| Group II introns self-splice via a two-step mechanism: cleavage at the 5′ splice site via branch formation or hydrolysis 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 the first. Using a bi-partite assay that bypasses the first step, free 5 ′ exon and either linear-3′ exon or lariat-3 ′ exon were used to analyze the kinetics of exon ligation. We then created a novel tri-partite assay separating the 3′ splice site from the bulk of the intron, creating a nick in domain six. In this system, a truncated linear intron (nucleotides 1–881) mediates exon ligation between two 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 six nor the identity of nucleotides flanking the 3′ splice site are critical for accurate 3′ splice site choice. The multiple turnover kcat (0.14 min−1) is slower than the single turnover kobs (0.6–0.7 min−1), consistent with rate-limiting product release under steady state conditions. Decreased single turnover rates at lower pHs were more consistent with loss of catalytic activity than with rate-limiting chemistry. Binding of the 3′ substrate (Km = 2.6 μM) was improved by changing a long-range A:U base pair involving the last intronic nucleotide (the γ-γ ′ interaction) to G:C (Km (3′substrate) = 1 μM). Preliminary analysis of an assay for spliced exon reopening (the reverse of exon ligation) was also begun.; To study the tertiary structure of ai5γ, a site-specific radionucleotide probe technique was also developed. A single phosphorous-32 atom was placed at the 5′ terminus of the intron, the RNA was incubated with 5′ exon under splicing conditions, then frozen to allow for radionuclide decay and radiolysis damage accumulation. Reverse transcription was used to identify affected intronic regions. |
