SEQUENCE REQUIREMENTS OF THE HAMMERHEAD RNA SELF-CLEAVAGE REACTION
SEQUENCE REQUIREMENTS OF THE HAMMERHEAD RNA SELF-CLEAVAGE REACTION
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DOI：
10.1021/bi00499a018
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发表时间：
1990-11-27
期刊：
影响因子：
2.9
通讯作者：
UHLENBECK, OC
中科院分区：
文献类型：
--
作者：
RUFFNER, DE;STORMO, GD;UHLENBECK, OC
一个以前的特点锤头状催化RNA组成的24个核苷酸的底物和19个核苷酸的核酶被用来进行广泛的诱变研究。测定了21种不同底物突变和24种不同核酶突变的切割率。只有一个的三个同源保守的碱基对，但在中央核心的所有九个保守的单链残基是需要自我切割。在大多数情况下，这些突变并没有改变锤头鲨组装成双分子复合物的能力。在少数情况下，突变锤头没有组装，这似乎是在替代底物或核酶二级结构的突变稳定的结果。突变底物和突变核酶的所有组合的活性低于相应的单一突变，这表明锤头包含很少的，如果有的话，可替换的三级相互作用中发现的tRNA。从这项工作中产生的改进的共识锤头被用来确定潜在的锤头存在于各种大肠杆菌基因序列。
A previously well-characterized hammerhead catalytic RNA consisting of a 24-nucleotide substrate and a 19-nucleotide ribozyme was used to perform an extensive mutagenesis study. The cleavage rates of 21 different substrate mutations and 24 different ribozyme mutations were determined. Only one of the three phylogenetically conserved base pairs but all nine of the conserved single-stranded residues in the central core are needed for self cleavage. In most cases the mutations did not alter the ability of the hammerhead to assemble into a bimolecular complex. In the few cases where mutant hammerheads did not assemble, it appeared to be the result of the mutation stabilizing in alternate substrate or ribozyme secondary structure. All combinations of mutant substrate and mutant ribozyme were less active than the corresponding single mutations, suggesting that the hammerhead contains few, if any, replaceable tertiary interactions as are found in tRNA. The refined consensus hammerhead resulting from this work was used to identify potential hammerheads present in a variety of Escherichia coli gene sequences.