Kinetic and thermodynamic characterization of the reaction pathway of box H/ACA RNA-guided pseudouridine formation

Abstract

The box H/ACA RNA-guided pseudouridine synthase is a complicated ribonucleoprotein enzyme that recruits substrate via both the guide RNA and the catalytic subunit Cbf5. Structural studies have revealed multiple conformations of the enzyme, but a quantitative description of the reaction pathway is still lacking. Using fluorescence correlation spectroscopy, we here measured the equilibrium dissociation constants and kinetic association and dissociation rates of substrate and product complexes mimicking various reaction intermediate states. These data support a sequential model for substrate loading and product release regulated by the thumb loop of Cbf5. The uridine substrate is first bound primarily through interaction with the guide RNA and then loaded into the active site while progressively interacted with the thumb. After modification, the subtle chemical structure change from uridine to pseudouridine at the target site triggers the release of the thumb, resulting in an intermediate complex with the product bound mainly by the guide RNA. By dissecting the role of Gar1 in individual steps of substrate turnover, we show that Gar1 plays a major role in catalysis and also accelerates product release about 2-fold. Our biophysical results integrate with previous structural knowledge into a coherent reaction pathway of H/ACA RNA-guided pseudouridylation.

Pseudouridine: Still mysterious, but never a fake (uridine)!

“…55 The snoRNA (called sRNA in Archaea) acts as guide for the protein components with Nop10 and the C synthase NAP57 (higher eukaryotes) or Cbf5 (yeast, Archaea) as minimal requirements. 56,57 Non-essential components Gar1 and L7Ae (or Nhp2 in Eukarya) are involved in catalysis and product release 58 or in substrate binding by interaction with Nop10 [59][60][61] , respectively. Cbf5 is also capable of guide RNA free catalysis, the activity of which is increased by Gar1 and Nop10.…”

RNA size is a critical factor for U-containing substrate selectivity and permanent pseudouridylated product release during the RNA:Ψ-synthase reaction catalyzed by box H/ACA sRNP enzyme at high temperature

“…Gar1 was found to make additional interactions with the Cbf5 b7b10 loop, inducing the enzyme to adopt an open state which facilitates substrate loading and product release [27,33]. Formation of the substrate-bound complex changes the positioning of the thumb as it contacts the substrate RNA, and thus this conformation represents the closed state of the enzyme [27,33].…”

“…Gar1 was found to make additional interactions with the Cbf5 b7b10 loop, inducing the enzyme to adopt an open state which facilitates substrate loading and product release [27,33]. Formation of the substrate-bound complex changes the positioning of the thumb as it contacts the substrate RNA, and thus this conformation represents the closed state of the enzyme [27,33]. Biochemical studies based on the in vitro reconstitution of catalytically active particles showed that Gar1 is not required for complete modification of an excess of substrate RNAs in singleturnover reactions [17], but its presence influences substrate placement [34] and enhances the rate of J formation [17,35].…”

“…It is proposed to influence the open-to-closed switch of the Cbf5 b7b10 thumb and facilitates product release, but additionally to stimulate catalysis [27,33].…”

“…Functional studies on H/ACA RNP enzyme were frequently conducted with short substrates, whose length was restricted to the interaction sequence with the pseudouridylation pocket, and were based on reactions carried out in vitro at low temperatures, which are thus not representative of conditions in vivo within the host hyperthermophilic organisms [27,33]. Here, we compared at high temperature the association of one H/ACA RNP enzyme from the hyperthermophilic archaeon P. abyssi with two substrate RNAs of variable length, and studied their turnover.…”