tRNA Modification by S-Adenosylmethionine:tRNA Ribosyltransferase-Isomerase (original) (raw)
Related papers
Organic Letters, 2000
The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) catalyzes the penultimate step in the biosynthesis of the tRNA nucleoside queuosine, a unique ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor. The use of AdoMet in this way is fundamentally new to the chemistry of this important biological cofactor. We report here the first mechanistic studies of this remarkable enzyme, and we propose a chemical mechanism for the reaction consistent with our experimental observations.
Nucleic Acids Research, 1978
In this work, the first example of chemicgl synthesis of oligoribonucleo- tide containing the hypermodified nucleoside N -/N-threonylcarbonyl/- adenosine /t6A/ is presented. Synthesis of the heptamer C-C-C-A-U-t6A-A IX, the sequence of which is related to the anticodon loop of the initiator tRNA from yellow lupine, was achieved by: /i/ phosphotriester block synthesis of suitably protected heptamer VI containing an adenosine unit with a free exo-NH2 group, /ii/ highly effective "one-flask" procedure for the transformation of the free exo-NH2 group of adenosine unit of heptamer VI into a N,N'-disubstituted urea system of t6A of heptamer VII /hypermodification/, and /iii/ final deprotect- ion of VIII /3296 total yield/ with the use of a new approach for simultaneous hydrogenolysis /PdO-hydrogen-pyridine/ of the p-nitrobenzyl group and 2,2,2- trichloroethyl groups from carboxyl function of t6A and internucleotide phosphates respectively.
A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNAAsp QUC anticodon
Nucleic Acids Research, 2004
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 modi®ed 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.
Identification of the Rate-Determining Step of tRNA-Guanine Transglycosylase from Escherichia coli
Biochemistry, 2009
The modified RNA base queuine (7-(4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7deazaguanine) occurs in tRNA via a unique base exchange process catalyzed by tRNA-guanine transglycosylase (TGT). Previous studies have suggested the intermediacy of a covalent TGT-RNA complex. To exist on the reaction pathway, this covalent complex must be both chemically and kinetically competent. Chemical competence has been demonstrated by the crystal structure studies of Xie et al. (Nature Structural Biology (2003) 10, 781-788); however, evidence of kinetic competence had not yet been established. The studies reported here unequivocally demonstrate that the TGT-RNA covalent complex is kinetically capable of occurring on the TGT reaction pathway. These studies further suggest that product RNA dissociation from the enzyme is overall rate-limiting in the steady-state. Interestingly, studies comparing RNA with a 2′-deoxyriboside at the site of modification suggest a role for the 2′-hydroxyl group in stabilizing the growing negative charge on the nucleophilic aspartate (264) as the glycosidic bond to the aspartate is broken during covalent complex breakdown. Over one hundred chemically distinct modified bases are known to occur in RNA, the majority of which occur in tRNA (1). Of these, queuine (Q) stands out for several reasons. Structurally, queuine is the only modified base that is not a purine or pyrimidine analog. Instead, queuine features a pyrrolo-pyrimidine heterocyclic scaffold that is further elaborated through exocyclic chemical modifications. Perhaps most interestingly, the mechanism of incorporation of queuine into tRNA is unique. The queuine base is post-transcriptionally introduced into tRNA via a transglycosylation reaction that is catalyzed by tRNA-guanine transglycosylase (TGT) (Figure 1). Among the known modified bases, only pseudouridine is installed in an analogous manner whereby pseudouridine synthase breaks and reforms the glycosidic bond to the uracil (2). As with pseudouridine synthase, the chemical mechanism of the TGT reaction has been studied for some time. Two distinct mechanisms have been proposed and subsequently investigated for the TGT catalyzed base-exchange reaction. It was first envisaged that a dissociative mechanism, involving the intermediacy of an oxocarbonium ion, could drive the cleavage of
Chemistry & Biodiversity, 2005
