Thg1 Research Articles

Thg1 family 3'-5' RNA polymerases as tools for targeted RNA synthesis.

Members of the 3'-5' RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), catalyze templated synthesis of RNA in the reverse direction to all other known 5'-3' RNA and DNA polymerases. The discovery of enzymes capable of this reaction raised the possibility of exploiting 3'-5' polymerases for posttranscriptional incorporation of nucleotides to the 5'-end of nucleic acids without ligation, and instead by templated polymerase addition. To date, studies of these enzymes have focused on nucleotide addition to highly structured RNAs, such as tRNA and other noncoding RNAs. Consequently, general principles of RNA substrate recognition and nucleotide preferences that might enable broader application of 3'-5' polymerases have not been elucidated. Here, we investigated the feasibility of using Thg1 or TLPs for multiple nucleotide incorporation to the 5'-end of a short duplex RNA substrate, using a templating RNA oligonucleotide provided in trans to guide 5'-end addition of specific sequences. Using optimized assay conditions, we demonstrated a remarkable capacity of certain TLPs to accommodate short RNA substrate-template duplexes of varying lengths with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to eight bases to 5'-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5'-end labeling tool.

Recognition of tRNAHis in an RNase P-Free Nanoarchaeum.

The 5' extra guanosine with 5'-monophosphate at position -1 (G-1) of tRNAHis (p-tRNAHis) is a nearly universal feature that establishes tRNAHis identity. G-1 is either genome encoded and retained after processing by RNase P (RNase P) or posttranscriptionally incorporated by tRNAHis guanylyltransferase (Thg1) after RNase P cleavage. However, RNase P is not found in the hyperthermophilic archaeum Nanoarchaeum equitans; instead, all of its tRNAs, including tRNAHis, are transcribed as leaderless tRNAs with 5'-triphosphate (ppp-tRNAs). How N. equitans histidyl-tRNA synthetase (NeHisRS) recognizes its cognate tRNA (NetRNAHis) is of particular interest. In this paper, we show that G-1 serves as the major identity element of NetRNAHis, with its anticodon performing a similar role, though to a lesser extent. Moreover, NeHisRS distinctly preferred p-tRNAHis over ppp-tRNAHis (~5-fold difference). Unlike other prokaryotic HisRSs, which strongly prefer tRNAHis with C73, this enzyme could charge tRNAsHis with A73 and C73 with nearly equal efficiency. As a result, mutation at the C73-recognition amino acid residue Q112 had only a minor effect (<2-fold reduction). This study suggests that NeHisRS has evolved to disregard C73, but it still maintains its evolutionarily preserved preference toward tRNAHis with 5'-monophosphate. IMPORTANCE Mature tRNAHis has, at its 5'-terminus, an extra guanosine with 5'-monophosphate, designated G-1. G-1 is the major recognition element for histidyl-tRNA synthetase (HisRS), regardless of whether it is of eukaryotic or prokaryotic origin. However, in the hyperthermophilic archaeum Nanoarchaeum equitans, all its tRNAs, including tRNAHis, are transcribed as leaderless tRNAs with 5'-triphosphate. This piqued our curiosity about whether N. equitans histidyl-tRNA synthetase (NeHisRS) prefers tRNAHis with 5'-triphosphate. We show herein that G-1 is still the major recognition element for NeHisRS. However, unlike other prokaryotic HisRSs, which strongly prefer tRNAHis with C73, this enzyme shows almost the same preference for C73 and A73. Most intriguingly, NeHisRS still prefers 5'-monophosphate over 5'-triphosphate. It thus appears that the preference of HisRS for tRNAHis with 5'-monophosphate emerged very early in evolution.

Fidelity of base-pair recognition by a 3'-5' polymerase: mechanism of the Saccharomyces cerevisiae tRNAHis guanylyltransferase.

