Ruffner D E, Stormo G D, Uhlenbeck O C
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
Biochemistry. 1990 Nov 27;29(47):10695-702. doi: 10.1021/bi00499a018.
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 an 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.
一个先前已充分表征的锤头状催化RNA，由一个24个核苷酸的底物和一个19个核苷酸的核酶组成，用于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速率。自我切割仅需要三个系统发育保守碱基对中的一个，但需要中央核心中所有九个保守单链残基。在大多数情况下，突变不会改变锤头状结构组装成双分子复合物的能力。在少数突变型锤头状结构无法组装的情况下，似乎是突变稳定了替代底物或核酶二级结构的结果。突变底物和突变核酶的所有组合都比相应的单突变活性低，这表明锤头状结构中几乎没有（如果有的话）像tRNA中那样可替换的三级相互作用。这项工作产生的优化共有锤头状结构用于识别多种大肠杆菌基因序列中存在的潜在锤头状结构。
