Zaug A J, McEvoy M M, Cech T R
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Biochemistry. 1993 Aug 10;32(31):7946-53. doi: 10.1021/bi00082a016.
In the cyanobacterium Anabaena, the precursor to tRNA(Leu) has a 249-nucleotide group I intron inserted between the wobble and second bases of the anticodon; the intron self-splices during transcription in vitro [Xu, M. Q., Kathe, S. D., Goodrich-Blair, H., Nierzwicki-Bauer, S. A., & Shub, D. A. (1990) Science 250, 1566-1570]. By studying splicing of isolated pre-tRNA, we confirm that splicing occurs by the two-step transesterification mechanism characteristic of group I introns, resulting in excision of the intron and accurate ligation of the 5' and 3' exons. The first step, guanosine-dependent cleavage of the phosphodiester bond at the 5' splice site, occurs with kcat congruent to 14 min-1 and kcat/Km = 5 x 10(4) M-1 min-1 (32 degrees C, 15 mM MgCl2), unexpectedly efficient for a small group I intron. (kcat/Km is comparable to that of the Tetrahymena pre-rRNA intron, and kcat is an order of magnitude higher than any previously reported for a group I intron). The second step, ligation of the exons, is so slow (k = 0.3 min-1) that it is rate-limiting for splicing in vitro except at very low guanosine concentrations. Disruption of the base pairs that make up the anticodon stem of the tRNA dramatically reduces the rate of the first step of splicing, while compensatory mutations that restore base pairing generally restore activity. We suggest that the very short P1 helix of this pre-tRNA, with only three base pairs preceding the 5' splice site, is unstable without the additional base pairs in the anticodon stem.(ABSTRACT TRUNCATED AT 250 WORDS)
在蓝细菌鱼腥藻中，tRNA(Leu)的前体在反密码子的摆动碱基和第二个碱基之间插入了一个249个核苷酸的I类内含子；该内含子在体外转录过程中进行自我剪接[徐，M.Q.，凯瑟，S.D.，古德里奇 - 布莱尔，H.，涅尔兹维基 - 鲍尔，S.A.，& 舒布，D.A.（1990年）《科学》250，1566 - 1570]。通过研究分离的前体tRNA的剪接，我们证实剪接是通过I类内含子特有的两步转酯反应机制发生的，导致内含子的切除以及5'和3'外显子的精确连接。第一步，5'剪接位点处磷酸二酯键的鸟苷依赖性切割，其催化常数约为14分钟^-1，催化常数与米氏常数的比值为5×10^4 M^-1分钟^-1（32℃，15 mM MgCl2），对于一个小的I类内含子来说效率出乎意料地高。（催化常数与米氏常数的比值与嗜热四膜虫前体rRNA内含子相当，催化常数比之前报道的任何I类内含子高一个数量级）。第二步，外显子的连接非常缓慢（k = 0.3分钟^-1），以至于它是体外剪接的限速步骤，除非在非常低的鸟苷浓度下。构成tRNA反密码子茎的碱基对的破坏显著降低了剪接第一步的速率，而恢复碱基配对的补偿性突变通常会恢复活性。我们认为，这种前体tRNA非常短的P1螺旋，在5'剪接位点之前只有三个碱基对，在没有反密码子茎中额外碱基对的情况下是不稳定的。（摘要截短至250字）
