Quantitative enzymatic hydrolysis of tRNAs (original) (raw)
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Purification and Characterization of the tRNA-processing Enzyme RNase BN
Journal of Biological Chemistry, 2000
RNase BN, a tRNA-processing enzyme previously shown to be required for the 3-maturation of certain bacteriophage T4-encoded tRNAs, was overexpressed and purified to near homogeneity from Escherichia coli. The purified enzyme, which is free of nucleic acid, is an ␣ 2-dimer with a molecular mass of ϳ65 kDa. RNase BN displays a number of unusual catalytic properties compared with the other exoribonucleases of E. coli. The enzyme is most active at pH 6.5 in the presence of Co 2؉ and high concentrations of monovalent salts. It is highly specific for tRNA substrates containing an incorrect residue within the universal 3-CCA sequence. Thus, tRNA-CU and tRNA-CA are effective substrates, whereas intact tRNA-CCA, elongated tRNA-CCA-Cn, phosphodiesterase-treated tRNA, and the closely related tRNA-CC are essentially inactive as substrates. RNA or DNA oligonucleotides also are not substrates. These data indicate that RNase BN has an extremely narrow substrate specificity. However, since tRNA molecules with incorrect residues within the-CCA sequence are not normally produced in E. coli, the role of RNase BN in uninfected cells remains to be determined.
The use of cloned tRNA genes for the purification and measurement of specific tRNAs
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1984
We have previously reported the ability of a cloned tRNAi Met gene (pt145) to bind tRNAi met specifically [5l. In this paper, we show that a pBR322 plasmid containing the tRNA As" gene of Xenopus (pt38 -donated by Stuart Ciarkson) will specifically bind to mouse tRNA AS" when total mouse tRNA, extracted from uninduced Friend erythroleukemia cells, is hybridized to the gene probe. Oue-dimensional electrophoresis of the hybridizing tRNA in 20% polyacrylamide reveals one major band and several small-molecular-weight minor bands. The hybridizing tRNA has been identified as tRNA A~n by partial RNA sequencing and the detection of both the Q base and t6A. The steady-state concentration of tRNA Asn in the uninduced Friend cell was determined by hybridizing tRNA labeled in vitro to pt38. 1% of the total tRNA hybridized, representing 0.017 pg tRNAASn/cell. The fraction of newly synthesized tRNA representing tRNA A~a or tRNAi Met was also determined by hybridizing tRNA labeled in vivo to either pt38 or pt145, respectively. 0.96% and 0.85% of the tRNA hybridized to pt38 and pt145, respectively.
Methods, 2008
Here we describe the many applications of acid urea polyacrylamide gel electrophoresis (acid urea PAGE) followed by Northern blot analysis to studies of tRNAs. Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Among the examples described are (i) analysis of the effect of mutations in the Escherichia coli initiator tRNA on its aminoacylation and formylation; (ii) evidence of orthogonality of suppressor tRNAs in mammalian cells and yeast; (iii) analysis of aminoacylation specificity of an archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNA Pro with cysteine, but does not aminoacylate archaeal tRNA Cys with cysteine; (iv) identification and characterization of the AUAdecoding minor tRNA Ile in archaea; and (v) evidence that the archaeal minor tRNA Ile contains a modified base in the wobble position different from lysidine found in the corresponding eubacterial tRNA.
LC-MS based quantification of 2′-ribosylated nucleosides Ar(p) and Gr(p) in tRNA
Chemical Communications, 2011
RNA nucleosides are often naturally modified into complex non-canonical structures with key biological functions. Here we report LC-MS quantification of the Ar(p) and Gr(p) 2 0-ribosylated nucleosides in tRNA using deuterium labelled standards, and the first detection of Gr(p) in complex fungi.
