Elsevier

Bioorganic Chemistry

Volume 31, Issue 1, February 2003, Pages 24-43
Bioorganic Chemistry

Biosynthesis of the 7-deazaguanosine hypermodified nucleosides of transfer RNA

https://doi.org/10.1016/S0045-2068(02)00513-8Get rights and content

Abstract

Transfer RNA (tRNA) is structurally unique among nucleic acids in harboring an astonishing diversity of post-transcriptionally modified nucleoside. Two of the most radically modified nucleosides known to occur in tRNA are queuosine and archaeosine, both of which are characterized by a 7-deazaguanosine core structure. In spite of the phylogenetic segregation observed for these nucleosides (queuosine is present in Eukarya and Bacteria, while archaeosine is present only in Archaea), their structural similarity suggested a common biosynthetic origin, and recent biochemical and genetic studies have provided compelling evidence that a significant portion of their biosynthesis may in fact be identical. This review covers current understanding of the physiology and biosynthesis of these remarkable nucleosides, with particular emphasis on the only two enzymes that have been discovered in the pathways: tRNA-guanine transglycosylase (TGT), which catalyzes the insertion of a modified base into the polynucleotide with the concomitant elimination of the genetically encoded guanine in the biosynthesis of both nucleosides, and S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA), which catalyzes the penultimate step in the biosynthesis of queuosine, the construction of the carbocyclic side chain.

Introduction

The post-transcriptional processing of transfer RNA (tRNA) involves a number of functionally distinct events essential for tRNA maturation [1], [2], [3], [4]. The phenomenon of nucleoside modification is perhaps the most remarkable of these events, and results in a wealth of structural changes to the canonical nucleosides [4]. Although other RNA species also exhibit varying degrees of nucleoside modification, it is only in the tRNA that a rich structural diversity is realized.

Nucleoside modification typically occurs to ∼10% of the nucleosides in a particular tRNA, but can involve as many as 25% of the nucleosides [4]. Over 80 modified nucleosides have been characterized [4], many of which are conserved across broad phylogenetic boundaries. The nature of nucleoside modification varies from simple methylation of the base or ribose ring to extensive “hypermodification” of the canonical bases, the later of which can result in radical structural changes and involve multiple enzymatic steps to complete. With the realization that many modifications are conserved in phylogenetically diverse organisms, and that an impressive amount of genetic information codes for tRNA modifying enzymes (an estimated 1% of the total genome in bacteria such as Salmonella typhimurium) [5], an appreciation for the importance of modified nucleosides to the basic physiology of the cell has emerged. It is thought that modifications located outside of the anticodon region function in general to maintain the structural integrity of the tRNA and serve as identity determinants for the myriad interactions involving tRNA [4]. In contrast, modifications within and around the anticodon are proposed to play a direct role in increasing translational efficiency and/or fidelity [6], [7], and in specific cases, to serve as recognition determinants or anti-determinants for aminoacyl-tRNA synthetases [8], [9]. In spite of the importance of modified nucleosides in tRNA, the contributions that specific modifications make to tRNA function are well established in only a few cases [4], [5], and our understanding of the biosynthesis of the various modified nucleosides is, in the main, rudimentary.

Arguably the most remarkable modifications known to occur in tRNA are the 7-deazaguanosine nucleosides queuosine (Q, Fig. 1) and archaeosine (G*, Fig. 1). Both nucleosides share the unusual 7-deazaguanosine core, but differ in the extent of further elaboration of this core structure; queuosine is characterized by a cyclopentendiol ring appended to (7-aminomethyl)-7-deazaguanosine [10], [11], which in some mammalian tRNAs is glycosylated with galactose or mannose at the C5 hydroxyl [12], while archaeosine possesses an amidine functional group at the 7-position [13]. Queuosine and its derivatives occur exclusively at position 34 (the wobble position) in the anticodons of tRNAs coding for the amino acids asparagine, aspartic acid, histidine, and tyrosine [14]. Each of these tRNAs posses the anticodon sequence GUN (positions 34–36), where N can be any nucleotide. Queuosine is ubiquitous throughout eukaryotic and bacterial phyla (with the exception of the tRNA of yeast and mycoplasma), but is absent from the tRNA of the Archaea. In marked contrast, archaeosine is present only in the Archaea, where it is found in the majority of archaeal tRNA specifically at position 15 in the dihydrouridine loop (D-loop) [15], a site not modified in any tRNA outside of the archaeal domain. As with queuosine modification, all of the tRNA in which archaeosine is found originally contain a genetically encoded G at the site of modification.

A definitive picture of queuosines biochemical function or functions has yet to emerge, but research over the last 30 years has established a strong connection between queuosine and a variety of physiological phenomena. In Eukarya, for example, the developmental stages of a cell are closely correlated with the extent of queuosine modification in the tRNA [16]; the relevant tRNAs of mature tissue exhibit full modification with queuosine while undermodification of tRNA is uniformly observed in developmental stages associated with cell proliferation and differentiation [16], [17], [18], [19]. Potentially related is the observation that neoplastic transformation and development are correlated with the appearance of (Q)-tRNA [17], [20], [21], [22]. So widespread is this later observation that it has been suggested that the extent of (Q)-tRNA be used for histopathological grading of malignancies [21]. Queuosine has also recently been shown to be essential in the biosynthesis of tyrosine in animals [23].