The tRNAHis guanylyltransferase (Thg1) was originally discovered in Saccharomyces cerevisiae where it catalyzes 3′–5′ addition of a single nontemplated guanosine (G−1) to the 5′ end of tRNAHis. In addition to this activity, S. cerevisiae Thg1 (SceThg1) also catalyzes 3′–5′ polymerization of Watson–Crick (WC) base pairs, utilizing nucleotides in the 3′-end of a tRNA as the template for addition. Subsequent investigation revealed an entire class of enzymes related to Thg1, called Thg1-like proteins (TLPs). TLPs are found in all three domains of life and preferentially catalyze 3′–5′ polymerase activity, utilizing this unusual activity to repair tRNA, among other functions. Although both Thg1 and TLPs utilize the same chemical mechanism, the molecular basis for differences between WC-dependent (catalyzed by Thg1 and TLPs) and non-WC-dependent (catalyzed exclusively by Thg1) reactions has not been fully elucidated. Here we investigate the mechanism of base-pair recognition by 3′–5′ polymerases using transient kinetic assays, and identify Thg1-specific residues that play a role in base-pair discrimination. We reveal that, regardless of the identity of the opposing nucleotide in the RNA “template,” addition of a non-WC G−1 residue is driven by a unique kinetic preference for GTP. However, a secondary preference for forming WC base pairs is evident for all possible templating residues. Similar to canonical 5′–3′ polymerases, nucleotide addition by SceThg1 is driven by the maximal rate rather than by NTP substrate affinity. Together, these data provide new insights into the mechanism of base-pair recognition by 3′–5′ polymerases.

Analysis of GTP addition in the reverse (3'-5') direction by human tRNAHis guanylyltransferase.

Human tRNAHis guanylyltransferase (HsThg1) catalyzes the 3′–5′ addition of guanosine triphosphate (GTP) to the 5′-end (−1 position) of tRNAHis, producing mature tRNAHis. In human cells, cytoplasmic and mitochondrial tRNAHis have adenine (A) or cytidine (C), respectively, opposite to G−1. Little attention has been paid to the structural requirements of incoming GTP in 3′–5′ nucleotidyl addition by HsThg1. In this study, we evaluated the incorporation efficiencies of various GTP analogs by HsThg1 and compared the reaction mechanism with that of Candida albicans Thg1 (CaThg1). HsThg1 incorporated GTP opposite A or C in the template most efficiently. In contrast to CaThg1, HsThg1 could incorporate UTP opposite A, and guanosine diphosphate (GDP) opposite C. These results suggest that HsThg1 could transfer not only GTP, but also other NTPs, by forming Watson–Crick (WC) hydrogen bonds between the incoming NTP and the template base. On the basis of the molecular mechanism, HsThg1 succeeded in labeling the 5′-end of tRNAHis with biotinylated GTP. Structural analysis of HsThg1 was also performed in the presence of the mitochondrial tRNAHis. Structural comparison of HsThg1 with other Thg1 family enzymes suggested that the structural diversity of the carboxy-terminal domain of the Thg1 enzymes might be involved in the formation of WC base-pairing between the incoming GTP and template base. These findings provide new insights into an unidentified biological function of HsThg1 and also into the applicability of HsThg1 to the 5′-terminal modification of RNAs.

Plant mitochondria use two pathways for the biogenesis of tRNA His.

All tRNAHis possess an essential extra G–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G–1 is added by a tRNAHis guanylyl transferase. In prokaryotes, G–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G–1 we find here that both maturation pathways can be used. Indeed, tRNAHis with or without a G–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNAHis precursors at both positions G+1 and G–1. The G–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNAHis without G–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNAHis with a G–1 are recovered. This shows that a previously unreported tRNAHis guanylyltransferase activity is present in plant mitochondria.