Enzymatic hydrolysis of N-substituted aminoacyl-tRNA
Proceedings of the National Academy of Sciences of the United States of America, 1967
The mechanism of the release of polypeptide chains from the ribosome-messenger RNA complex is not fully understood. It has been reported that free polypeptide chains are formed in cell-free protein-synthesizing systems, directed by polyribonucleotides, only if these polynucleotides contain statistically high frequencies of UAA codons.'-5 However, we do not know how the chain is released from the tRNA ribosome-mRNA complex after interruption of the translation by the UAA triplet. This release implies a hydrolysis of the ester bond between polypeptide and tRNA which could be catalyzed by a specific enzyme. The search for such an enzymatic activity necessitates the use of the relatively unstable polypeptidyl-tRNA's. It is difficult and laborious to prepare them in measurable quantities. In contrast, the chemically N-substituted aminoacyl-tRNA's, although having similar characteristics in other respects, are stable and readily synthesized.6' I An enzyme capable of hydrolyzing this ester linkage between N-acetylamino-acids and tRNA's has now been found in extracts of Escherichia coli. This enzyme was partially purified and several of its characteristics were studied. The enzyme also catalyzes the hydrolysis of di-phenylalanyl-tRNA and N-substituted oligopeptidyl-tRNA's. Material.-C'4-amino acids were obtained from the Commissariat a l'Energie Atomique (France); E. coli B tRNA, from General Biochemicals; crystalline pancreatic DNase and RNase, from Mann Research Laboratories; snake venom phosphodiesterase, from British Drug Houses Ltd.; T1 RNase, from Sigma Corp. E. coli leucine-specific tRNA of about 50% purity was a gift from Dr. M. Yaniv; and a sample of H3-diphenylalanyl tRNA, from Dr. C. Ganoza. The tRNA was charged with different C"4-amino acids in the presence of an E. coli 105,000 X g supernatant. The C'4-aminoacyl-tRNA was acetylated with acetic anhydride, as described by Haenni and Chapeville.7 In all cases it was shown that after acetylation all amino groups of the tRNA-bound amino acids were substituted. When serine and threonine are used it is possible that the OH groups also react with acetic anhydride, forming the corresponding esters. C'4-diphenylalanyl-tRNA was prepared according to Nakamoto and Kolakofsky8 by incubating C14-phenylalanyl-tRNA in the presence of ribosomes and 105,000 X g supernatant without addition of GTP. C'4-polylysyl-tRNA was prepared from an incubation mixture of E. coli ribosomes with C'4-lysyl-tRNA, poly A, GTP, and E. coli supernatant. Methods.-Analysis of the degradation products of N-acetylaminoacyl-tRNA: For most of the N-acetylaminoacyl-tRNA's, the method described below for N-acetylleucyl-tRNA was used. N-acetylleucine, leucine, N-acetylleucyladenosine (obtained after digestion of N-acetylleucyl-tRNA with pancreatic ribonuclease), and N-acetylleucyl-tRNA were separated by paper electrophoresis (Fig. 1). Under the same conditions, after treatment with RNase T1, two N-acetylleucyloligonucleotides were separated, one of which migrates with N-acetylleucine (Fig. 6). If a similar mixture had to be analyzed, both N-acetylleucyloligonucleotides would be converted to N-acetylleucyladenosine by treatment with pancreatic RNase before electrophoresis. N-acetylleucyl-tRNA, N-acetylleucyladenylate (N-acetylleucyl AMP, obtained after digestion with purified venom phosphodiesterase of N-acetylleucyl-tRNA), N-acetylleucyladenosine, and 2079
Chemical and Conformational Diversity of Modified Nucleosides Affects tRNA Structure and Function
Biomolecules
RNAs are central to all gene expression through the control of protein synthesis. Four major nucleosides, adenosine, guanosine, cytidine and uridine, compose RNAs and provide sequence variation, but are limited in contributions to structural variation as well as distinct chemical properties. The ability of RNAs to play multiple roles in cellular metabolism is made possible by extensive variation in length, conformational dynamics, and the over 100 post-transcriptional modifications. There are several reviews of the biochemical pathways leading to RNA modification, but the physicochemical nature of modified nucleosides and how they facilitate RNA function is of keen interest, particularly with regard to the contributions of modified nucleosides. Transfer RNAs (tRNAs) are the most extensively modified RNAs. The diversity of modifications provide versatility to the chemical and structural environments. The added chemistry, conformation and dynamics of modified nucleosides occurring at the termini of stems in tRNA's cloverleaf secondary structure affect the global three-dimensional conformation, produce unique recognition determinants for macromolecules to recognize tRNAs, and affect the accurate and efficient decoding ability of tRNAs. This review will discuss the impact of specific chemical moieties on the structure, stability, electrochemical properties, and function of tRNAs.
