New methods to investigate ATP requirement for pre-mRNA splicing: inhibition by hexokinase/glucose or an ATP-binding site blocker.
Addition of yeast hexokinase and glucose at various time points of the pre-mRNA splicing reaction rapidly depleted ATP and inhibited further progress of the reaction, indicating that ATP is required for both the first and second steps of splicing. ATP analogues, p-fluorosulfonylbenzoyl-5'-adenosine (FSBA) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), which can modify amino acids at the ATP-binding site of a protein, inactivated the splicing activity of the nuclear extract. While the inactivation by the former was irreversible, the splicing activity was complemented by a Micrococcal nuclease-treated extract. This ATP analogue (FSBA) may be a useful tool for identification of ATP-dependent splicing factors.
Tatei
K
K
Department of Biology, Faculty of Science, Kyushu University, Fukuoka.
Kimura
K
K
Ohshima
Y
Y
D000255:Adenosine Triphosphate / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006593:Hexokinase / Q000494:pharmacology; D008722:Methods; D058914:Purinergic Antagonists; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis
D058914:Purinergic Antagonists; D012333:RNA, Messenger; D000255:Adenosine Triphosphate; D006593:Hexokinase; D005947:Glucose