tRNA is best known for its function as amino acid carrier in the translation process, using the anticodon loop in the recognition process with mRNA. However, the impact of tRNA on cell function is much wider, and mutations in tRNA can lead to a broad range of diseases. Although the cloverleaf structure of tRNA is wellknown based on X-ray-diffraction studies, little is known about the dynamics of this fold, the way structural dynamics of tRNA is influenced by the modified nucleotides present in tRNA, and their influence on the recognition of tRNA by synthetases, ribosomes, and other biomolecules. One of the reasons for this is the lack of good synthetic methods to incorporate modified nucleotides in tRNA so that larger amounts become available for NMR studies. Except of 2'-O-methylated nucleosides, only one other sugar-modified nucleoside is present in tRNA, i.e., 2'-O-b-d-ribofuranosyl nucleosides. The T loop of tRNA often contains charged modified nucleosides, of which 1-methyladenosine and phosphorylated disaccharide nucleosides are striking examples. A protecting-group strategy was developed to introduce 1-methyladenosine and 5''-O-phoshorylated 2'-O-(b-dribofuranosyl)-b-d-ribofuranosyladenine in the same RNA fragment. The phosphorylation of the disaccharide nucleoside was performed after the assembly of the RNA on solid support. The modified RNA was characterized by mass-spectrometry analysis from the RNase T1 digestion fragments. The successful synthesis of this T loop of the tRNA of Schizosaccharomyces pombe initiator tRNA Met will be followed by its structural analysis by NMR and by studies on the influence of these modified nucleotides on dynamic interactions within the complete tRNA.
Catalytic antisense RNAs produced by incorporating ribozyme cassettes into cDNA
Gene, 1991
A simple strategy is described for the generation of catalytic hammerhead-type ribozymes (Rz) that can be used as highly specific endoribonucleases to cleave a particular target RNA. The technique requires that a cloned cDNA fragment is available which encodes at least a part of the target RNA. About 25 different restriction recognition sequences can be utilized to incorporate specifically designed DNA cassettes into the cDNA. Besides some nucleotides which are specific for a certain restriction site, the DNA cassettes contain a sequence corresponding to the catalytic domain of the hammerhead Rz and, optionally, selectable marker genes, that are removable. The resulting recombinant DNA constructs permit the in vitro and in vivo synthesis of novel 'catalytic antisense RNAs' or 'antisense Rz (AZ)', which combine two features: (i) they bind like antisense RNA to their specific substrate RNA, and (ii) they cleave their target as hammerhead Rz do. The utility of the strategy to generate Rz was demonstrated experimentally by incorporating a synthetic SalI-specific DNA ribozyme (Rz) cassette into a unique Sal1 site of a cloned cDNA fragment of plum pox virus (PPV), which is a single-stranded positive sense plant RNA virus, belonging to the group of potyviruses. The resulting AZ constructs delivered AZ that were directed against the PPV ( + ) or (-) RNA, respectively, which cleaved their corresponding target RNAs in the expected manner. Besides the synthetic Rz cassette, a comparable S&-specific Rz cassette, that had been prepared from a specifically designed plasmid and that contained the tet gene inserted into the sequence of the catalytic domain of the Rz, was also incorporated into the S&I site of the PPV cDNA. The catalytic activity of the resulting AZ was maintained, though the much larger sequence of the marker gene was inserted into the catalytic domain.
Detection of queuosine and queuosine precursors in tRNAs by direct RNA sequencing
Nucleic Acids Research
Queuosine (Q) is a complex tRNA modification found in bacteria and eukaryotes at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall...
Biomolecules, 2017
QueF enzymes catalyze the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ₀) to 7-aminomethyl-7-deazaguanine (preQ₁) in the biosynthetic pathway to the tRNA modified nucleoside queuosine. The QueF-catalyzed reaction includes formation of a covalent thioimide intermediate with a conserved active site cysteine that is prone to oxidation in vivo. Here, we report the crystal structure of a mutant of Bacillus subtilis QueF, which reveals an unanticipated intramolecular disulfide formed between the catalytic Cys55 and a conserved Cys99 located near the active site. This structure is more symmetric than the substrate-bound structure and exhibits major rearrangement of the loops responsible for substrate binding. Mutation of Cys99 to Ala/Ser does not compromise enzyme activity, indicating that the disulfide does not play a catalytic role. Peroxide-induced inactivation of the wild-type enzyme is reversible with thior...