An embarrassment of riches: the enzymology of RNA modification

Abstract

The maturation of transfer RNA (tRNA) involves extensive chemical modification of the constituent nucleosides and results in the introduction of significant chemical diversity to tRNA. Many of the pathways to these modified nucleosides are characterized by chemically complex transformations, some of which are unprecedented in other areas of biology. To illustrate the scope of the field, recent progress in understanding the enzymology leading to the formation of two distinct classes of modified nucleosides, the thiouridines and queuosine, a 7-deazaguanosine, is reviewed. In particular, recent data validating the involvement of several proposed intermediates in the formation of thiouridines are discussed, including two key enzyme intermediates and the activated tRNA intermediate. The discovery and mechanistic characterization of a new enzyme activity in the queuosine pathway is discussed.

Figure 1

Examples of modified nucleosides found in tRNA. a) Simple modifications that involve relatively small structural changes to the parent nucleoside. b) Complex or hypermodifications that involve significant structural changes to the parent nucleoside.

Figure 2

Proposed mechanism for the formation of thiouridine. The mechanism is explicit for E. coli ThiI catalyzed formation of s⁴U, but the overall chemistry is relevant for MnmA catalyzed formation of s²U. Two potential routes to sulfur insertion have been proposed and are compatible with all the experimental data. In a direct sulfur transfer the nucleophilic, terminal sulfur of the enzyme persulfide (shown on Cys⁴⁵⁶) attacks the activated adenylated uridine (red path) to form a covalent, disulfide linked protein-tRNA adduct. Subsequent nucleophilic attack of a second active-site Cys residue (Cys³⁴⁴) on the Cys-sulfur of the disulfide completes sulfur transfer to the tRNA. In a free bisulfide mechanism to sulfur transfer (blue path) the second active-site Cys residue attacks the enzyme persulfide at the internal sulfur, releasing free bisulfide, which subsequently attacks the activated adenylated uridine. In both routes the breakdown of the tetrahedral intermediates releases AMP and leaves the enzyme in the oxidized disulfide form. Reduction of the disulfide prepares the enzyme for another round of trans-persulfidation from IscS. Although persulfide formation is shown prior to tRNA and ATP binding, formation of the adenylated uridine has been shown to occur with MnmA without sulfur transfer from IscS.

Figure 3

a) The reaction catalyzed by the QueF enzyme and it’s role in the biosynthesis of queuosine. b) The proposed mechanism for reduction of the nitrile group of preQ₀ to the aminomethyl group of preQ₁. After binding of preQ₀ the active-site Cys residue (Cys⁵⁵ in Bacilis subtilis QueF) attacks the nitrile group to form a thioimide (III). Binding of the first equivalent of NADPH and hydride transfer gives the thiohemiaminal IV followed by the dissociation of NADP⁺. Binding of the second equivalent of NADPH is followed by breakdown of IV to give the imine V. The second hydride is then transferred to generate preQ₁, followed by dissociation of NADP⁺ and preQ₁ from the enzyme. By keeping the intermediate thiohemiaminal form until after the binding of the second NADPH hydrolysis of the intermediate imine is avoided.

Figure 4

a) The MiaB catalyzed thiomethylation of i⁶A to give ms²i⁶A. b) The reaction catalyzed by the Tyw1 enzyme in the wyosine pathway. The co-substrate(s) in the Tyw1 reaction has not yet been identified.