Naturally occurring dual recognition of tRNAHis substrates with and without a universal identity element

ABSTRACTThe extra 5ʹ guanine nucleotide (G-1) on tRNAHis is a nearly universal feature that specifies tRNAHis identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNAHis guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNAHis (CetRNAnHis) retains a genome-encoded G-1. Our study showed that this eukaryote possesses a histidyl-tRNA synthetase (CeHisRS) gene encoding two distinct HisRS isoforms that differ only at their N-termini. Most interestingly, its mitochondrial tRNAHis (CetRNAmHis) lacks G-1, a scenario never observed in any organelle. This tRNA, while lacking the canonical identity element, can still be efficiently aminoacylated in vivo. Even so, addition of G-1 to CetRNAmHis strongly enhanced its aminoacylation efficiency in vitro. Overexpression of CeHisRS successfully bypassed the requirement for yeast THG1 in the presence of CetRNAnHis without G-1. Mutagenesis assays showed that the anticodon takes a primary role in CetRNAHis identity recognition, being comparable to the universal identity element. Consequently, simultaneous introduction of both G-1 and the anticodon of tRNAHis effectively converted a non-cognate tRNA to a tRNAHis-like substrate. Our study suggests that a new balance between identity elements of tRNAHis relieves HisRS from the absolute requirement for G-1.

Translational Regulation of Plant Response to High Temperature by a Dual-Function tRNAHis Guanylyltransferase in Rice

As sessile organisms, plants have evolved numerous strategies to acclimate to changes in environmental temperature. However, the molecular basis of this acclimation remains largely unclear. In this study we identified a tRNAHis guanylyltransferase, AET1, which contributes to the modification of pre-tRNAHis and is required for normal growth under high-temperature conditions in rice. Interestingly, AET1 possibly interacts with both RACK1A and eIF3h in the endoplasmic reticulum. Notably, AET1 can directly bind to OsARF mRNAs including the uORFs of OsARF19 and OsARF23, indicating that AET1 is associated with translation regulation. Furthermore, polysome profiling assays suggest that the translational status remains unaffected in the aet1 mutant, but that the translational efficiency of OsARF19 and OsARF23 is reduced; moreover, OsARF23 protein levels are obviously decreased in the aet1 mutant under high temperature, implying that AET1 regulates auxin signaling in response to high temperature. Our findings provide new insights into the molecular mechanisms whereby AET1 regulates the environmental temperature response in rice by playing a dual role in tRNA modification and translational control.

Chemical footprinting and kinetic assays reveal dual functions for highly conserved eukaryotic tRNAHis guanylyltransferase residues

tRNAHis guanylyltransferase (Thg1) adds a single guanine to the -1 position of tRNAHis as part of its maturation. This seemingly modest addition of one nucleotide to tRNAHis ensures translational fidelity by providing a critical identity element for the histidyl aminoacyl tRNA synthetase (HisRS). Like HisRS, Thg1 utilizes the GUG anticodon for selective tRNAHis recognition, and Thg1-tRNA complex structures have revealed conserved residues that interact with anticodon nucleotides. Separately, kinetic analysis of alanine variants has demonstrated that many of these same residues are required for catalytic activity. A model in which loss of activity with the variants was attributed directly to loss of the critical anticodon interaction has been proposed to explain the combined biochemical and structural results. Here we used RNA chemical footprinting and binding assays to test this model and further probe the molecular basis for the requirement for two critical tRNA-interacting residues, His-152 and Lys-187, in the context of human Thg1 (hThg1). Surprisingly, we found that His-152 and Lys-187 alanine-substituted variants maintain a similar overall interaction with the anticodon region, arguing against the sufficiency of this interaction for driving catalysis. Instead, conservative mutagenesis revealed a new direct function for these residues in recognition of a non-Watson-Crick G-1:A73 bp, which had not been described previously. These results have important implications for the evolution of eukaryotic Thg1 from a family of ancestral promiscuous RNA repair enzymes to the highly selective enzymes needed for their essential function in tRNAHis maturation.

Genetic Interactions and Molecular Evolution of the Duplicated Genes ICARUS2 and ICARUS1 Help Arabidopsis Plants Adapt to Different Ambient Temperatures.

