Substrate analogs that trap the 2'-phospho-ADP-ribosylated RNA intermediate of the Tpt1 (tRNA 2'-phosphotransferase) reaction pathway.

Autor: Dantuluri S; Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA., Abdullahu L; Department of Chemistry, McGill University, Montreal, Quebec H3A0B8, Canada., Munir A; Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA., Katolik A; Department of Chemistry, McGill University, Montreal, Quebec H3A0B8, Canada., Damha MJ; Department of Chemistry, McGill University, Montreal, Quebec H3A0B8, Canada., Shuman S; Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
Jazyk: angličtina
Zdroj: RNA (New York, N.Y.) [RNA] 2020 Apr; Vol. 26 (4), pp. 373-381. Date of Electronic Publication: 2020 Jan 13.
DOI: 10.1261/rna.074377.119
Abstrakt: The enzyme Tpt1 removes an internal RNA 2'-PO 4 via a two-step reaction in which: (i) the 2'-PO 4 attacks NAD + to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Because step 2 is much faster than step 1, the ADP-ribosylated RNA intermediate is virtually undetectable under normal circumstances. Here, by testing chemically modified nucleic acid substrates for activity with bacterial Tpt1 enzymes, we find that replacement of the ribose-2'-PO 4 nucleotide with arabinose-2'-PO 4 selectively slows step 2 of the reaction pathway and results in the transient accumulation of high levels of the reaction intermediate. We report that replacing the NMN ribose of NAD + with 2'-fluoroarabinose (thereby eliminating the ribose O2″ nucleophile) results in durable trapping of RNA-2'-phospho-(ADP-fluoroarabinose) as a "dead-end" product of step 1. Tpt1 enzymes from diverse taxa differ in their capacity to use ara-2″F-NAD + as a substrate.
(© 2020 Dantuluri et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
Databáze: MEDLINE
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