tRNA ^His guanylyltransferase (THG1), a unique 3′-5′ nucleotidyl transferase, shares unexpected structural homology with canonical 5′-3′ DNA polymerases

Abstract

All known DNA and RNA polymerases catalyze the formation of phosphodiester bonds in a 5′ to 3′ direction, suggesting this property is a fundamental feature of maintaining and dispersing genetic information. The tRNA His guanylyltransferase (Thg1) is a member of a unique enzyme family whose members catalyze an unprecedented reaction in biology: 3′-5′ addition of nucleotides to nucleic acid substrates. The 2.3-Å crystal structure of human THG1 (hTHG1) reported here shows that, despite the lack of sequence similarity, hTHG1 shares unexpected structural homology with canonical 5′-3′ DNA polymerases and adenylyl/guanylyl cyclases, two enzyme families known to use a two-metal-ion mechanism for catalysis. The ability of the same structural architecture to catalyze both 5′-3′ and 3′-5′ reactions raises important questions concerning selection of the 5′-3′ mechanism during the evolution of nucleotide polymerases. G-1 addition | reverse polymerase | tRNA modificationA ll nucleotide polymerases, including DNA and RNA polymerases, reverse transcriptase, and telomerase, catalyze nucleotide addition in the 5′ to 3′ direction. The reaction involves the nucleophilic attack of a polynucleotide terminal 3′-OH onto the α-phosphate of an incoming nucleotide, followed by release of the pyrophosphate moiety. Although the 5′ to 3′ direction has been adopted by all polymerases and transferases described to date, there is one notable exception: the enzyme tRNA His guanylyltransferase (Thg1). Thg1 catalyzes the highly unusual 3′-5′ addition of a single guanine to the 5′-end of tRNA His (1, 2). This reaction is an obligatory step in the maturation of this tRNA because the extra 5′ base, G −1 , constitutes a primary identity element for the aminoacyl-tRNA synthetase (HisRS) that attaches the amino acid histidine to the 3′-end of the tRNA (3-9). Thg1 is thus essential for maintaining the fidelity of protein synthesis. Consistent with the critical nature of the G −1 residue, THG1 is an essential gene in yeast and RNAi-mediated silencing of the Thg1 homolog in human cells results in severe cell-cycle progression and growth defects (2, 10, 11). Thg1 is widely conserved throughout eukarya, and Thg1 homologs are present in many archaea and bacteria.In eukarya, G −1 addition occurs opposite a universally conserved A 73 and thus is the result of a nontemplated 3′-5′ addition reaction. In addition, yeast Thg1 catalyzes a second reaction in vitro, extending tRNA substrates in the 3′-5′ direction in a template-directed manner driven by Watson-Crick pairing (12). Thg1 enzymes in archaea also catalyze template-dependent 3′-5′ addition, but do not catalyze nontemplated G −1 addition (13), suggesting that the templated 3′-5′ addition reaction likely represents an ancestral activity of the earliest Thg1 family members.The 3′-5′ addition of G −1 to tRNA His occurs via three chemical reactions, all catalyzed by Thg1 (2, 14) (Fig. 1). First, the 5′-monophosphorylated tRNA that results from RNase P cleavage of pre-tRNA His is activated using ATP, creating a 5...

Doing it in reverse: 3′-to-5′ polymerization by the Thg1 superfamily

“…Remarkably, although the sequence of Thg1 shows no obvious similarities to canonical polymerases, the recently solved crystal structure of the human Thg1 (hTHG1) enzyme exhibits striking structural homology with 59-to-39 DNA polymerases, including the placement of residues critical for catalysis (Hyde et al 2010). These findings suggest that 59-to-39 and 39-to-59 polymerization activities may be evolutionarily related.…”

Crystal structure of a reverse polymerase

“…1) (18). A small, helical subdomain located 20-30 Å from the nucleotide binding site also contains highly conserved residues critical to catalysis (3,17), and may be a key determinant of tRNA binding.…”

“…Based on mutational experiments, Hyde et al (3) suggest that the dGTP nucleotide bound to human Thg1 may correspond to the position of ATP used in the initial activation step that produces the adenylylated intermediate. The triphosphate portion of a second bound nucleotide is also observed in the active site (3), where it coordinates a number of basic residues important to catalytic efficiency in the yeast homolog (17).…”

“…This G −1 addition step is a phosphodiester bond formation reaction that operates in a unique 3′-5′ direction, opposite to all conventional DNA and RNA polymerases. In PNAS, Hyde et al (3) unveil crystal structures of a Thg1 enzyme that catalyzes G −1 addition. The structures show that the catalytic domain of Thg1 shares both a common architecture and a two-metal ion-dependent mechanism with canonical 5′-3′ DNA polymerases.…”

“…Moreover, superpositions of Thg1 with DNA polymerase locate three common carboxylate groups in the predicted active site. Using a cocrystal structure of Thg1 with bound dGTP together with metal ion soaking experiments, Hyde et al (3) further show that these catalytic residues bridge to the substrate α-and β-phosphates via two divalent metal ions that are oriented similarly to those bound in substrate complexes of T7 DNA polymerase (16). Thus, both the palm domain fold and classic two-metal ion mechanism for 5′-3′ nucleotide addition by DNA polymerases appear to be conserved in the 3′-5′ phosphodiester bond formation reaction catalyzed by Thg1.…”