Bedouelle H, Carter P, Waye M M, Winter G, Lowe D M, Wilkinson A J, Fersht A R
Biochimie. 1985 Jul-Aug;67(7-8):737-43. doi: 10.1016/s0300-9084(85)80161-9.
The gene encoding the enzyme tyrosyl tRNA synthetase from Bacillus stearothermophilus has been systematically altered using synthetic oligonucleotides as mutagens. The construction of mutations has been facilitated by using strains of bacteria defective in mismatch repair and also by utilising a genetic marker in the M13 strain (such as an amber mutation, or an EcoK or EcoB site) which allows selection for the progeny of M13 replication derived from the minus (mutagenized) strand. Several mutations have been constructed in the ATP binding site to elucidate the roles of individual residues in catalysis and substrate binding and it has even been possible to construct mutants which have improved affinity for ATP. Mutations in various surface lysine and arginine residues have allowed us to identify potential contacts with the tRNA, and indicate that a cluster of basic residues close to the C-terminus of the enzyme probably makes important interactions with the tRNA.
利用合成寡核苷酸作为诱变剂，对嗜热脂肪芽孢杆菌中编码酪氨酰tRNA合成酶的基因进行了系统改造。通过使用错配修复缺陷的细菌菌株，以及利用M13菌株中的遗传标记（如琥珀突变、EcoK或EcoB位点）来促进突变的构建，这些标记允许从负链（诱变链）衍生的M13复制后代进行选择。已在ATP结合位点构建了多个突变，以阐明各个残基在催化和底物结合中的作用，甚至有可能构建出对ATP亲和力更高的突变体。对各种表面赖氨酸和精氨酸残基的突变使我们能够确定与tRNA的潜在接触，并表明靠近酶C末端的一簇碱性残基可能与tRNA发生重要相互作用。
