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Self-splicing of the group I intron from Anabaena pre-tRNA: requirement for base-pairing of the exons in the anticodon stem.

Abstract：

: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)

journal_name

Biochemistry

journal_title

Biochemistry

authors

Zaug AJ,McEvoy MM,Cech TR

doi

10.1021/bi00082a016

subject

Has Abstract

pub_date

1993-08-10 00:00:00

pages

7946-53

issue

31

eissn

0006-2960

issn

1520-4995

journal_volume

32

pub_type

杂志文章

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