(1/46) Yeast asparagine (Asn) tRNA without Q base promotes eukaryotic frameshifting more efficiently than mammalian Asn tRNAs with or without Q base.
In this study, we compare the efficiency of Asn tRNA from mammalian sources with and without the highly modified queuosine (Q) base in the wobble position of its anticodon and Asn tRNA from yeast, which naturally lacks Q base, to promote frameshifting. Interestingly, no differences in the ability of the two mammalian Asn tRNAs to promote frameshifting were observed, while yeast tRNA(ASn)(-Q) promoted frameshifting more efficiently than its mammalian counterparts in both rabbit reticulocyte lysates and wheat germ extracts. The shiftability of yeast Asn tRNA is therefore not due, or at least not completely, to the lack of Q base and most likely the shiftiness resides in structural differences elsewhere in the molecule. However, we cannot absolutely rule out a role of Q base in frameshifting as wheat germ extracts and a lysate depleted of most of its tRNA and supplemented with calf liver tRNA contain both Asn tRNA with or without Q base. (+info)
(2/46) Increased expression of queuosine synthesizing enzyme, tRNA-guanine transglycosylase, and queuosine levels in tRNA of leukemic cells.
Queuosine is a modified nucleoside located at the first position of the tRNA anticodon, which is synthesized by tRNA-guanine transglycosylase (TGT). Although the levels of queuosine in cancer cells have been reported to be lower than those in normal cells, the expression levels of TGT remain to be determined. We determined the expression levels of a subunit of TGT (TGT60KD). Contrary of our expectations, the results revealed higher levels of expression of TGT60KD than that in normal cells, and the level of queuosine in the tRNA fraction corresponded with that of TGT60KD expression. These results suggest the possibilities that the expression levels of TGT60KD regulate TGT activity and the levels of queuosine, and that TGT60KD plays significant roles in carcinogenesis. To our knowledge, this is a first report of increased expression levels of TGT60KD in human cancer cells. (+info)
(3/46) An unexpected absence of queuosine modification in the tRNAs of an Escherichia coli B strain.
The post-transcriptional processing of tRNAs decorates them with a number of modified bases important for their biological functions. Queuosine, found in the tRNAs with GUN anticodons (Asp, Asn, His, Tyr), is an extensively modified base whose biosynthetic pathway is still unclear. In this study, it was observed that the tRNA(Tyr) from Escherichia coli B105 (a B strain) migrated faster than that from E. coli CA274 (a K-12 strain) on acid urea gels. The organization of tRNA(Tyr) genes in E. coli B105 was found to be typical of the B strains. Subsequent analysis of tRNA(Tyr) and tRNA(His) from several strains of E. coli on acid urea gels, and modified base analysis of tRNA preparations enriched for tRNA(Tyr), showed that E. coli B105 lacked queuosine in its tRNAs. However, the lack of queuosine in tRNAs was not a common feature of all E. coli B strains. The tgt and queA genes in B105 were shown to be functional by their ability to complement tgt and queA mutant strains. These observations suggested a block at the step of the biosynthesis of preQ(1) (or preQ(0)) in the B105 strain. Interestingly, a multicopy vector harbouring a functional tgt gene was toxic to E. coli B105 but not to CA274. Also, in mixed cultures, E. coli B105 was readily competed out by the CA274 strain. The importance of these observations and this novel strain (E. coli B105) in unravelling the mechanism of preQ(1) or preQ(0) biosynthesis is discussed. (+info)
(4/46) Identification of four genes necessary for biosynthesis of the modified nucleoside queuosine.
Queuosine (Q) is a hypermodified 7-deazaguanosine nucleoside located in the anticodon wobble position of four amino acid-specific tRNAs. In bacteria, Q is produced de novo from GTP via the 7-deazaguanosine precursor preQ1 (7-aminoethyl 7-deazaguanine) by an uncharacterized pathway. PreQ1 is subsequently transferred to its specific tRNA by a tRNA-guanine transglycosylase (TGT) and then further modified in situ to produce Q. Here we use comparative genomics to implicate four gene families (best exemplified by the B. subtilis operon ykvJKLM) as candidates in the preQ1 biosynthetic pathway. Deletions were constructed in genes for each of the four orthologs in Acinetobacter. High pressure liquid chromatography analysis showed the Q nucleoside was absent from the tRNAs of each of four deletion strains. Electrospray ionization mass spectrometry confirmed the absence of Q in each mutant strain. Finally, introduction of the Bacillus subtilis ykvJKLM operon in trans complemented the Q deficiency of the two deletion mutants that were tested. Thus, the products of these four genes (named queC, -D, -E, and -F) are essential for the Q biosynthetic pathway. (+info)
(5/46) The promoter of the tgt/sec operon in Escherichia coli is preceded by an upstream activation sequence that contains a high affinity FIS binding site.