Understanding how plants adapt to ambient temperatures has become a major challenge prompted by global climate change. This has led to the identification of several genes regulating the thermal plasticity of plant growth and flowering time. However, the mechanisms accounting for the natural variation and evolution of such developmental plasticity remain mostly unknown. In this study, we determined that natural variation at ICARUS2 (ICA2), which interacts genetically with its homolog ICA1, alters growth and flowering time plasticity in relation to temperature in Arabidopsis (Arabidopsis thaliana). Transgenic analyses demonstrated multiple functional effects for ICA2 and supported the notion that structural polymorphisms in ICA2 likely underlie its natural variation. Two major ICA2 haplogroups carrying distinct functionally active alleles showed high frequency, strong geographic structure, and significant associations with climatic variables related to annual and daily fluctuations in temperature. Genome analyses across the plant phylogeny indicated that the prevalent plant ICA genes encoding two tRNAHis guanylyl transferase 1 units evolved ∼120 million years ago during the early divergence of mono- and dicotyledonous clades. In addition, ICA1/ICA2 duplication occurred specifically in the Camelineae tribe (Brassicaceae). Thus, ICA2 appears to be ubiquitous across plant evolution and likely contributes to climate adaptation through modifications of thermal developmental plasticity in Arabidopsis.

The Role of 3' to 5' Reverse RNA Polymerization in tRNA Fidelity and Repair.

The tRNAHis guanylyltransferase (Thg1) superfamily includes enzymes that are found in all three domains of life that all share the common ability to catalyze the 3′ to 5′ synthesis of nucleic acids. This catalytic activity, which is the reverse of all other known DNA and RNA polymerases, makes this enzyme family a subject of biological and mechanistic interest. Previous biochemical, structural, and genetic investigations of multiple members of this family have revealed that Thg1 enzymes use the 3′ to 5′ chemistry for multiple reactions in biology. Here, we describe the current state of knowledge regarding the catalytic features and biological functions that have been so far associated with Thg1 and its homologs. Progress toward the exciting possibility of utilizing this unusual protein activity for applications in biotechnology is also discussed.

A Temporal Order in 5'- and 3'- Processing of Eukaryotic tRNAHis.

For flawless translation of mRNA sequence into protein, tRNAs must undergo a series of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 3′-terminal CCA sequence that includes the site of aminoacylation, the additional 5′-G-1 position is a unique feature of most histidine tRNA species, serving as an identity element for the corresponding synthetase. In eukaryotes including yeast, both 3′-CCA and 5′-G-1 are added post-transcriptionally by tRNA nucleotidyltransferase and tRNAHis guanylyltransferase, respectively. Hence, it is possible that these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences associated with CCA and G-1-addition to yeast cytosolic tRNAHis, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNAHis transcripts independent of the presence of G-1; however, tRNAHis guanylyltransferase clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes G-1 incorporation in S. cerevisiae. Evidently, the 3′-CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition.

Molecular mechanism of substrate recognition and specificity of tRNAHis guanylyltransferase during nucleotide addition in the 3'-5' direction.

The tRNAHis guanylyltransferase (Thg1) transfers a guanosine triphosphate (GTP) in the 3′–5′ direction onto the 5′-terminal of tRNAHis, opposite adenosine at position 73 (A73). The guanosine at the −1 position (G−1) serves as an identity element for histidyl-tRNA synthetase. To investigate the mechanism of recognition for the insertion of GTP opposite A73, first we constructed a two-stranded tRNAHis molecule composed of a primer and a template strand through division at the D-loop. Next, we evaluated the structural requirements of the incoming GTP from the incorporation efficiencies of GTP analogs into the two-piece tRNAHis. Nitrogen at position 7 and the 6-keto oxygen of the guanine base were important for G−1 addition; however, interestingly, the 2-amino group was found not to be essential from the highest incorporation efficiency of inosine triphosphate. Furthermore, substitution of the conserved A73 in tRNAHis revealed that the G−1 addition reaction was more efficient onto the template containing the opposite A73 than onto the template with cytidine (C73) or other bases forming canonical Watson–Crick base-pairing. Some interaction might occur between incoming GTP and A73, which plays a role in the prevention of continuous templated 3′–5′ polymerization. This study provides important insights into the mechanism of accurate tRNAHis maturation.

