Gordon P M, Sontheimer E J, Piccirilli J A
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology, The University of Chicago, 5841 South Maryland Avenue, MC1028, Chicago, Illinois 60637, USA.
Biochemistry. 2000 Oct 24;39(42):12939-52. doi: 10.1021/bi001089o.
The ai5gamma group II intron from yeast excises itself from precursor transcripts in the absence of proteins. When a shortened form of the intron containing all but the 3'-terminal six nucleotides is incubated with an exon 1 oligonucleotide and a 3' splice site oligonucleotide, a nucleotidyl transfer reaction occurs that mimics the second step of splicing. As this tripartite reaction provides a means to identify important functional groups in 3' splice site recognition and catalysis, we establish here a minimal kinetic framework and demonstrate that the chemical step is rate-limiting. We use this framework to characterize the metal ion specificity switch observed previously upon sulfur substitution of the 3'-oxygen leaving group and to elucidate by atomic mutagenesis the role of the neighboring 2'-OH in catalysis. The results suggest that both the 3'-oxygen leaving group and the neighboring 2'-OH are important ligands for metal ions in the transition state but not in the ground state and that the 2'-OH may play an additional role in transition state stabilization by donating a hydrogen bond. Metal specificity switch experiments combined with quantitative analysis show that the Mn(2+) that interacts with the leaving group binds to the ribozyme with the same affinity as the metal ion that interacts with the neighboring 2'-OH, raising the possibility that a single metal ion mediates interactions with the 2'- and 3'-oxygen atoms at the 3' splice site.
来自酵母的ai5gamma II型内含子在没有蛋白质的情况下从前体转录本中自我切除。当将包含除3'末端六个核苷酸外所有序列的缩短形式的内含子与外显子1寡核苷酸和3'剪接位点寡核苷酸一起孵育时，会发生一种核苷酸转移反应，该反应模拟剪接的第二步。由于这种三方反应提供了一种识别3'剪接位点识别和催化中重要功能基团的方法，我们在此建立了一个最小动力学框架，并证明化学步骤是限速步骤。我们使用这个框架来表征先前在3'-氧离去基团硫取代时观察到的金属离子特异性转换，并通过原子诱变阐明相邻2'-OH在催化中的作用。结果表明，3'-氧离去基团和相邻的2'-OH在过渡态而非基态时都是金属离子的重要配体，并且2'-OH可能通过提供氢键在过渡态稳定中发挥额外作用。金属特异性转换实验与定量分析相结合表明，与离去基团相互作用的Mn(2+)与核酶结合的亲和力与与相邻2'-OH相互作用的金属离子相同，这增加了单个金属离子介导与3'剪接位点处2'-和3'-氧原子相互作用的可能性。
