The sequential 2',3'-cyclic phosphodiesterase and 3'-phosphate/5'-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent

Abstract

The RNA ligase RtcB splices broken RNAs with 5′-OH and either 2′,3′-cyclic phosphate or 3′-phosphate ends. The 3′-phosphate ligase activity requires GTP and entails the formation of covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates. There are currently two models for how RtcB executes the strand sealing step. Scheme 1 holds that the RNA 5′-OH end attacks the 3′-phosphorus of the N(3′)pp(5′)G end to form a 3′,5′-phosphodiester and release GMP. Scheme 2 posits that the N(3′)pp(5′)G end is converted to a 2′,3′-cyclic phosphodiester, which is then attacked directly by the 5′-OH RNA end to form a 3′,5′-phosphodiester. Here we show that the sealing of a 2′,3′-cyclic phosphate end by RtcB requires GTP, is contingent on formation of the RtcB–GMP adduct, and involves a kinetically valid RNA(3′)pp(5′)G intermediate. Moreover, we find that RtcB catalyzes the hydrolysis of a 2′,3′-cyclic phosphate to a 3′-phosphate at a rate that is at least as fast as the rate of ligation. These results weigh in favor of scheme 1. The cyclic phosphodiesterase activity of RtcB depends on GTP and the formation of the RtcB–GMP adduct, signifying that RtcB guanylylation precedes the cyclic phosphodiesterase and 3′-phosphate ligase steps of the RNA splicing pathway.