Biochemical analysis of human tRNAHis guanylyltransferase in mitochondrial tRNAHis maturation

Mitochondria contain their own protein synthesis machinery, which includes mitochondrial tRNA maturation. It has been suggested that mammalian mitochondrial tRNAHis (mtRNAHis) is matured by post-transcriptional addition of guanosine at the −1 position (G−1), which serves as an identity element for mitochondrial histidyl-tRNA synthetase. However, the exact maturation process of mammalian mtRNAHis remains unclear. In cytoplasmic tRNAHis (ctRNAHis) maturation, tRNAHis guanylyltransferase (Thg1) adds a GTP onto the 5′-terminal of ctRNAHis and then removes the 5′-pyrophosphate to yield the mature 5′-monophospholylated G-1-ctRNAHis (pG-1-ctRNAHis). Although mammalian Thg1 is localized to both the cytoplasm and mitochondria, it remains unclear whether mammalian Thg1 plays a role in mtRNAHis maturation in mitochondria. Here, we demonstrated that human Thg1 (hThg1) catalyzes the G-1 addition reaction for both human ctRNAHis and mtRNAHis through recognition of the anticodon. While hThg1 catalyzed consecutive GTP additions to mtRNAHisin vitro, it did not exhibit any activity toward mature pG-1-mtRNAHis. We further found that hThg1 could add a GMP directly to the 5′-terminal of mtRNAHis in a template-dependent manner, but fungal Thg1 could not. Therefore, we hypothesized that acceleration of the pyrophosphate removal activity before or after the G-1 addition reaction is a key feature of hThg1 for maintaining a normal 5′-terminal of mtRNAHis in human mitochondria. This study provided a new insight into the differences between tRNAHis maturation in the cytoplasm and mitochondria of humans.

Crystal structure of tRNAHis guanylyltransferase from Saccharomyces cerevisiae

tRNA maturation involves several steps, including processing, splicing, CCA addition, and posttranscriptional modifications. tRNAHis guanylyltransferase (Thg1) is the only enzyme known to catalyze templated nucleotide addition in the 3′–5′ direction, unlike other DNA and RNA polymerases. For a better understanding of its unique catalytic mechanism at the molecular level, we determined the crystal structure of GTP-bound Thg1 from Saccharomyces cerevisiae at the maximum resolution of 3.0 Å. The structure revealed the enzyme to have a tetrameric conformation that is well conserved among different species, and the GTP molecule was clearly bound at the active site, coordinating with two magnesium ions. In addition, two flexible protomers at the potential binding site (PBS) for tRNAHis were observed. We suggest that the PBS of the tetramer could also be one of the sites for interaction with partner proteins.

A mutation in the THG1L gene in a family with cerebellar ataxia and developmental delay

Autosomal-recessive cerebellar atrophy is usually associated with inactivating mutations and early-onset presentation. The underlying molecular diagnosis suggests the involvement of neuronal survival pathways, but many mechanisms are still lacking and most patients elude genetic diagnosis. Using whole exome sequencing, we identified homozygous p.Val55Ala in the THG1L (tRNA-histidine guanylyltransferase 1 like) gene in three siblings who presented with cerebellar signs, developmental delay, dysarthria, and pyramidal signs and had cerebellar atrophy on brain MRI. THG1L protein was previously reported to participate in mitochondrial fusion via its interaction with MFN2. Abnormal mitochondrial fragmentation, including mitochondria accumulation around the nuclei and confinement of the mitochondrial network to the nuclear vicinity, was observed when patient fibroblasts were cultured in galactose containing medium. Culturing cells in galactose containing media promotes cellular respiration by oxidative phosphorylation and the action of the electron transport chain thus stimulating mitochondrial activity. The growth defect of the yeast thg1Δ strain was rescued by the expression of either yeast Thg1 or human THG1L; however, clear growth defect was observed following the expression of the human p.Val55Ala THG1L or the corresponding yeast mutant. A defect in the protein tRNAHis guanylyltransferase activity was excluded by the normal in vitro G-1 addition to either yeast tRNAHis or human mitochondrial tRNAHis in the presence of the THG1L mutation. We propose that homozygosity for the p.Val55Ala mutation in THG1L is the cause of the abnormal mitochondrial network in the patient fibroblasts, likely by interfering with THG1L activity towards MFN2. This may result in lack of mitochondria in the cerebellar Purkinje dendrites, with degeneration of Purkinje cell bodies and apoptosis of granule cells, as reported for MFN2 deficient mice.

