Archaeal Elp3 catalyzes tRNA wobble uridine modification at C5 via a radical mechanism - PubMed (original) (raw)

Archaeal Elp3 catalyzes tRNA wobble uridine modification at C5 via a radical mechanism

Kiruthika Selvadurai et al. Nat Chem Biol. 2014 Oct.

Abstract

Approximately 25% of cytoplasmic tRNAs in eukaryotic organisms have the wobble uridine (U34) modified at C5 through a process that, according to genetic studies, is carried out by the eukaryotic Elongator complex. Here we show that a single archaeal protein, the homolog of the third subunit of the eukaryotic Elongator complex (Elp3), is able to catalyze the same reaction. The mechanism of action by Elp3 described here represents unprecedented chemistry performed on acetyl-CoA.

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Figures

Figure 1

Figure 1. In vitro reconstitution of _Min_Elp3 activity

(a) Schematic view of the domain structure of _Min_Elp3, similar to the structure for human Elp3 shown in Supplementary Figure 1a. The CxxxxCxxC motif that coordinates a [4Fe-4S] cluster is highlighted. (b) Denaturing PAGE analysis of tRNA products from the Elp3-catalyzed reactions. SM, size markers. (c) RP-HPLC chromatograms of the digests of tRNA products from the reactions without (top) and with (bottom) Elp3. The Elp3-produced product cm5U is indicated by an arrow. (d) ESI-LC/MS analyses of cm5U from the Elp3-catalyzed reaction (left) and the synthetic compound (right).

Figure 2

Figure 2. Chromatographic and spectrometric analyses of the consumption of cofactors and the production of 5′-dA

(a) RP-HPLC chromatograms of the reactions differing in components, starting with the one containing only the cofactors (SAM and acetyl-CoA) in the reaction buffer (panel 1). Missing components were added one at a time (panels 2 and 3) until all the components required for the reaction were included (panel 4). (b) ESI-LC/MS analyses of the resulting 5′-dA from the reactions in the presence of the deuterated tRNA (top) and the deuterated acetyl-CoA (bottom).

Figure 3

Figure 3. Proposed mechanism of the Elp3-catalyzed reaction

B−, unidentified general base.

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