Kinetic analysis of 3′-5′ nucleotide addition catalyzed by eukaryotic tRNAHis guanylyltransferase

Fidelity of protein synthesis largely depends on the ability of aminoacyl-tRNA synthetases to distinguish between cognate and non-cognate tRNA substrates. However, due to similar secondary and tertiary structures of all tRNAs, proper substrate recognition must be facilitated by identity elements present within the primary sequence of tRNAs. Many characterized identity elements are located in either the anticodon, or in the aminoacyl-acceptor stem, with the discriminator nucleotide (N73) often playing a significant role (1). In most organisms, the histidyl-tRNA synthetase (HisRS)1 recognizes a single guanosine residue located at the −1 position (G−1) of tRNAHis as a critical identity element (2-7). In E. coli, G−1 is genomically encoded and retained in the primary transcript following ribonuclease P (RNase P) cleavage of the 5′ leader sequence from the precursor tRNAHis; a similar mechanism is predicted to incorporate G−1 in most bacteria and some archaea (8). Cleavage of the pre-tRNA in this case results in a 5′-monophosphorylated tRNAHis containing a G−1:C73 Watson-Crick (WC) base pair. However, in eukaryotes the G−1 residue is not genomically encoded and therefore this mechanism of incorporating the G−1 identity element cannot occur. Instead, G−1 is added post-transcriptionally via an unusual 3′-5′ addition reaction, as first described in Schizosaccharomyces pombe and Drosophila melanogaster (9). In these organisms, the additional residue is added opposite a universally conserved adenosine at position 73, resulting in a non-WC G−1:A73 base pair at the top of the acceptor stem. The enzyme that catalyzes post-transcriptional G−1 addition, the tRNAHis guanylyltransferase (Thg1), was first discovered in Saccharomyces cerevisiae, where it was demonstrated that controlled depletion of Thg1 correlates with accumulation of tRNAHis lacking G−1, reduction in aminoacylated tRNAHis, and unexpected accumulation of m5C modifications at positions 48 and 50 (10, 11). In yeast, the THG1 gene is essential for optimal growth, although overexpression of HisRS and its cognate tRNAHis can suppress the lethal growth phenotype of the yeast deletion strain (12). In accordance with the essential role for G−1 in tRNAHis recognition, Thg1 is highly conserved throughout eukaryotes, which nearly all lack a genomically encoded G−1 residue (10). Interestingly, Thg1 orthologs referred to as Thg1-like proteins (TLPs) have been identified in some bacteria and archaea that also contain a genomically encoded G−1, where TLPs have been implicated in tRNA 5′-end repair reactions (13-15) The molecular mechanism of Thg1 is of extreme interest because the Thg1 enzymes are the only known enzymes that catalyze nucleotide addition in the 3′-5′ direction. However, the mechanism of 3′-5′ addition cannot be inferred through identifiable sequence similarity between Thg1 enzymes and any other known enzyme family. Yeast Thg1 (yThg1) adds G−1 to a 5′ monophosphorylated tRNAHis (p-tRNAHis) species generated from RNase P cleavage of the precursor tRNA, and selective recognition of tRNAHis is ensured through recognition of the His anticodon (GUG) (16). G−1 addition to the physiologically-relevant p-tRNAHis substrate proceeds via a three step reaction (Fig. 1A). First, yThg1 activates the 5′ end of p-tRNAHis using ATP in an adenylylation step, resulting in formation of an adenylylated intermediate (App-tRNAHis) containing a 5′-5′ phosphoanhydride bond. The presence of the App-activated intermediate was first demonstrated with partially purified yeast enzyme, and confirmed later using purified yThg1, once the identity of the gene was known (10, 17). In both cases, incorporation of [α-32P]-AMP from labeled ATP into tRNAHis was confirmed by P1 and snake venom nuclease analysis of reaction products, and release of the labeled AMP upon addition of G−1 was also demonstrated. In the second step of the G−1 addition reaction, yThg1 utilizes the 3′-hydroxyl of GTP to attack the intermediate in a nucleotidyl transfer step, releasing AMP and resulting in addition of a single GTP to the 5′ end of the tRNA (pppG−1p-tRNAHis). Third, yThg1 removes pyrophosphate from the G−1 residue in a pyrophosphate removal step, yielding mature pG−1-containing tRNAHis (pG−1p-tRNAHis). Thg1 can also add nucleotides to a 5′-triphosphorylated tRNAHis (ppp-tRNAHis) in the absence of ATP in vitro (Fig. 1B) (10). The ppp-tRNAHis substrate is presumed to mimic the adenylylated intermediate, allowing Thg1 to bypass the adenylylation step when acting on a substrate containing a pre-activated 5′ end. Figure 1 Two pathways for G−1 addition to tRNAHis In addition to non-WC G−1 addition that occurs in vivo in yeast with A73-containing tRNAHis, the natural substrate in eukaryotes, yThg1 also catalyzes multiple rounds of nucleotide addition in a strictly template-dependent 3′-5′ polymerase reaction with a C73-containing tRNAHis variant in vitro (18). This reverse polymerase activity forms G:C and C:G WC base pairs, and while yThg1 does not polymerize A:U and U:A base pairs with these substrates, other Thg1 family enzymes have the ability to polymerize any of the four WC base pairs (13). Although an in vivo function for 3′-5′ polymerization in yeast remains unknown, Thg1 family enzymes from every domain of life catalyze the WC-dependent reverse polymerase activity, suggesting that this may be an ancestral activity of the earliest Thg1 family members (13, 14). The 3′-5′ polymerase activity is likely exploited by TLPs to facilitate tRNA 5′-end repair and a 5′-tRNA editing reaction that occurs in the mitochondria of lower eukaryotes (13, 19). Despite the lack of identifiable sequence similarity between Thg1 and any other known enzyme, the putative active site of human Thg1 (hThg1) shares significant structural similarity with canonical WC-dependent 5′-3′ DNA/RNA polymerases. This suggests the possibility of a common evolutionary origin between 5′-3′ and 3′-5′ nucleotide addition enzymes (20) The long-term goal of this work is to determine the complete molecular mechanism for the unusual enzymatic activity catalyzed by Thg1. The hThg1 structure suggested that Thg1 utilizes two-metal-ion catalysis, a feature also shared among canonical DNA/RNA nucleotidyl transferase enzymes (21, 22), and alanine-scanning mutagenesis previously identified 19 residues that are important for the overall G−1 addition reaction (23). However, the precise roles of the metal ions and of individual Thg1 residues in the 3′-5′ addition reaction were not addressed by these earlier studies. To fully understand the complex, multistep reaction catalyzed by yThg1, we developed single turnover kinetic assays that allow isolation and characterization of each of the three individual chemical steps that occur during addition of G−1 to tRNAHis in yeast (adenylylation, nucleotidyl transfer and pyrophosphate removal). Here we use this approach to construct a kinetic framework for the non-WC-dependent G−1 addition reaction catalyzed by yThg1, which is the first in-depth biochemical investigation into the molecular mechanism of any Thg1 family member. We also assign functional roles for nine highly conserved yThg1 residues that participate in the mechanism of G−1 addition.