Natural Variation Identifies ICARUS1, a Universal Gene Required for Cell Proliferation and Growth at High Temperatures in Arabidopsis thaliana.

Plants are highly sensitive to environmental changes and even small variations in ambient temperature have severe consequences on their growth and development. Temperature affects multiple aspects of plant development, but the processes and mechanisms underlying thermo-sensitive growth responses are mostly unknown. Here we exploit natural variation in Arabidopsis thaliana to identify and characterize novel components and processes mediating thermo-sensitive growth responses in plants. Phenotypic screening of wild accessions identified several strains displaying pleiotropic growth defects, at cellular and organism levels, specifically at high ambient temperatures. Positional cloning and characterization of the underlying gene revealed that ICARUS1 (ICA1), which encodes a protein of the tRNAHis guanylyl transferase (Thg1) superfamily, is required for plant growth at high temperatures. Transcriptome and gene marker analyses together with DNA content measurements show that ICA1 loss-of-function results in down regulation of cell cycle associated genes at high temperatures, which is linked with a block in G2/M transition and endoreduplication. In addition, plants with mutations in ICA1 show enhanced sensitivity to DNA damage. Characterization of additional strains that carry lesions in ICA1, but display normal growth, shows that alternative splicing is likely to alleviate the deleterious effects of some natural mutations. Furthermore, analyses of worldwide and regional collections of natural accessions indicate that ICA1 loss-of-function has arisen several times independently, and that these occur at high frequency in some local populations. Overall our results suggest that ICA1-mediated-modulation of fundamental processes such as tRNAHis maturation, modify plant growth responses to temperature changes in a quantitative and reversible manner, in natural populations.

Saccharomyces cerevisiae Thg1 Uses 5′-Pyrophosphate Removal To Control Addition of Nucleotides to tRNAHis

In eukaryotes, the tRNAHis guanylyltransferase (Thg1) catalyzes 3′–5′ addition of a single guanosine residue to the −1 position (G–1) of tRNAHis, across from a highly conserved adenosine at position 73 (A73). After addition of G–1, Thg1 removes pyrophosphate from the tRNA 5′-end, generating 5′-monophosphorylated G–1-containing tRNA. The presence of the 5′-monophosphorylated G–1 residue is important for recognition of tRNAHis by its cognate histidyl-tRNA synthetase. In addition to the single-G–1 addition reaction, Thg1 polymerizes multiple G residues to the 5′-end of tRNAHis variants. For 3′–5′ polymerization, Thg1 uses the 3′-end of the tRNAHis acceptor stem as a template. The mechanism of reverse polymerization is presumed to involve nucleophilic attack of the 3′-OH from each incoming NTP on the intact 5′-triphosphate created by the preceding nucleotide addition. The potential exists for competition between 5′-pyrophosphate removal and 3′–5′ polymerase reactions that could define the outcome of Thg1-catalyzed addition, yet the interplay between these competing reactions has not been investigated for any Thg1 enzyme. Here we establish transient kinetic assays to characterize the pyrophosphate removal versus nucleotide addition activities of yeast Thg1 with a set of tRNAHis substrates in which the identity of the N–1:N73 base pair was varied to mimic various products of the N–1 addition reaction catalyzed by Thg1. We demonstrate that retention of the 5′-triphosphate is correlated with efficient 3′–5′ reverse polymerization. A kinetic partitioning mechanism that acts to prevent addition of nucleotides beyond the −1 position with wild-type tRNAHis is proposed.

