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

Abstract

The tRNA(His) guanylyltransferase (Thg1) family of enzymes comprises members from all three domains of life (Eucarya, Bacteria, Archaea). Although the initial activity associated with Thg1 enzymes was a single 3'-to-5' nucleotide addition reaction that specifies tRNA(His) identity in eukaryotes, the discovery of a generalized base pair-dependent 3'-to-5' polymerase reaction greatly expanded the scope of Thg1 family-catalyzed reactions to include tRNA repair and editing activities in bacteria, archaea, and organelles. While the identification of the 3'-to-5' polymerase activity associated with Thg1 enzymes is relatively recent, the roots of this discovery and its likely physiological relevance were described ≈ 30 yr ago. Here we review recent advances toward understanding diverse Thg1 family enzyme functions and mechanisms. We also discuss possible evolutionary origins of Thg1 family-catalyzed 3'-to-5' addition activities and their implications for the currently observed phylogenetic distribution of Thg1-related enzymes in biology.

FIGURE 1.

Chemically equivalent mechanisms of 5′-to-3′ and 3′-to-5′ nucleic acid synthesis. (A) 5′-to-3′ pathway of nucleotide addition catalyzed by canonical DNA/RNA polymerases. The reaction involves attack of the 3′-hydroxyl of the growing polynucleotide chain on the 5′-triphosphate of the incoming NTP, with release of pyrophosphate (PP_i). (B) Alternative 3′-to-5′ pathway for polynucleotide synthesis catalyzed by Thg1 family enzymes. The reversal of functional groups with respect to the nucleic acid and NTP substrates results in the net extension of the nucleic acid chain in the opposite direction to extension by other DNA/RNA polymerases.

FIGURE 2.

Multiple mechanisms for specifying tRNA^His identity. (A) tRNA^His identity in several groups of α-proteobacteria, including Rhodobacterales and Caulobacterales. In these species, tRNA^His genes lack an encoded G₋₁, and the precursor tRNA (5′ leader sequence indicated in blue) is cleaved by RNase P to leave the tRNA without the usual G₋₁ residue. The HisRS in these organisms is atypical and recognizes alternative identity elements in the α-proteobacterial tRNA^His for G₋₁-independent aminoacylation. (B) tRNA^His identity in E. coli and chloroplasts. tRNA^His genes in these cases are encoded with a G₋₁ residue in the 5′ leader sequence (indicated in blue), which is retained following 5′ end processing by RNase P to yield the G₋₁-containing tRNA that is the substrate for aminoacylation by HisRS. Many archaea and all bacteria, with the exception of the few α-proteobacterial species described in A, encode G₋₁ in their tRNA^His genes and therefore presumably obtain the G₋₁ identity element by this mechanism. (C) tRNA^His identity in eukaryotes. In eukaryotes, tRNA^His genes do not contain the G₋₁ identity element in the precursor sequence (indicated in blue), and the residue is added post-transcriptionally by Thg1, after 5′ end processing by RNase P. A small number of eukaryotic species lack an identifiable Thg1 enzyme in their genomes; the G₋₁-status and its requirement for tRNA^His identity have not been investigated in these species.

FIGURE 3.

Three-step mechanism for G₋₁ addition to tRNA^His by yeast Thg1. First, the 5′-monophosphorylated tRNA^His is activated by adenylylation using ATP. In the second step, the G₋₁ nucleotide (in the form of GTP) is transferred to the activated 5′ end, releasing AMP. In the third step, the 5′-pyrophosphate is removed from the G₋₁ nucleotide to yield the monophosphorylated G₋₁-containing tRNA^His that is the optimal substrate for HisRS.

FIGURE 4.

