Inhibition of the group I ribozyme splicing by NADP+.

The coenzyme NADP+ (nicotinamide adenine dinucleotide phosphate) and its analogs were tested for inhibition of the self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td). Of all compounds examined, the 3'-NADP was the most potent inhibitor and the order of inhibitory efficiency for compounds tested was as follows: 3'-NADP+ > NADP+ > NADP(+)-dialdehyde > NADPH > 1,N6-etheno-NADP+. Increasing guanosine concentration up to 40 microM overcame the suppression of self-splicing by NADP+ up to 76% of the level of normal splicing but didn't recover the full splicing activity. Similarly, Mg2+ also served to restore the splicing activity by about 90% at 25 mM concentration above which the splicing started to decline. The kinetic analysis showed that NADP+ acts as a mixed type non-competitive inhibitor for the td intron RNA with a Ki of 4.1 mM. The specificity of the splicing inhibition by NADP+ is predominantly due to increases in Km and decreases in kcat values. The results indicate that the inhibition by NADP+ was guanosine and Mg2+ dependent.