ヌクレオチドを3^|^apos;末端から5^|^apos;末端方向へと伸長する酵素の作用原理

During tRNA maturation tRNAHis guanylyltransferase (Thg1) catalyzes the untemplated 3'-5' addition of a G to the tRNA 5'-end. This additional guanylate provides the major identity element for histidyl-tRNA synthetase to recognize its cognate tRNAHis. Thg1 is a structural homolog of canonical 5'-3' polymerases. Here, we report the structures of Candida albicans Thg1 with its substrates tRNA, or ATP, or GTP. Two tRNAs bind to the tetrameric enzyme complex, but interaction of three subunits is required for tRNA positioning and catalytic activity. The tRNA substrate enters the Thg1 active site from the opposite direction compared to canonical 5'-3' polymerases, indicating that the directionality of nucleotide polymerization is strongly related to the directionality of substrate access. Structural and biochemical data support a reaction model of Thg1 that catalyzes reverse nucleotide addition.

Structural Studies of a Bacterial tRNAHIS Guanylyltransferase (Thg1)-Like Protein, with Nucleotide in the Activation and Nucleotidyl Transfer Sites

All nucleotide polymerases and transferases catalyze nucleotide addition in a 5′ to 3′ direction. In contrast, tRNAHis guanylyltransferase (Thg1) enzymes catalyze the unusual reverse addition (3′ to 5′) of nucleotides to polynucleotide substrates. In eukaryotes, Thg1 enzymes use the 3′–5′ addition activity to add G−1 to the 5′-end of tRNAHis, a modification required for efficient aminoacylation of the tRNA by the histidyl-tRNA synthetase. Thg1-like proteins (TLPs) are found in Archaea, Bacteria, and mitochondria and are biochemically distinct from their eukaryotic Thg1 counterparts TLPs catalyze 5′-end repair of truncated tRNAs and act on a broad range of tRNA substrates instead of exhibiting strict specificity for tRNAHis. Taken together, these data suggest that TLPs function in distinct biological pathways from the tRNAHis maturation pathway, perhaps in tRNA quality control. Here we present the first crystal structure of a TLP, from the gram-positive soil bacterium Bacillus thuringiensis (BtTLP). The enzyme is a tetramer like human THG1, with which it shares substantial structural similarity. Catalysis of the 3′–5′ reaction with 5′-monophosphorylated tRNA necessitates first an activation step, generating a 5′-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5′-end. Consistent with earlier characterization of human THG1, we observed distinct binding sites for the nucleotides involved in these two steps of activation and nucleotidyl transfer. A BtTLP complex with GTP reveals new interactions with the GTP nucleotide in the activation site that were not evident from the previously solved structure. Moreover, the BtTLP-ATP structure allows direct observation of ATP in the activation site for the first time. The BtTLP structural data, combined with kinetic analysis of selected variants, provide new insight into the role of key residues in the activation step.

Genome sequence and analysis of a broad-host range lytic bacteriophage that infects the Bacillus cereus group

BackgroundComparatively little information is available on members of the Myoviridae infecting low G+C content, Gram-positive host bacteria of the family Firmicutes. While numerous Bacillus phages have been isolated up till now only very few Bacillus cereus phages have been characterized in detail.ResultsHere we present data on the large, virulent, broad-host-range B. cereus phage vB_BceM_Bc431v3 (Bc431v3). Bc431v3 features a 158,618 bp dsDNA genome, encompassing 239 putative open reading frames (ORFs) and, 20 tRNA genes encoding 17 different amino acids. Since pulsed-field gel electrophoresis indicated that the genome of this phage has a mass of 155-158 kb Bc431v3 DNA appears not to contain long terminal repeats that are found in the genome of Bacillus phage SPO1.ConclusionsBc431v3 displays significant sequence similarity, at the protein level, to B. cereus phage BCP78, Listeria phage A511 and Enterococcus phage ØEF24C and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Based on these data we suggest that Bc431v3 should be included as a member of the Spounavirinae; however, because of all the diverse taxonomical information has been addressed recently, it is difficult to determine the genus. The Bc431v3 phage contains some highly unusual genes such as gp143 encoding putative tRNAHis guanylyltransferase. In addition, it carries some genes that appear to be related to the host sporulation regulators. These are: gp098, which encodes a putative segregation protein related to FstK/SpoIIIE DNA transporters; gp105, a putative segregation protein; gp108, RNA polymerase sigma factor F/B; and, gp109 encoding RNA polymerase sigma factor G.