和5环上的信息结合以前2和4环上的突变数据[Siwkowski，A.，Shippy，R.和Hampel，A.(1997)BioChemical 36,3930−3940]完成了对发夹状核酶−底物复合体所有必需环内的每个碱基的全面突变/动力学分析，并为所提出的催化机制的发展奠定了基础。
A comprehensive analysis of base preferences for all positions in loops 1 and 5 of the hairpin ribozyme−substrate complex was carried out using acis-ribozyme tethered to substrate by a pentapyrimidine loop. Ribozyme−substrate molecules were mutated to contain each of the three non-native base variations at each of the eight positions within these loops. Catalytic activity was measured for each mutant and compared to the activity of the original native sequence. This was the first time all base positions in these loops have been mutated to all variants and kinetically characterized. Various effects were found, ranging from invariant base positions to those with nearly complete tolerance of any base change. Two positions resulted in cleavage rates below the lower limit of accurate quantification for all non-wild-type base substitutions. These positions are G8in the ribozyme and Gs6in the substrate. When A10was substituted with a pyrimidine, self-cleavage activity fell below the lower limit of detection while the remaining positions showed varying base preferences. The information reported here on loops 1 and 5 combined with previous mutagenesis data on loops 2 and 4 [Siwkowski, A., Shippy, R., and Hampel, A. (1997)Biochemistry36, 3930−3940] completed a comprehensive mutational/kinetic analysis of every base position located within all the required loops of the hairpin ribozyme−substrate complex and allowed for the development of a mechanism for catalysis which is proposed.