OXOPAP assay: For selective amplification of aminoacylated tRNAs from total cellular fractions
Methods, 2008
Transfer RNA (tRNA) plays a pivotal role in protein synthesis within cells, where it is recognized by one cognate aminoacyl-tRNA synthetase, in competition with the remaining non-cognate synthetases, and esterified with an amino acid. For many years the levels of tRNA aminoacylation, in a given population of cellular RNA, have been analyzed using methods that include northern analysis and/or oxidation techniques to separate aminoacylated from non-aminoacylated species. In the present report we describe an approach recently developed by us that combines oxidation-protection with polyadenylation and PCR. The OXOPAP approach permits the amplification of tRNA species that are nearly identical and that evade differential identification by more classical northern hybridization methods. Our approach also allows the identification of aminoacylatable ''naïve'' species, where no prior knowledge of sequence content is necessary for amplification.
Functional assay of tRNA molecules transcribed from a purified gene
Nucleic Acids Research, 1982
Purified tRNA genes are expressed when microinjected into the nucleus of X.laevis oocytes. In this paper we describe a method to assay the capacity to be aminoacylated of the tRNA transcribed in the frog oocytes. The method exploits the radiochemical purity of the transcript and relies on the binding of aminoacyl-tRNA but not of uncharged tRNA to purified elongation factor EF-Tu. We also present some preliminary results on several single point mutants of tRNA ro from Caenorhabditis elegans. We show that nucleotide 73 of tRNAPro can be substituted by any other nucleotide without loss of acceptor activity. A double mutant, causing transition from G45G46 to A45A46 has lost acceptor activity. Also inactive is a mutant carrying an insertion of a single base in the anticodon loop.
Bulletin of the Chemical Society of Japan, 2007
This report characterizes the structure and function of four modified nucleosides first identified by myself or members of my research group in thermophilic bacteria and animal mitochondria over the past 30 years. I identified 2-thioribothymidine (s 2 T) (or 5-methyl-2-thiouridine (m 5 s 2 U)) in 1974 at position 54 in the T loop of tRNAs from an extreme thermophile, Thermus thermophilus, as well as related thermophiles, and a good deal of evidence has shown that it is required for thermostabilization of tRNAs, functioning at high temperatures. The functional roles and the biosynthetic pathway of s 2 T are outlined here. My research group has identified three novel modified nucleosides, 5-formylcytidine (f 5 C), N-(uridine-5-ylmethyl)taurine (m 5 U), and N-(2-thiouridine-5-ylmethyl)taurine (m 5 s 2 U), at the first anticodon position of mitochondrial (mt) tRNAs from higher animals (f 5 C is present in tRNA Met , m 5 U in tRNA Leu (UUR) and tRNA Trp , and m 5 s 2 U in tRNA Gln , tRNA Lys , and tRNA Glu). Their chemical structures were determined and their functional roles in translation were examined. Additionally, f 5 C at the first anticodon position of mt tRNA Met plays a crucial role in decoding the AUA codon (decoded as isoleucine in the universal genetic code system) as methionine, which will form the second topic of this article. Defective modification of m 5 U and m 5 s 2 U at the first anticodon position in human mt tRNA Leu (UUR) and tRNA Lys , respectively, is the result of point mutations in these tRNAs in mitochondria. It is strongly suggested that these defects are the direct cause of two mitochondrial diseases, MELAS (mitochondrial encephalomyopathies, encephalopathy, lactic acidosis, and stroke-like episodes) and MERRF (myoclonus epilepsy associated with ragged-red fibers). Their molecular mechanisms are also discussed here. These studies would serve to highlight again the importance of modified tRNA nucleosides in the structure and function of tRNAs.
Enzymatic Formation of Modified Nucleosides in tRNA: Dependence on tRNA Architecture
Journal of Molecular Biology, 1996
Information is still quite limited concerning the structural requirements in 1 Laboratoire d'Enzymologie tRNA molecules for their post-transcriptional maturation by base and du CNRS, 1 avenue de la ribose modification enzymes. To address this question, we have chosen as Terrasse, F-91198 the model system yeast tRNA Asp that has a known three-dimensional Gif-sur-Yvette, France structure and the in vivo modifying machinery of the Xenopus laevis oocyte 2 Department of Microbiology able to act on microinjected tRNA precursors. We have systematically University of Umeå compared the modification pattern of wild-type tRNA Asp with that of a S-901 87 Umeå, Sweden series of structural mutants (21 altogether) altered at single or multiple positions in the D-, T-and the anticodon branch, as well as in the variable 3 Unité Propre de Recherche region. The experimental system allowed us to analyze the effects of ''Structure des structural perturbations in tRNA on the enzymatic formation of modified Macromolécules Biologiques et nucleosides at 12 locations scattered over the tRNA cloverleaf.