Tri-partite assay for studying exon ligation by the ai5 gamma group II intron

Group II introns self-splice via a two-step mechanism: cleavage at the 5' s plice site followed by exon ligation at the 3' splice site. The second step has been difficult to study in vitro because it is generally faster than t he first. Herein we describe development and partial kinetic characterizati on of a ge novel assay for studying the second step in isolation. In this s ystem, a truncated linear intron (nucleotides 1-881) mediates exon ligation between two oligonucleotide substrates: a 19 nt 5' exon and a 3' substrate consisting of the last 6 nucleotides of the intron plus a 6 nucleotide 3' exon. We found that neither the exact structure of domain 6 nor the identit y of nucleotides flanking the 3' splice site is critical for accurate 3' sp lice site choice by the ai5 gamma group II intron. The multiple turnover k( cat) (0.14 min(-1)) is slower than the single turnover k(obs) (0.6-0.7 min( -1)), consistent with rate-limiting product release under steady-state cond itions. Decreased single turnover rates at lower pHs were more consistent w ith loss of catalytic activity than with rate-limiting chemistry. Binding o f the 3' substrate (K-m = 2.6 mu M) could be improved by changing a long-ra nge A:U base pair involving the last intronic nucleotide (the gamma-gamma' interaction) to G:C (K-m(3' substrate)= 1 mu M).