Yeast Thg1 catalyzes template-dependent 3′-to-5′ polymerase activity. Utilizing tRNA^His variant substrates that contain C₇₃ instead of the wild-type A₇₃, yeast Thg1 catalyzes sequential addition of up to three G-residues to the 5′ end of the tRNA. The reverse polymerase reaction only occurs in the presence of the correct Watson-Crick base-pairing nucleotide (GTP in the case of the C₇₃-tRNA). For multiple nucleotide additions, the 5′-triphosphorylated end resulting from the previous nucleotide addition is the activated end for attack by the 3′-hydroxyl of the subsequent nucleotide, as shown in detail in brackets.

FIGURE 5.

Thg1 and canonical 5′-to-3′ polymerases share significant structural similarity. A model of the active site of T7 DNA polymerase (A; Protein Data Bank identification 1T7P) and human Thg1 (B; Protein Data Bank 3OTB), each co-crystallized with bound dNTP, reveals that both enzymes share a similar secondary structure characteristic of the polymerase palm domain, including two metal ions that are critical for catalysis by each enzyme. The bound nucleotide visible in the T7 DNA polymerase structure reveals the position of the incoming dNTP, poised for attack by the 3′-hydroxyl of the elongating primer (data not shown). The bound nucleotide observed in the hThg1 structure is believed, based on kinetic and mutational data, to mimic the position of the nucleotide used to activate the 5′ end of the tRNA, consistent with the reversed orientation of the nucleotide substrates for 5′-to-3′ and 3′-to-5′ polymerization.

FIGURE 6.

Phylogenetic reconstruction depicting the evolutionary relationships among members of the Thg1 superfamily. Numbers in parentheses refer to the number of sequences used in the construction of the maximum likelihood (ML) phylogenetic tree (Supplemental Fig. S1) upon which the figure is based. Numbers along edges are bootstrap values (see below). Names of phyla or supergroups in each clade are listed. The branching positions of the four Dictyostelium discoideum TLPs (DdiTLP1 to 4; see text) are indicated by arrows. Methodology: Thg1 and TLP sequences were retrieved in December 2011 using the BLASTP program (Altschul et al. 1997) from the nr database at the NCBI (http://www.ncbi.nlm.nih.gov/). BLAST outputs were checked manually to identify homologs (no arbitrary cut-off e-value was used). The corresponding sequences were aligned using MAFFT v6.857b (Katoh et al. 2002). The resulting alignments were then inspected visually and refined manually using the ED program from the MUST package (Philippe 1993). Prior to phylogenetic analyses, regions of doubtful homology were removed manually from the alignments using NET from the MUST package (data sets are available on request). ML phylogenetic trees were computed with PHYML 3.0 (Guindon and Gascuel 2003) using the LG model (Le and Gascuel 2008) and a γ correction to take into account the heterogeneity of the evolutionary rates across sites (using four discrete classes of sites and an estimated α parameter). Branch robustness of the resulting trees was estimated using the nonparametric bootstrap procedure implemented in PHYML using 100 replicates of the original data set and the same parameters as for tree reconstruction. In order to obtain further insights into the evolutionary history of these proteins, an accurate phylogenetic analysis was performed using the same methods as outlined above with a subset of Thg1/TLP homologs reflecting the taxonomic diversities of this gene family.

FIGURE 7.

tRNA 5′ editing repairs encoded mismatches found in some mitochondrial tRNAs. The mitochondrial tRNA 5′ editing reaction comprises at least two activities. First, up to three mismatched nucleotides are removed from the 5′ ends of tRNA species, such as the mt-tRNA^Asp from A. castellanii shown here, by (an) unidentified enzyme(s). A 5′-monophosphate-containing repair intermediate species is generated that lacks the mismatches, although the precise biochemical structure of this intermediate has not been determined. In the second step, the aminoacyl acceptor stem is repaired using 3′-to-5′ polymerase activity, with 3′ end nucleotides (shown in red) serving as the template for addition of the correct Watson-Crick pairing NTPs. The biochemical activities catalyzed by two TLPs from D. discoideum are consistent with a role for TLPs in this step of the editing reaction.