Although there is considerably less data available on the role(s) of queuosine in bacterial physiology, what is known is nonetheless striking. Escherichia coli mutants defective in queuosine biosynthesis exhibit an apparently normal phenotype during favorable growth conditions, even out-growing a wild-type control strain in mixed population experiments [24], [25]. Upon entry into stationary growth phase, however, the viability of the (Q)-strain drops dramatically, such that after a short time the number of viable (Q)-cells comprise less than 1% of the control. Recent studies with the pathogenic bacterium Shigella flexneri have identified tRNA-guanine transglycosylase (TGT), a key enzyme in queuosine biosynthesis (vide infra), as the gene product of vacC [26], a virulence-associated chromosomal locus associated with epithelial cell invasion. Among the physiological characteristics of the vacC mutant were significantly decreased levels of key invasion-associated proteins, as well as reduced capacity for provoking keratoconjunctivitis. When the cloned tgt gene from E. coli was introduced into the vacC mutant, the virulence phenotype was fully restored.

While the molecular basis for these disparate phenomena is unknown, the presence of queuosine in the anticodon is compatable with a role in modulating translational fidelity. Indeed, translational effects unique to individual tRNAs have been observed both in vitro and in vivo. For example, the -1 frameshifting events essential for correct translation of the retroviral Gag-Pol-Pro polypeptides of HTLV-1 and BLV appear to be dependent on (Q)-tRNAAsn [27], [28], [29]. A similar dependance on tRNA hypermodification has been observed with the analogous frame shifting events in HIV and Rous sarcoma virus, where frame shifting has been shown to be more efficient with a tRNAPhe that lacks the hypermodified nucleoside wyebutoxine (Wye) normally present in the anticodon loop [28]. In other experiments, (Q)-tRNATyr has been shown to exhibit amber suppresser activity in both a context-dependent and independent manner [14], [30], [31], while queuosine in tRNAHis eliminates the codon bias exhibited by the (Q)-tRNAHis for the degenerate codons in vivo [32]. Furthermore, the loss of pathogenicity observed in S. flexneri tgt mutants has been traced primarily to mistranslation of the virulence factor virF [33], a transcription factor responsible for up-regulation of a suite of virulence associated proteins. Finally, the role of queuosine in mammalian tyrosine biosynthesis appears to be related to the (Q)-tRNA dependent mistranslation of mRNA coding for the enzyme phenylalanine hydroxylase.

Interestingly, a growing body of evidence also suggests that queuine, the free base of queuosine, may exert important effects on eukaryotic physiology independent of its role in tRNA. It has been shown, for example, that queuine is essential in HeLe cells for relieving hypoxic stress [34], and queuine has been implicated in lactate dehydrogenase isoenzyme distribution, protein phosphorylation patterns [35], [36], and in the modulation of cell proliferation and signaling pathways [37], [38], [39], [40], [41].

The molecular physiology of archaeosine has yet to be investigated, but its location at position-15 in tRNA has led to speculation that it may function in stabilizing the tertiary structure of the tRNA [13], an especially critical role given the prevalence of thermophiles within the Archaea. This proposal is based on global interactions between the D-loop and the T-stem and loop [42] that are conserved in the tertiary structure of all tRNA. Specifically, the nucleotide at position-15 (always a purine) base pairs with a pyrimidine at position-48 (the junction of the T-stem and variable loop), and is involved in a stacking interaction with nucleotide-59 (usually a purine). Loss of this structural element triggers complete denaturation of the tRNA to random coil [43]. Thus, stabilization of the D-loop/T-stem interaction is essential to maintenance of tRNA tertiary and secondary structure. Archaeosine may contribute to the stability of this structural element through electrostatic interactions of the positively charged formamidine group, which based on the crystal structure of yeast tRNAPhe [44] should sit in a cleft of high negative electrostatic potential containing the 5-phosphate groups from nucleotides 7, 14, and 49.

Section snippets

Biosynthesis of queuosine and archaeosine

The biosynthesis of the 7-deazaguanosines is only partially understood (Fig. 2), and has been studied primarily in the context of queuosine formation. Whole organism incorporation experiments with Salmonella typhimuruim established that GTP is the probable primary precursor in the biosynthesis of queuosine. In these experiments positive incorporation into queuosine was observed with [2-14C]guanine, but not with [8-14C]guanine [45]. These results were interpreted to implicate loss of carbon-8

The TGT enzymes

The TGTs are a particularly intriguing family of enzymes. They are the only enzymes involved in RNA modification that catalyze the replacement of a genetically encoded base with a modified base [5], and their phylogenetic segregation is correlated with unique modified base substrate specificity and disparate tRNA recognition elements.

TGT activity was first reported in the early 1970s [58], [59], and purification of the relevant enzymes from eukaryotic and bacterial sources followed over the

The QueA enzyme

The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) catalyzes the penultimate step in the biosynthesis of queuosine, the formation of oQ via the addition of an epoxycyclopentandiol ring to preQ1 (Fig. 4). The recombinant enzyme was isolated and its activity characterized [53] following the identification of an operon in E. coli containing the tgt gene along with three unidentified ORFs, one of which (designated queA) complemented a mutation in Q biosynthesis after the TGT

Future directions

While significant progress has been made in recent years in elucidating details of the biosynthesis of queuosine and archaeosine, the majority of the biosynthetic pathways still remain unknown. It is clear from a cursory examination of the structural changes that occur in the biosynthesis of these nucleosides that the steps yet to be discovered are likely to be as remarkable as those already known. For example, while the cyclohydrolases offer good biochemical precedent for the incorporation of

Acknowledgements

Special acknowledgement is made to Helga Kersten (Universität Erlangen), who provided encouragement and important plasmid stocks when my laboratory initiated work in this area, and to the current and past members of the lwata-Reuyl group who contributed to some of the work described in this review. Acknowledgement is also made to Squire Booker (Pennsylvania State University) and Wilfred van der Donk (University of Illinois) for fruitful discussions on the chemistry of ribonucleotide reductases.

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