The tgt/sec operon in E. coli consists of five genes: queA, tgt, ORF12, secD, and secF. QueA and Tgt participate in the biosynthesis of the hypermodified t-RNA nucleoside Queuosine, whereas SecD and SecF are involved in protein secretion. Examination of the promoter region of the operon showed structural similarity to promoter regions of the rrn-operons. An upstream activation sequence (UAS) containing a potential binding site for the factor of inversion stimulation (FIS) was found. Gel retardation assays and DNaseI footprinting indicated, that FIS binds specifically and with high affinity to a site centred at position -58. Binding of FIS caused bending of the DNA, as deduced from circular permutation analysis. Various 5' deletion mutants of the promoter region were constructed and fused to a lacZ reporter gene to determine the influence of the UAS element on the promoter strength. An approximately two-fold activation of the promoter by the UAS element was observed. (+info)
(6/46) A truncated aminoacyl-tRNA synthetase modifies RNA.
Aminoacyl-tRNA synthetases are modular enzymes composed of a central active site domain to which additional functional domains were appended in the course of evolution. Analysis of bacterial genome sequences revealed the presence of many shorter aminoacyl-tRNA synthetase paralogs. Here we report the characterization of a well conserved glutamyl-tRNA synthetase (GluRS) paralog (YadB in Escherichia coli) that is present in the genomes of >40 species of proteobacteria, cyanobacteria, and actinobacteria. The E. coli yadB gene encodes a truncated GluRS that lacks the C-terminal third of the protein and, consequently, the anticodon binding domain. Generation of a yadB disruption showed the gene to be dispensable for E. coli growth in rich and minimal media. Unlike GluRS, the YadB protein was able to activate glutamate in presence of ATP in a tRNA-independent fashion and to transfer glutamate onto tRNA(Asp). Neither tRNA(Glu) nor tRNA(Gln) were substrates. In contrast to canonical aminoacyl-tRNA, glutamate was not esterified to the 3'-terminal adenosine of tRNA(Asp). Instead, it was attached to the 2-amino-5-(4,5-dihydroxy-2-cyclopenten-1-yl) moiety of queuosine, the modified nucleoside occupying the first anticodon position of tRNA(Asp). Glutamyl-queuosine, like canonical Glu-tRNA, was hydrolyzed by mild alkaline treatment. Analysis of tRNA isolated under acidic conditions showed that this novel modification is present in normal E. coli tRNA; presumably it previously escaped detection as the standard conditions of tRNA isolation include an alkaline deacylation step that also causes hydrolysis of glutamyl-queuosine. Thus, this aminoacyl-tRNA synthetase fragment contributes to standard nucleotide modification of tRNA. (+info)
(7/46) A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNAAsp QUC anticodon.
Escherichia coli encodes YadB, a protein displaying 34% identity with the catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-binding domain. We show that YadB is a tRNA modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the tRNA(Asp) QUC anticodon. This conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate tRNA(Asp) isolated from an E.coli tRNA-guanosine transglycosylase minus strain deprived of the capacity to exchange guanosine 34 with queuosine. Structural mimicry between the tRNA(Asp) anticodon stem and the tRNA(Glu) amino acid acceptor stem in prokaryotes encoding YadB proteins indicates that the function of these tRNA modifying enzymes, which we rename glutamyl-Q tRNA(Asp) synthetases, is conserved among prokaryotes. (+info)
(8/46) From cyclohydrolase to oxidoreductase: discovery of nitrile reductase activity in a common fold.
The enzyme YkvM from Bacillus subtilis was identified previously along with three other enzymes (YkvJKL) in a bioinformatics search for enzymes involved in the biosynthesis of queuosine, a 7-deazaguanine modified nucleoside found in tRNA(GUN) of Bacteria and Eukarya. Genetic analysis of ykvJKLM mutants in Acinetobacter confirmed that each was essential for queuosine biosynthesis, and the genes were renamed queCDEF. QueF exhibits significant homology to the type I GTP cyclohydrolases characterized by FolE. Given that GTP is the precursor to queuosine and that a cyclohydrolase-like reaction was postulated as the initial step in queuosine biosynthesis, QueF was proposed to be the putative cyclohydrolase-like enzyme responsible for this reaction. We have cloned the queF genes from B. subtilis and Escherichia coli and characterized the recombinant enzymes. Contrary to the predictions based on sequence analysis, we discovered that the enzymes, in fact, catalyze a mechanistically unrelated reaction, the NADPH-dependent reduction of 7-cyano-7-deazaguanineto7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of queuosine. We report here in vitro and in vivo studies that demonstrate this catalytic activity, as well as preliminary biochemical and bioinformatics analysis that provide insight into the structure of this family of enzymes. (+info)