archaeosine and 7-deazaguanosine

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.

Topics: Catalysis; Guanosine; Nucleoside Q; Nucleosides; Pentosyltransferases; RNA, Transfer

The presence of the 7-deazaguanosine derivative archaeosine (G(+)) at position 15 in tRNA is one of the diagnostic molecular characteristics of the Archaea. The biosynthesis of this modified nucleoside is especially complex, involving the initial production of 7-cyano-7-deazaguanine (preQ(0)), an advanced precursor that is produced in a tRNA-independent portion of the biosynthesis, followed by its insertion into the tRNA by the enzyme tRNA-guanine transglycosylase (arcTGT), which replaces the target guanine base yielding preQ(0)-tRNA. The enzymes responsible for the biosynthesis of preQ(0) were recently identified, but the enzyme(s) catalyzing the conversion of preQ(0)-tRNA to G(+)-tRNA have remained elusive. Using a comparative genomics approach, we identified a protein family implicated in the late stages of archaeosine biosynthesis. Notably, this family is a paralog of arcTGT and is generally annotated as TgtA2. Structure-based alignments comparing arcTGT and TgtA2 reveal that TgtA2 lacks key arcTGT catalytic residues and contains an additional module. We constructed a Haloferax volcanii DeltatgtA2 derivative and demonstrated that tRNA from this strain lacks G(+) and instead accumulates preQ(0). We also cloned the corresponding gene from Methanocaldococcus jannaschii (mj1022) and characterized the purified recombinant enzyme. Recombinant MjTgtA2 was shown to convert preQ(0)-tRNA to G(+)-tRNA using several nitrogen sources and to do so in an ATP-independent process. This is the only example of the conversion of a nitrile to a formamidine known in biology and represents a new class of amidinotransferase chemistry.

Topics: Amidinotransferases; Archaeal Proteins; Guanosine; Haloferax volcanii; Pentosyltransferases; Recombinant Proteins; RNA, Archaeal; RNA, Transfer; Structural Homology, Protein

Queuosine (Q) and archaeosine (G(+)) are hypermodified ribonucleosides found in tRNA. Q is present in the anticodon region of tRNA(GUN) in Eukarya and Bacteria, while G(+) is found at position 15 in the D-loop of archaeal tRNA. Prokaryotes produce these 7-deazaguanosine derivatives de novo from GTP through the 7-cyano-7-deazaguanine (pre-Q(0)) intermediate, but mammals import the free base, queuine, obtained from the diet or the intestinal flora. By combining the results of comparative genomic analysis with those of genetic studies, we show that the first enzyme of the folate pathway, GTP cyclohydrolase I (GCYH-I), encoded in Escherichia coli by folE, is also the first enzyme of pre-Q(0) biosynthesis in both prokaryotic kingdoms. Indeed, tRNA extracted from an E. coli DeltafolE strain is devoid of Q and the deficiency is complemented by expressing GCYH-I-encoding genes from different bacterial or archaeal origins. In a similar fashion, tRNA extracted from a Haloferax volcanii strain carrying a deletion of the GCYH-I-encoding gene contains only traces of G(+). These results link the production of a tRNA-modified base to primary metabolism and further clarify the biosynthetic pathway for these complex modified nucleosides.

Topics: Cluster Analysis; Comparative Genomic Hybridization; Computational Biology; Escherichia coli; Escherichia coli Proteins; GTP Cyclohydrolase; Guanosine; Haloferax volcanii; Nucleoside Q; Phylogeny; RNA, Bacterial; RNA, Transfer

Archaeosine is a novel derivative of 7-deazaguanosine found in transfer RNAs of most organisms exclusively in the archaeal phylogenetic lineage and is present in the D-loop at position 15. We show that this modification is formed by a posttranscriptional base replacement reaction, catalyzed by a new tRNA-guanine transglycosylase (TGT), which has been isolated from Haloferax volcanii and purified nearly to homogeneity. The molecular weight of the enzyme was estimated to be 78 kDa by SDS-gel electrophoresis. The enzyme can insert free 7-cyano-7-deazaguanine (preQ0 base) in vitro at position 15 of an H. volcanii tRNA T7 transcript, replacing the guanine originally located at that position without breakage of the phosphodiester backbone. Since archaeosine base and 7-aminomethyl-7-deazaguanine (preQ1 base) were not incorporated into tRNA by this enzyme, preQ0 base appears to be the actual substrate for the TGT of H. volcanii, a conclusion supported by characterization of preQ0 base in an acid-soluble extract of H. volcanii cells. Thus, this novel TGT in H. volcanii is a key enzyme for the biosynthetic pathway leading to archaeosine in archaeal tRNAs.

Topics: Amino Acid Sequence; Animals; Cattle; Guanosine; Halobacteriaceae; Models, Chemical; Molecular Sequence Data; Nucleic Acid Conformation; Pentosyltransferases; RNA, Bacterial; RNA, Transfer; RNA, Transfer, Lys; Saccharomyces cerevisiae; Sequence Alignment; Substrate Specificity